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Classical Media

Reagents

Serum

Stem Cell Products

Serum-Free Media

Process Supplements

Specialty Waters

Custom Products

Rapid Response Production

Thermo Scientific HyClone Cell Culture Products and Capabilities

2010/2011

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©2010 Thermo Fisher Scientific Inc. All rights reserved. Pluronic F68 is a trademark of BASF Corp. PER.C6 is a trademark of Crucell NV. High Five is a trademark of Invitrogen Corp. Pluronic F68 is a trademark of BASF Corp. Dow Corning is a trademark of Dow Corning Corporation. High Five is a trademark of Invitrogen Corp. Sigmacote is a registered trademark of Sigma Chemical Co. Tween 80 is a registered trademark of ICI Americas, Inc. Celligen Plus is a registered trademark of New Brunswick Scientific. High Five is a trademark of Invitrogen Corp. C-Flex is a registered trademark of Saint-Gobain. All other trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. Specifications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details.

Cell Culture & bioProcessing 925 West 1800 SouthLogan, UT84321

in americas/asia435-792-8000800-492-5663 toll-free435-792-8001 fax

in europe+32 53 85 75 59+32 53 85 74 31 fax

www.thermo.com/hyclonewww.thermo.com/perbio

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1

Introduct

ion

Thermo Scientific HyClone Media and Sera Table of Contents

Introduction

About HyClone® Media Products..............................................................................3

A History of Innovation and Quality ..........................................................................4

Commitment to Quality..............................................................................................5

Quality Control and Product Release Testing ..........................................................6

Design and Production Control and RawMaterials ................................................7

Validation Qualifications............................................................................................8

Facilities......................................................................................................................9

New Expansion of Manufacturing Operations For Single-UseBioProcessing Containers........................................................................................12

Product Line Overview ............................................................................................13

Thermo Scientific HyClone Media Product Listing

Capabilities ..............................................................................................................14

Classical Liquid Media ............................................................................................16

HyQPAK™ Convenience Products ..........................................................................20

Classical Dry Powdered Media ..............................................................................22

HyQ-RS™ Reduced SerumMedia ..........................................................................25

Stem Cell Media, Supplements, Reagents and Cells ..........................................26

About AdvanceSTEM™ ......................................................................................26

Murine Stem Cell Products ................................................................................27

Primogenix® Cells ................................................................................................28

Mesenchymal Stem Cell Products......................................................................29

CET Mesenchymal Stem Cells ............................................................................32

CET Somatic Stem Cells ......................................................................................33

HyStem Hydrogels ..............................................................................................34

Serum-Free Media Platforms..................................................................................35

CHO Cell Culture Platform ..................................................................................36

Hybridoma and Myeloma Cell Culture Platform ..............................................39

HEK 293 and PER.C6® Cell Culture Platform......................................................43

Insect Cell Culture Platform ................................................................................47

Viral Vaccine Cell Culture Platform ....................................................................49

Process Supplements ..............................................................................................50

Nutrient Media Supplements ................................................................................53

Reagents and Cell Dissociation Products ..............................................................54

Process Liquids, Buffers and Salts ........................................................................55

Antibiotics and Selection Agents ..........................................................................58

Microcarriers............................................................................................................60

Antifoam ..................................................................................................................63

CustomMedia Products ..........................................................................................65

Rapid Response Production ....................................................................................66

Thermo Scientific HyClone Sera Product Listing

About HyClone Sera Products ................................................................................67

Fetal Bovine Serum (FBS) ........................................................................................69

Defined Fetal Bovine Serum, US Origin............................................................69

Characterized Fetal Bovine Serum, US Origin..................................................69

Standard Fetal Bovine Serum, US Origin..........................................................70

New Zealand Foetal Bovine Serum ..................................................................70

New Zealand Standard Foetal Bovine Serum..................................................70

Characterized Australian Foetal Bovine Serum................................................71

Australian Standard Foetal Bovine Serum ......................................................71

USDA Tested Fetal Bovine Serum ....................................................................71

Research Grade Fetal Bovine Serum, South American Origin ........................72

Specialty Fetal Bovine Serum ................................................................................75

Super Low IgG Fetal Bovine Serum, US Origin ................................................75

Charcoal/Dextran Treated Fetal Bovine Serum, US Origin..............................75

Dialyzed Fetal Bovine Serum, US Origin ..........................................................76

Lipid-Reduced Fetal Bovine Serum, US Origin ................................................76

Super Safe New Zealand Irradiated Fetal Bovine Serum ..............................76

Embryonic Stem (ES) Cell Screened Fetal Bovine Serum, US Origin ..............77

HumanMesenchymal Stem Cell Screened Fetal Bovine Serum, US Origin ......77

Tetracycline Screened Fetal Bovine Serum, US Origin ....................................77

Insect Cell Screened Fetal Bovine Serum, US Origin ......................................78

Bovine Calf Serum™................................................................................................80

Iron-Supplemented Bovine Calf Serum, US Origin ..........................................80

Bovine Calf Serum, US Origin ..........................................................................80

Cosmic Calf™ Serum, US Origin ......................................................................80

Bovine Growth Serum, US Origin......................................................................81

Newborn Bovine Calf Serum, US Origin ..........................................................81

New Zealand Newborn Bovine Calf Serum ....................................................81

New Zealand Iron-Supplemented Newborn Serum ........................................82

New Zealand Bovine Growth Serum................................................................82

New Zealand Cosmic Calf Serum ....................................................................82

Fetal Bovine Serum Alternatives ............................................................................85

FetalClone® I, US Origin ....................................................................................85

FetalClone II, US Origin......................................................................................85

FetalClone III, US Origin ....................................................................................85

Alpha Calf™ Fraction ........................................................................................86

Sera Products ..........................................................................................................88

Donor Adult Bovine Serum, US Origin..............................................................88

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Thermo Scientific HyClone Media and Sera Table of Contents

Adult Bovine Serum, New Zealand Origin ......................................................88

Donor Equine Serum, US Origin........................................................................88

Donor Equine Serum, US Origin........................................................................88

Donor Bovine Serum, New Zealand Origin ......................................................89

Porcine Serum, New Zealand Origin ................................................................89

EMEA Conforming Serum Products........................................................................91

EMEA/CVMP/743 /00 and EMEA/CPMP/BWP/1793 /02 Conforming ..........91

EMEA/CPMP/BWP/1793 /02 Conforming ......................................................92

Thermo Scientific HyClone BioProcess Containers

Labtainers™ ......................................................................................................93

General Cell Culture Protocols

Passage of Adherent Cell Lines (Subculture) ..................................................96

Viability Assay....................................................................................................97

HyQTase™ and Trypsinization Protocol ............................................................98

Hemocytometer Counting..................................................................................99

Generation of Growth Curve ..........................................................................100

Plating Efficiency..............................................................................................101

Cryopreservation of Cells ................................................................................102

Thawing Cryopreserved Cells ........................................................................103

Application Specific Technical Information—Protocols

CDM4CHO™ ....................................................................................................104

SFM4CHO™ ....................................................................................................105

SFM4CHO-A™ ................................................................................................106

SFM4CHO-Utility ............................................................................................107

CDM4NS0™ ....................................................................................................108

CDM4MAb™....................................................................................................109

ADCF-MAb™....................................................................................................110

SFM4MAb™ ....................................................................................................111

SFM4MAb-Utility ............................................................................................112

LS1000™ ..........................................................................................................113

LS250™ ............................................................................................................113

CDM4PERMAb™ ............................................................................................114

CDM4Retino™ ................................................................................................115

CDM4HEK293™ ..............................................................................................116

SFM4Transfx-293™ ........................................................................................117

SFM4HEK293™................................................................................................118

SFM4MegaVir™ ..............................................................................................119

SFX-Insect™ ....................................................................................................120

SFX-Insect rAutographa ..................................................................................121

SFX-Insect Production......................................................................................121

SFM4Insect ......................................................................................................122

Cell Boost™ Process Supplement Preparation ..............................................123

HyQSphere™Microcarriers—Siliconizing Glassware..................................124

HyQSphere Microcarriers—Cell Culture Initiation ......................................125

HyQSphere Microcarriers—Harvesting Cells from 200 mL MicrocarrierCultures ............................................................................................................126

HyQSphere Microcarriers-Microcarrier Sampling Technique ......................127

HyQSphere (HLX II-170) Microcarriers Cell Culture Initiation ......................128

HyQSphere (HLX II-170) Microcarriers Harvesting Cells MicrocarrierCulture ..............................................................................................................129

HyQSphere (HLX II-170) Microcarriers Microcarrier Sampling Technique ..130

AdvanceStem Protocols ..................................................................................131

Application Specific Technical Information—Application NotesStem Cell Protocols

Chinese Hamster Ovary Cells for the Production of RecombinantGlycoproteins ..................................................................................................133

Lipids in Cell Culture Media............................................................................135

Adherent Growth of CHO Cells in SFM4CHO-A Medium Using Corning®CellBIND® Surface Culture Flasks ..................................................................138

Metabolically Designed Serum-Free Media For HEK293 and PER.C6Cell Lines ..........................................................................................................139

Production of Poliovirus Using Vero Cells Cultured in SFM4MegaVir..........142

A Novel Process for rProtein Production Using BEVS....................................144

Growth Comparison Studies Between FBS and Other Serum Products ......148

Cell Lines Grown Successfully in FetalClone I, II, and III ..............................153

Heat Inactivation—Are YouWasting Your Time? ........................................154

Viral Safety in Serum for Cell Culture Use ....................................................158

Single-Use Bioreactor (S.U.B.) for Vero Cell Culture on Microcarriers ........165

Formulations........................................................................................................168

Standard BPC Packaging Options for Liquid Media/Buffers ..................212

Standard Terms and Conditions of Sale ......................................................220

Acronyms ............................................................................................................223

Product Index (in alphabetical order) ..........................................................225

Product Index (by part number) ......................................................................229

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Introduct

ion

About Thermo Scientific HyClone Products

Thermo Scientific HyClone Cell Culture products and BioProcessing systems accelerateproductivity for life-science research and protein-based drug production. Widely knownfor more than 30 years as the premier supplierof quality sera for cell culture, these classicalmedia, serum-free and protein-free media,process liquids and flexible, single-use BioProcess Container systems set industrystandards for quality and innovation. Withdecades of experience in optimizing cell culture performance Thermo Scientific HyCloneproduct experts understand your researchneeds. Our expertise in cell culture science canfacilitate your research to maximize results.

Mission

Our reputation and success is built on our ability to provide high-quality products manufactured according to specification, ontime, every time. We understand the needs ofour customers and the industry, and have builtour quality systems, manufacturing processes,and products in response to those requirements.

Our mission is to be the leading global supplierof quality solutions for challenges faced by thebiopharmaceutical industry and animal cell culture-based researchers. By applying our expertise in animal cell nutrition as it influencescell function and product yield through the production and delivery of liquid media andprocess liquids, powdered cell culture media,reagents and liquid handling systems, we operate a business that expresses value to our customers, suppliers, distributors and employees through:• Scientific and Technical Excellence• Integrity• Teamwork• Honesty• Respect• Trust• Continuous Improvement• Creativity• Innovation• Personal Growth• Corporate Growth• Productivity

Company Overview

We are a global manufacturer of sera,liquid and powdered media, process liquids,and liquid handling systems to support research and production of biopharmaceuticals. Thermo Scientific HyClone products continue to be produced with consistency and quality at our main facilities located in Logan, Utah.

The primary Logan campus includes manufacturing facilities for sera, dry powdered media, liquid media, process liquids and disposable BioProcess Container(BPC) in addition to warehouses and a maindistribution center. In addition, our Researchand Product Development, as well as DesignEngineering, are also centered in Logan.

Other American operations include multipleblood collection and serum processing sites at abattoirs throughout the United States,Canada and South America.

Global markets are serviced through serumprocessing sites in New Zealand and Australia,a liquid media and BPC production site inCramlington, England, and a liquid media production facility in Beijing, China. International sales and distribution offices andwarehouses are located in Aalst, Belgium;Hong Kong; and Beijing, China.

The Logan manufacturing facility for HyCloneCell Culture and BioProcessing products is fullycompliant with current Good ManufacturingPractices (cGMP) for Medical Devices, ISO9001:2000, and ISO 13485:2003 guidelines asdescribed in 21 C.F.R. §820 et seq.

Logan, Utah facilities


4

A History of Innovation and Quality

A History of Innovation and Quality

A commitment to providing researchers glob-ally with the most innovative tools to facilitateand progress research provides the foundationfor all Thermo Scientific Cell Culture andBioProcessing products.

As a global manufacturer of sera, liquid andpowdered media, process liquids and handlingsystems to support research and production ofbiopharmaceuticals, Thermo Scientific HyCloneproducts continue to be manufactured to be thehighest and most consistent quality product.

For more than 40 years, Thermo ScientificHyClone Sera products have been remaineddedicated to the advancement of sciencethrough the support of cell culture and acomprehensive commitment to academia,research and industry.

As a leading supplier of serum, media andother cell culture reagents, we strive to keepprovide innovations and advances in cell cultureand to continually expand our knowledge andexpertise in the major areas of research.

With decades of experience in optimizing cellculture performance, these product expertsunderstand research needs. Our expertise incell culture science can facilitate your researchto maximize results.

Continuing to Lead

With innovations in serum processing,disposable liquid manufacturing, high-speedparticle reduction of dry powdered media, andspecialized products for stem cell research,we have evolved into a world leader inmanufacturing media, reagents, sera andBioProcess Container systems.

Meeting the Growing Demandsof the Industry

Since the bioprocessing industry moved intothe 21st century, we have responded to theneeds of customers by significantlyexpanding manufacturing operations.

From our state-of-the-art powdered mediamicronization facility to our revolutionarysingle-use disposable manufacturing technology,our organization is well positioned to providethe products and delivery systems neededfor customers to increase manufacturingefficiency and accelerate product delivery tothe marketplace.

To meet the ever-increasing needs of ourcustomers, we have recently expanded ourWater For Injection (WFI) Quality water systems.

We also partner with our customers in processdevelopment. Whether these requirementsinclude customized liquid delivery systems ormedia optimized for a particular cell line orapplication, these expert development staff istrained to assess these exacting requirementsand provide effective solutions.

Whether a customer's processes are in basicresearch, process development or in full-scaleproduction, our products include a completeline of classical, serum-free, protein-free andcustom media formulations in liquid andpowder forms to meet their needs.

Thermo Scientific HyClone cell culture media,buffers, balanced salt solutions and reagentsare manufactured with the same attentionto detail as our legacy serum products. Weprovide these products in convenient deliverysystems ranging from small volume bottlesto large volumes inour industry-leadingBioProcess Container Systems.

From production to quality control to finalproduct packaging, every care is taken toensure the final products our customers receiveare of the highest quality and consistency.

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Introduct

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Commitment to Quality

All Thermo Scientific HyClone products areproduced according to our Quality System,which is based on compliance with currentGood Manufacturing Practices (cGMP) forMedical Devices and ISO 9001:2000 and ISO13485:2003 guidelines.

We regularly obtain completed and signedanimal origin questionnaires from all suppliersof our raw materials and contact components.We can determine which (if any) ingredients ina medium formulation are of animal origin or, ifthey have come in contact with animal-derivedmaterials during processing. Additionally, we canidentify the tissue, species and country of originof those materials that are animal in origin.

As part of our commitment to quality wemaintain a database in Logan, Utah thatcontains supplier information on mode ofproduction and processing steps that reduce therisk of adventitious animal agents. Through ourEnterprise Resource Planning (ERP) softwaresystem, we are able to generate formula-specificreports on all ingredients and componentslisted in a given Bill of Materials.

With heightened concerns in the industryregarding animal origin components, We havetaken the leadership role to obtainCertificates of Suitability from the EuropeanPharmacopoeia for all serum products.Selected information described on this pagecan be shared with customers subject toappropriate confidentiality assurances.

Quality Manual

Quality Assurance policies required for cGMP,ISO 9001:2000 and ISO 13485:2003compliance are summarized in our QualityManual. This manual is available for reviewduring customer audits of our Logan facilities.

Audits

We are subject to inspection by severalregulatory agencies including the Food andDrug Administration and the United StatesDepartment of Agriculture. Continuedconformance with ISO 9001:2000 and ISO13485:2003 is confirmed via biannual auditsby SGS International Certification Services.Customers from around the world areinvited to visit the Thermo Fisher Scientificmanufacturing facilities for HyClone products(under appropriate confidentiality agreements).

Document Control and Quality Records

In addition to our other quality systems, wemaintain a validated electronic documentmanagement system which controls allrevisions, approvals and document security.Quality records are maintained to demonstratecompliance with specified requirements.


6

Examples ofLiquid MediaRelease Criteria

Examples ofPowdered MediaRelease Criteria

Appearance AppearancepH pHOsmolality OsmolalityEndotoxin EndotoxinSterility Solubility

Growth PromotionMoisture

Examples of Bovine SerumRelease CriteriaEndotoxin (Limulus Amebocyte Lysate—Gel Clotor EP)Hemoglobin (spectrophotometric)Sterility Testing (Current USP and EP)

Bacteria and FungiVirus Testing (9CFR 113.53 or Full EMEA)

Fluorescent AntibodyBluetongueBovine AdenovirusBovine ParvovirusBovine Respiratory Syncytial VirusBovine Viral Diarrhea VirusRabiesReovirus

Cytopathogenic AgentsHemadsorbing Agents

Mycoplasma (Large Volume Direct Culture, HoechstStain, or EP)

In order to meet customers' unique testingrequirements, additional quality control testingis available on a custom basis. We haveaudited and reserve the right to utilizequalified, approved third-party testing facilitiesas needed.

We take pride in providing the highest qualityproducts and services to assure conformanceto customer requirements and appropriateregulatory standards. In order to maintainthese standards, all of our products and rawmaterial components must pass strict QualityControl testing. In addition, our productspecifications define and stipulate the finishedpart inspection necessary for acceptabilityand release of each lot of finished product.

The finished product release tests andacceptability levels for custom productapplications are jointly determined with ourcustomers. For standard products we developexclusively, appropriate release tests andspecifications are established.

Each product specification includes validatedrelease criteria and in-process testing. Onlythose products that conform to these stringentrequirements are released for sale. A detailedCertificate of Analysis (COA) that includes theproduct attributes, specifications and actualtest results is provided with each lotmanufactured. The release criteria listed to theright are examples of testing performed toensure quality products.

Product Specifications

Thermo Scientific HyClone media and serumproducts are manufactured to meet our highstandards for consistency, traceability andquality. Product specifications are thoroughlydefined for each lot and adherence to our rigidprocedures for product manufacturing, testingand handling are well-documented.

Our business operations are coordinatedthrough an integrated ERP computer systemthat drives planning, manufacture, release andshipment of every product. Under this system,unique part numbers and specifications areestablished for all raw materials andcomponents, process intermediates andfinished products.

Each part number has a correspondingspecification document. Defined within thespecification are the product name, labelinginformation, packaging, formulation, referenceprocedures for manufacture or testing, releasetesting and criteria for acceptance, storage,and shipping conditions, expirationinformation and all information to bereported on the COA.

Quality Control and Product Release Testing

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Introduct

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Design and Production Control and Raw Materials

Design Control and New ProductDevelopment

Design and development of new productsundergoes a Stage-Gate Process, which isunder the control and supervision of GateCommittees. The Gate Committees arecross-functional committees made up ofrepresentatives from our Research andProduct Development, Marketing, Sales,Engineering, Operations and Quality Controland Assurance departments.

The Gate Committees are responsible formanaging resources and evaluating theviability of projects based on deliverables,resource requirements, risk and return oninvestment. Once a project is approved, theCommittee chooses a team leader and assistsin organizing a cross-functional project team.

The project team, under the direction of theteam leader, carries out all aspects of the newproduct development process through eachstage of development. While the bulk of thisdevelopmental work resides in Research andProduct Development or Engineering, teammembers are cross-functional, ensuring asmooth transition from research anddevelopment through scale-up, production ofpilot and qualification lots, product validationand documentation. The end result is a qualityproduct that meets customer specifications.

Production Control

Our production control processes ensurelot-to-lot consistency and product qualitywhile establishing full manufacturingtraceability for each lot. Product ControlDocuments (PCDs) transmit information frommanufacturing specifications into productiondrivers and lot history records.

Production orders are generated within the ERPcomputer system where information fromcontrolled Bills of Material merge with orderedlot sizes to generate pick lists and batch recipedocuments.

Requests for serum production are initiated bythe Production Coordinator and are submittedto the Master Scheduler. The MasterScheduler then combines and locks theappropriate production ticket assembled from

the approved PCD, the approved QualityControl Test Report and the approved Bill ofMaterials. The PCD becomes the stepwisemanufacturing record that physicallyaccompanies the lot through production.

In addition to listing production steps, thePCD includes spaces for operator responsesand sign-offs to track and document eachprocedure. Such response spaces includecheck-offs for additions of ingredients(with confirmed amounts and component lotnumbers), measurements, adjustments andprocess procedures.

We adhere to cGMP guidelines for properinitialing, dating, supervisor review and sign-offfor these documents.

Raw Materials

Only the highest quality raw materials areused in the manufacture of all our products.Each item has its own unique part number andcontrolled raw material specification. Thespecification document describes the product,the required grade or purity, the source and thecriteria for acceptance on incoming inspection.These specifications are provided to eachrespective supplier who must in turn certifythat their product meets this standard forqualification.

Our suppliers are rated and approved through asupplier certification program, which evaluateseach supplier for quality, availability,performance and service. Where possible,secondary and back-up suppliers for each rawmaterial or component are qualified. Customer-supplied or specified raw materials can beincorporated into a product formulation andare given their own unique part numbers.

Since Thermo Scientific HyClone products areused in the biopharmaceutical industry, thestandard procedure is to specify USP (UnitedStates Pharmacopoeia) or NF (National Formu-lary) grades where available. Multicompendialgrades such as EP, JP or others can also bespecified where available at an addedpremium. For those raw materials that do nothave pharmacopoeial monographs established,we specify chemicals that meet FCC (FoodChemical Codex), ACS (American ChemicalSociety) or other applicable grades. Any otherchemicals must meet minimum requirementsestablished by our Quality Assurance Department.

Incoming raw materials are quarantined for QCinspection and acceptance. This inspectionincludes a physical examination of the productand its packaging, and close scrutiny of thelabeling and accompanying Certificates ofAnalysis to ensure all specifications for grade,quality and purity are met. Raw materials aresampled for identity testing (where practical).

Before any lots are produced, all rawingredients of powdered media arecontrolled and confirmed via our bar codeverification system.

In addition to high-quality raw materials, weuse Water For Injection (WFI) quality water inthe manufacture of liquid media and processliquids. All plastic product contact materialsmeet requirements for USP Biological Class VItesting and physicochemical testing.

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Validation Qualifications

We qualify utilities equipment and validateproduction processes for all of our media,serum and BPC products. Our philosophy issummarized in our Validation ManagementStandard Operating Procedure.

Our validation team plans and supervises thequalification of key production equipment andthe validation of production processes incompliance with the principles of cGMP andISO 9001:2000 and ISO 13485:2003. Thevalidation team also identifies new productand process validation requirements, workswith the NPDT, product development andproduction personnel to create validationprotocols while documenting all validationactivities.

When sufficient validation runs have beencompleted to ensure reproducibility, avalidation summary is prepared. Summariesare reviewed and submitted for approval.Validated processes are revalidated when asignificant manufacturing change is made.

Media and other sterile-filtered liquid fills aswell as serum processing and filtration arevalidated twice-yearly using aseptic filling.At least one validation run of each validatedsterilization load configuration is performedannually. Other validated processes arereviewed annually.

Selected information described on this pagecan be shared with customers subject toappropriate confidentiality assurances.

Equipment Validation

Our validations are performed to assure that allproducts meet our quality standards andcomply with cGMP guidelines. Validationprovides opportunities to understand andimprove production processes and assures thatquality is built into the product. Validation ofproduction processes is based on thequalification of subordinate equipment.Equipment qualifications include the following:• Equipment Identification• Intended Use• Equipment Specifications• Support Utilities• List of Procedures Pertaining to

Proper Intended Use• Manufacturer's Manuals• Installation Qualification (IQ)• Operational Qualification (OQ)• Performance Qualification (PQ)• Training of User and Maintenance• Personnel• Summary• Approval by Production, Validation,

Quality Assurance and ProductDevelopment/Engineering as applicable

Validation Master Plans

Master plans are written for product types orproduction processes. Validation Projectprotocols are written as defined in theapplicable Validation Master Plan. Productvalidation demonstrates compliance withrelease criteria and approved marketing claims.

Processes are challenged and monitored duringvalidation. Changes in production processesare reviewed by Validation, ProductManagement and Quality Assurance todetermine if revalidation of the process isrequired before implementation of the change.

Validation Project protocols contain thefollowing:• Objective• Responsibility• Acceptance Criteria• Supplies• Equipment• Procedures• Procedure Approval• Results• Summary• Review and Approval• Attachments• References


9

Introduct

ion

FacilitiesMedia, Sera, Process Liquids, and Reagents

Liquid Manufacturing Suites

Thermo Scientific HyClone liquid media,reagents and process liquids aremanufactured in two filtration suites:1) the large volume production suite whereproduct is filled into disposableBioProcess Containers, and

2) the small volume suite where product isfilled into rigid bottles.

The liquid media filling areas maintain separateHEPA air handling systems. Each filling suite isenvironmentally monitored by Quality Control toensure the manufacturing areas remain withinspecified ranges.

Formulation, mixing and filtration of largevolume liquids take place in a Class 100,000cleanroom. The large volume liquid mediacore features stainless-steel tanks capable ofproducing lot sizes up to 10,000 L. Sterilizing0.2 Rm or 0.1 Rm filters, stainless-steelmanifolds and connections from gamma-irradiated BioProcess Containers to thosemanifolds are sterilized using steam-in-place(SIP). No aseptic connections are made,resulting in a true, closed-system fillingenvironment.

Small volume sterile liquids are filled utilizing a"fill-by-weight" system located in a Class1000-rated production suite in a Class 100hood. Equilibrated solutions are sterile filteredinto irradiated bottles using autoclaved fillingheads and equipment.

Powdered Media Facilities

We regularly produce powdered mediaproducts using ball mill technology in oursegregated Dry Powdered Media (DPM)Production Facility.

The facility has been specifically designedto control humidity and preventcross-contamination by chemical particulatesgenerated during the manufacturing process.

Each work area in the DPM production coreutilizes a separate air handling system,eliminating the worry of cross-contaminationbetween media formulations.

Continuous Micronization

Large volumes of DPM are also manufacturedin our dedicated animal component-free,state-of-the-art, continuous micronizationfacility. This facility has been designed withstrict environmental controls includingtemperature, humidity, pressure and dust andparticulates.

The facility optimizes gravity flow and vacuumtransfer, eliminating the need for fiber dustbags and powder filters associated withpositive pressure transfer systems.

Large pharmaceutical blenders, used inconjunction with the new micronizationcapabilities, produce homogenous lot sizesup to 6,500 kg. Accuracy of each mediumformulation is confirmed using a computerized,bar-coded weight verification system.

Sera Manufacturing Site

Our sera facilities are one of the reasons wehave become known as a technical leader inthe industry. Our serum filtration laboratoryaccommodates two 3,000 L stainless-steelpressure tanks to allow for larger serumlots and strengthens our ability to maintainlot-to-lot consistency.

Variability in FBS components is natural and isthe result of a variety of factors such as age offetus, geographical origin, etc. Large lot sizes,coupled with true-pool processing, temperthis natural serum component variability, thusincreasing consistency and quality of ThermoScientific HyClone Serum products.

Distribution Facilities

Our 50,000 square foot finished goods ware-house and distribution center is located on themain campus in Logan. Construction of thisfacility was the result of our rapid growth as aconsumables supplier to the biopharmaceuticalindustry and research institutions.

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This facility provides extensive storage spacefor finished products requiring variousconditions and temperatures. A -20°C freezerstores inventories of serum and other frozenproducts. A 2 to 8°C refrigerator maintainsinventories of liquid and powdered media.Dry goods and BPCs requiring ambient storage(15 to 30°C) are also warehoused within thebuilding.

Finished products are packaged for shipment inthe distribution portion of this building.

Packaging Facilities

When increasingly larger volumes of mediaand process liquids are required, we are poisedto respond. Liquid media, supplements andprocess liquids are available in lots up to10,000 L. BioProcess Containers (BPCs),available in sizes up to 900 L, provide excellentsolutions to large-volume liquid handling.

BPCs are single use, flexible plastic containersdesigned for the transport, processing andstorage of liquids for a wide range of biotech-nology and pharmaceutical applications. BPCsare manufactured in a Class 10,000 cleanroomunder cGMP guidelines and gamma irradiatedto assure sterility.

BPCs can be customized to meet many processneeds, regulatory requirements and facilityconstraints. In today's highly regulatedproduction environments, they offercost-effective and flexible packaging solutionsthat are adaptable to specific requirements. Inthe large volume liquid media core, BPCs arefilled using a closed system.

When small volumes of media are required, weproduce lots that range in size from 100 to4,000 L. These lots are dispensed into a varietyof packaging options including 50, 100, 500and 1,000 mL bottles as well as a wideselection of small-volume BPCs ranging in sizefrom 50 mL to 20 L.

Dry powdered medium products are packagedin a variety of standard sizes. Smaller sizes aredelivered in bottles, and larger units ()500 L)are packaged in plastic bags and rigid pails.

Standard packaging configurations include:

• 1 p 50 L bottle

• 1 p 100 L bottle

• 1 p 10 L bottle

• 1 p 500 L polybag/pail

Additional Sera Manufacturing Sites

Our New Zealand facility, designed to filter lotsizes of 2,500 liters, is consistent with themanufacturing facility in Logan. Because NewZealand's islands are protected from many out-side influences, both geographically and biolog-ically, they have the fewest reported bovinediseases in the world and have been deter-mined to be free from BSE and FMD. NewZealand has perhaps the healthiest and safestcattle herd in the world and their facility is atthe forefront of this industry.

Our Australian and Canadian facilities use1,000 L disposable filtration. As with NewZealand, Australia is an island continent, thusmaking animal disease control andmanagement easier than in most areas of theworld. Australia is considered to be free of BSEand FMD.

Most of New Zealand, Australian andCanadian serum products are filtered throughthree sequential 100 nm (0.1 Rm) pore-sizerated filters; however, any level of filtration canbe made available upon request.

European Manufacturing Site

In addition to the primary manufacturing anddistribution facilities in Logan, we operate acGMP manufacturing facility in Cramlington,UK. This facility produces liquid media, processliquids and BioProcess Container Systems forthe European market.

In December 2003, an $11 million expansion tothe Cramlington facility was completed. Thenew cGMP production facilities dramaticallyincrease capacity to support the growingdemand in Europe and provide manufacturingredundancy for U.S. operations.

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11

Introduct

ionCapacity has been expanded to allow for

production of liquids in lots up to 10,000 L.Production areas for animal derived componentfree (ADCF™) products feature dedicatedseparate ADCF equipment, air handlingand are accessed via segregated changeareas. Efficient material and process flowsare designed into the facility.

The cleanrooms in the production areasconsist of segregated production suites,including:• Class 100,000 liquid manufacturing• Class 10,000 BPC assembly• Class 1,000 filling areas

Production vessels in the traditional liquidmanufacturing suite range in size from 250 to10,000 L, and are duplicated up to 5000 L in theADCF area. Production is supported by the newWFI system, which produces 2,000 L per hour.

In addition to large-volume liquids,small-volume liquids packaged in 1,000 mLbottles are also manufactured at theCramlington facility in lot sizes up to 1,000 L.Small-volume liquids are filtered in a Class100 zone in the Class 1000 suite.

Each of the manufacturing and filtration suitesis tested and certified for acceptable levels ofparticulate every six to 12 months, with routineenvironmental monitoring to confirm certification.

Beijing, China Manufacturing Site

We have established the first foreign investedcell culture powder media repackaging andliquid media manufacturing facility in China.Consistent with the manufacturing facility inLogan, the Beijing facility utilizes a state-of-the-art designation concept, strictly followingcurrent Good Manufacturing Practices (cGMP)standards. This innovative facility provides thehighest quality products to Chinese academicand industrial customers.

The Beijing liquid media production facilityhas been designed with a total productionarea of 212 m2. Capacity of the facility is twolots per day—up to 250,000 L per year.

Packaging sizes filled in the facility range from125 mL to 1 L bottles, with lot sizes rangingfrom 100 to 1000 L. To ensure the highest levelof product quality, we use WFI Quality Waterto formulate our liquid media, and fill liquidproducts in a Class 100 laminar flow hoodwithin our Class 1000 cleanroom.

The biochemical products facility also featuresa dry powdered media (DPM) repackaging suitedesigned in accordance with cGMPrequirements. Our 70 m2 facility includes threeindividual packaging rooms, two levels ofchange rooms and a dust control system.The packaging suite is classified as Class100,000, and features temperature andhumidity control. Packaging sizes availablerange from 10 g pouches to 5 kg buckets tomeet a range of customer needs.


12

BioCenter—New Expansion of Manufacturing Operationsfor Single-Use BioProcessing Containers

Construction of the new BioCenter located inLogan, Utah finished in 2008. The 37,000 ft2

facility, is the first of a three-phase constructionplan that will create a 94,000 ft2 over the nextfour years, replacing and expanding BPC opera-tion in Logan. The BioCenter currently includesan administration building, sera and liquid-

media processing facility, powdered-media fa-cility, warehouse and the existing BPC facility.

The investment is a response to theincreasing demand for disposablebioprocessing technology among customersin the life-sciences research, biotechnologyand pharmaceutical industries. BioProcessContainers serve cell-culture applications aswell as the production of protein-baseddrugs, and are used in mixing, storage andtransportation of bioprocessing fluids and forbioreactors (used to grow cells, proteins andother biochemically active substances).

Products manufactured in the new facilityinclude the Thermo Scientific HyClone BPCline— ranging from simple bioprocesscontainers to complex bioprocessing systemsand custom designed units for a variety ofapplications. This offering includes sterilefluid-handling bags, bag manifold systems,tank liners, chambers, biopharmaceuticaltubing and other accessories, as well as therecently introduced Thermo Scientific HyCloneSingle-Use Bioreactor (S.U.B.) and Single-UseMixer (S.U.M.).


13

Introduct

ion

Product Line OverviewsMedia, Sera, Supplements, Reagents, and Process Liquids

Media

In addition to standard product offerings forclassical and published medium formulations,We develop and optimize leading-edge Animal-Derived Component Free (ADCF), serum-free,protein-free and chemically defined media. Wealso specialize in the manufacture of custommedia formulations for large-volume industrialapplications.

Additionally, we are privileged to supportendeavors in stem cell research. We arecommitted to providing researchers with thehighest-quality products to help in theunderstanding of these cells, which westrongly believe will better the humancondition by bringing new therapeutics andcures for human disease. To this end, we havedeveloped our new line of Thermo ScientificHyClone AdvanceSTEM cell culture products.

Utilizing expertise gained during 40 yearsas a leading cell culture supplier, the newAdvanceSTEM line of stem cell cultureproducts is developed specifically for applica-tions in stem cell research. The line comprisesnumerous products designed to support a widerange of applications such as the expansionand maintenance of undifferentiated murineembryonic and human adult mesenchymalstem cells to the directed differentiation ofsomatic stem cells into such cell types asneurons, adipocytes, chondrocytesand osteocytes.

The new line of Thermo Scientific HyCloneProcess Supplements has been developed tosupport demand in the area of fed-batch cellculture. Each supplement is developed throughthe Metabolic Pathway Designq approach toprovide specific nutrient combinations for avariety of applications, and have beensuccessfully tested in CHO, hybridoma,NS0, HEK 293, PER.C6 (Crucell NV) andother cell lines.

The fed-batch version of stirred-tank culturehas become most popular at large scales. Theprimary driver of this trend is based on addingnutrients to a batch culture in mid-run toincrease the quantity of product harvested.The prevalence of fed-batch over other modesis due to many practical factors such asreliability, ease of scalability and applicationlatitude.

Exacting manufacturing processes ensurethe consistency and quality of sterile-filteredliquid media whether packaged for laboratoryand research use in 500 mL and 1 L volumes orpackaged for industrial use in large-volumeBPCs. Intrinsic to our products are:• High quality raw materials of USPor multi-compendial grade used

• Manufactured using cGMP and ISO9001:2000 and ISO 13485:2003-certifiedprocesses

• Full traceability and documented origin ofall formula ingredients

• Validated and consistent productionprocesses

• Just in Time (JIT) and ready-to-useliquid products

• Complete QA/QC release and testing

Serum

Since our inception, we have pioneered effortsresulting in unique FBS collection and filtrationmethods. In fact, we led the way using triple100 nm (0.1 µm) filtration years before theprocedure became the standard in the industry.Later we directed our attention to reducingvirus levels and developing our unique serial40 nm (0.04 µm) filtration. This highlyretentive filtration method has been shownto significantly reduce the level of viralparticles as small as 60 nm.

We have recently expanded our specialty fetalbovine serum product line to better meet theneeds of academic and research cell culturistsby moving our filitration process for USDA

tested Sera, as well as our NZ Porcine Serumto a new state-of-the-art cGmp compliant andISO certified disposable facility located at ourprimary campus in Logan, Utah.

BioProcess Containers

The natural synergy of HyClone liquids andBioProcess Containers differentiates ourproduct offerings into integrated mediadelivery systems.

Product Lines

These products are categorized into thefollowing groupings:• Classical Liquid Media (pp. 16)• HyQPAK Convenience Packaging (pp. 20)• Classical Dry Powdered Media (pp. 22)• HyQ-RS Reduced SerumMedia (pp. 25)• AdvanceSTEM Cell Culture Media,Supplements, Reagents and Cells (pp. 26)

• Serum-Free Media (pp. 35)• Process Supplements (pp. 50)• Nutrient Media Supplements (pp. 53)• Reagents and Cell Dissociation Products(pp. 54)

• Process Liquids, Buffers and Salts (pp. 55)• Antibiotics and Selection Agents (pp. 58)• Microcarriers (pp. 60)• Antifoam (pp. 63)• CustomMedia Products (pp. 65)• Rapid Response Production (pp. 66)• Fetal Bovine Serum (FBS) (pp. 67)• Specialty Fetal Bovine Serum (pp. 75)• Bovine Calf Serum (pp. 80)• Fetal Bovine Serum Alternatives (pp. 85)• Serum Products (pp. 88)• EMEA Confirming Serum Products (pp. 91)• Labtainers (pp. 93)


14

CapabilitiesProduction, Customization and Optimization Services

Production of Liquids

We utilize state-of-the-art filtration and asepticfill technologies to manufacture liquid media,buffers and reagents. All facilities andprocesses are validated to ensure productsmeet not only our exacting quality standards,but also cGMP, ISO 9001:2000 and ISO13485:2003 guidelines.

Standard or custom liquid formulations areavailable in lot sizes up to 10,000 L. Dependingupon lot size and product application, theseproducts can be manufactured in our traditionalproduction suite that utilizes Clean-In-Place(CIP) and Steam-In-Place (SIP), or in theHyNetics suite using disposable surfaces.Water For Injection (WFI) Quality Water isavailable for manufacture of liquid products.

Liquid products are sterile-filtered usingvalidated microbe-retentive filters. Filter integritytests are performed pre- and post-filtration fortraditional filling systems, and post-filtration forsingle-use capsule filters.

Water For Injection System

Our manufacturing facility is located in theWasatch Mountain Range of Northern Utah.Thus, all of our process liquids begin with highquality source water free from many of theindustrial chemicals and pollutants that maybe common to more urbanized regions. Thepurification process in its state-of-the-art,audit friendly facility yields process liquids ofuncompromising quality and consistency.

From this pristine beginning, the water issubjected to a sequential purification processthat includes multi-media filtration, mineralreduction, reverse osmosis, continuousdeionization, UV light treatment and distillation.Our validated system is designed to meet allmanufacturing processes and testingrequirements of the current USP monograph forWFI. Routine monitoring and sampling plansinclude the following tests and specifications:

Additional tests routinely performed onpackaged WFI Quality Water include those forsterility, pH, ammonia, calcium, carbon dioxide,chloride, sulfate, particulate matter, oxidizablesubstances and heavy metals.

Large-capacity requirements for media, buffers,salts, or other process liquids that utilize waterfrom our WFI system are routinely produced inlots sizes of up to 10,000 L. Recent and futureexpansions to these systems will enableThermo Scientific HyClone Specialty WaterProducts to continue meeting the demands oftoday's large-scale biopharm manufacturers.

Production of Dry PowderedMedia

Dry powdered media are manufactured inaccordance with cGMP guidelines and usingISO 9001:2000-certified processes. High qualityraw materials are transferred from our cGMPstorage facility and assigned to the productionlot according to specification using thevalidated weight verification system.

Production personnel follow validatedprocedures to mill or micronize each lot beforeit is packaged. Each formulation is sampled forquality control testing and is labeled perspecification.

All steps in the production process fromweighingto cleaning have been completely validated bya skilled validation team. Validations arerepeated according to individual specifications.

Research & Product Development

Many biologically active products includingmonoclonal antibodies, recombinant proteinsand viruses for vaccines are produced usinganimal cell culture. The quantity andperformance of these biologicals directlycorrespond to the quality of cell culturereagents used in the manufacturing process.

Our Research and The Product Development(R&PD) team has led the industry incharacterizing sera, identifying critical cellculture components and developing a premierline of serum-free media.

Using the latest culture and analyticalequipment to determine the needs of cells, theR&PD team has developed media for a varietyof applications. Thermo Scientific HyCloneapplies its core competence in the nutrition ofanimal cells to develop media that enablesclients to meet their cell culture objectives.

By applying our understanding of themetabolism and characteristics of the majorcell platforms employed in modernmanufacturing processes, the R&PD facilityprovides services for optimized mediadevelopment. We offer a wide variety ofoptions for development collaboration to meetclients' specific needs for most cell types,culture configurations and selection/amplifica-tion systems.

Test SpecificationConductivity Meets current U.S.P.Bioburden P10 cfu/dlEndotoxin P0.25 EU/mL per LAL testTotal OrganicCarbon (TOC)

Maximum of 500 ppb

Coliforms FIO (reference limit of0 per 100 mL)

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Metabolic Pathway Design

Our R&PD group develops serum-free cellculture media utilizing Metabolic PathwayDesign. This development process balancesthe required nutrient supply against metabolicwaste accumulation, determines the effectiveconcentration of medium components, anddefines complexed lipids that facilitate stabilityand nutritional delivery to the cells.

By addressing these requirements, our R&PDteam develops serum-free, protein-free andchemically defined media without sacrificingthe performance traditionally achieved withserum-containing media. This process alsofacilitates the design of animal-derivedcomponent-free media.

Media Optimization

The development of Thermo Scientific HyCloneSerum-Free Media has been centered onsupporting the growth of the major cellplatformsused inanimal cell-basedmanufacturing.

A key to improved performance inbiopharmaceutical development andmanufacturing is identifying the optimalmedium. Our optimization program ensuresthat the medium selection process is asuccess.

Thermo Scientific HyClone standard serum-freemedia (SFM) formulations suitable for key cellculture platforms including Hybridoma/Myelona, Insect, Chinese Hamster Ovary (CHO),Gene Therapy (e.g., HEK293) and Viral Vaccine(e.g., Vero). While these standard serum-freemedia offer superior performance, in manycases unique cell line or process-specific crite-ria require further optimization to achieve thehighest level of success.

Combining our knowledge of cell culturerequirements with state-of-the-art analyticaland development equipment, we have theability to assess and meet the specific needsof our customers' cell culture processes.

Our optimization program thoroughly evaluatesthe nutrient demands of customers cell culturesystems, potential key component toxicities,necessary process specific supplementation,and many other critical cell culture elements.

Throughout the optimization program,recommended changes in medium compositionare carefully evaluated for manufacturingsuitability. This assessment ensures that thefinished, optimizedmedium can bemanufacturedaccording to customer requirements.

Rapid Response Production Facility

Customization and optimization prototypes forindustrial applications must be evaluatedquickly to facilitate final product selection andimplementation. In research settings, minormodifications of existing media and reagentformulations are necessary to accommodatespecific goals and objectives. To address these,the Rapid Response Production facility offerspremier media and reagents.

Our Rapid Response Production (RRP) facilityis designed to offer client-specific media andreagents with the shortest turnaround times.

Requests are typically manufactured withinseven days. The quality system for the RRPfacility is neither governed by cGMP guidelinesnor a regulated part of our manufacturingfacility. The manufacturing suite is capable ofpreparing lot sizes up to 200 L in liquid, and upto 20 kg dry powdered. Liquid lots are packagedin 100, 500, and 1000 mL bottles, or in ThermoScientific HyClone BPCs (100 L BPCmaximum). Dry powdered lots are packaged involume specific bottles or in bulk containers.

The Rapid Response Production facility offersthe highest quality products by followingconsistent and reproducible manufacturingpractices. Documentation for media andreagents is designed to providecomponent-specific tracking, as well asfacilitate technical transfer to our cGMPmanufacturing facility when appropriate.Sterility, pH, osmolality, and endotoxin testingis available for all liquid lots. Solubility,moisture, particle size, pH, and osmolalitytesting is available for all dry powdered lots.

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16

Product Line Overview

Classical Liquid Media

Since our beginning 40 years ago, we havedemonstrated our commitment to theadvancement of science by becoming apremier supplier of products to support cellculture. While we have achieved dramaticgrowth in the supply of large-scale productionvolumes of media for industry, our roots arein laboratory research and academia, andwe remain committed to providing superior,innovative products for these applications.

Our extensive line of high-quality ClassicalMedia formulations is offered in convenientpackaging configurations. All Classical Mediaare system-tested with Thermo ScientificHyClone sera to ensure product efficacy andhomogeneity.

From production to quality control and finalproduct packaging, we deliver the best in highquality media. Our Classical Media productscomprise the most commonly-used formulationsused in today's research market. Variations ofthese standard formulations are available on amade-to order basis.

All of our Logan media manufacturing facilitiesand state-of-the-art milling, formulating,filtration and aseptic filling processes arevalidated to ensure compliance with cGMP, ISO9001:2000, ISO 13485:2003, and our ownstringent quality standards.

Vertical integration of these manufacturingprocesses, such as hydration of media usingmilled formulations and supplier inspection andcertification programs helps ensure productefficacy, uniformity and consistency.

All products are formulated, milled, andblended into complete powdered mediaaccording to written, approved specifications.These bulk formulations are then eitherpackaged as ready-to-hydrate finished

powders, or transferred to the media hydrationfacility for hydration and sterile filtration intobottles or Thermo Scientific HyClone BPCs.Other capabilities and value-added featuresinclude:• Hydration using our WFI Quality Water

system for unsurpassed quality• Lot sizes of up to 2,000 L in our standard 500

mL and 1 L bottles• Lot sizes of up to 10,000 L in our flexible

BPCs in 5, 10, 20, 50, 100, and 200 L sizes(standard configuration or custom designed)

• 0.1 Rm filtration for all standard products• Thermo Scientific HyQPAK 6-packs of most

common formulations are now available

Media formulations are listed beginning on page 168.

Classical Liquid MediaDescription Part Number Packaging Storage TempBasal Medium Eagle with Earl's Balanced SaltsBME/EBSS with L-glutamine ● SH30157.01 500 mL 2-8°C

○ SH30157.02 1000 mL 2-8°CDulbecco's Modified Eagle's Medium (DMEM)DMEMwith High Glucose, with 4.0 mM L-glutamine, without Sodium Pyruvate ● SH30022.01 500 mL 2-8°C

● SH30022.FS 6 x 500 mL 2-8°C● SH30022.02 1000 mL 2-8°C● SH30022.LS 6 x 1000 mL 2-8°C

DMEMwith High Glucose, without L-glutamine and Sodium Pyruvate ● SH30081.01 500 mL 2-8°C● SH30081.FS 6 x 500 mL 2-8°C● SH30081.02 1000 mL 2-8°C● SH30081.LS 6 x 1000 mL 2-8°C○ SH30081.03 20 L Bag 2-8°C○ SH30081.04 10 L Bag 2-8°C

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Classical Liquid Media (continued)Description Part Number Packaging Storage TempDMEMwith High Glucose, with 4.0 mM L-glutaminewith Sodium Pyruvate

● SH30243.01 500 mL 2-8°C● SH30243.FS 6 x 500 mL 2-8°C● SH30243.02 1000 mL 2-8°C● SH30243.LS 6 x 1000 mL 2-8°C○ SH30243.03 10 L Bag 2-8°C○ SH30243.04 20 L Bag 2-8°C

DMEMwith High Glucose, with L-glutamine, HEPES,without Sodium Pyruvate

● SH30249.01 500 mL 2-8°C● SH30249.02 1000 mL 2-8°C

DMEMwith High Glucose, without Calcium, Magnesium ● SH30262.01 500 mL 2-8°C○ SH30262.02 1000 mL 2-8°C

DMEMwith High Glucose, with L-glutamine, without Phenol Red,Sodium Pyruvate

● SH30284.01 500 mL 2-8°C● SH30284.02 1000 mL 2-8°C

DMEMwith High Glucose, without L-glutamine, with Sodium Pyruvate ● SH30285.01 500 mL 2-8°C● SH30285.FS 6 x 500 mL 2-8°C● SH30285.02 1000 mL 2-8°C● SH30285.LS 6 x 1000 mL 2-8°C○ SH30285.03 20 L Bag 2-8°C○ SH30285.04 10 L Bag 2-8°C

DMEMwithout High Glucose, Modified, without Phenol Red, SodiumPyrvate, L-glutamine

○ SH30585.01 500 mL 2-8°C● SH30585.02 1000 mL 2-8°C

DMEMwith High Glucose, Modified, without Phenol Red,L-glutamine, with Sodium Pyruvate

● SH30604.01 500 mL 2-8°C● SH30604.02 1000 mL 2-8°C

DMEMwith High Glucose, Sodium Pyruvate, without L-glutamine,Methionine, Cystine

○ SH30606.01 500 mL 2-8°C○ SH30606.02 1000 mL 2-8°C

DMEM High Modified, with Sodium Pyruvate, without L-glutamine,with 25 mM HEPES

○ SH30563.01 500 mL 2-8°C○ SH30563.02 1000 mL 2-8°C

DMEMwith High Glucose, Sodium Pyruvate, without L-glutamineand Phosphate

○ SH30607.01 500 mL 2-8°C

DMEMwith Low Glucose, 4.0mM L-glutamine, and110 mg/Sodium Pyruvate

● SH30021.01 500 mL 2-8°C● SH30021.FS 6 x 500 mL 2-8°C● SH30021.02 1000 mL 2-8°C

DMEM/F12 1:1DMEM/F12 1:1, with 2.50 mM L-glutamine, 15 mM HEPES ● SH30023.01 500 mL 2-8°C

● SH30023.FS 6 x 500 mL 2-8°C● SH30023.02 1000 mL 2-8°C

DMEM/F12 1:1, without L-glutamine, with HEPES ● SH30126.01 500 mL 2-8°C● SH30126.FS 6 x 500 mL 2-8°C○ SH30126.02 1000 mL 2-8°C

DMEM/F12 1:1, with L-glutamine, HEPES, Sodium Pyruvate ● SH30261.01 500 mL 2-8°C● SH30261.02 1000 mL 2-8°C

DMEM/F12 1:1, with L-glutamine, without HEPES ● SH30271.01 500 mL 2-8°C● SH30271.FS 6 x 500 mL 2-8°C● SH30271.02 1000 mL 2-8°C

DMEM/F12 1:1, with L-glutamine, without HEPES, Phenol Red ● SH30272.01 500 mL 2-8°C● SH30272.02 1000 mL 2-8°C



18

Classical Liquid Media (continued)

Description Part Number Packaging Storage TempHam's Nutrient MixtureHam's F10, with L-glutamine ● SH30025.01 500 mL 2-8°C

○ SH30025.02 1000 mL 2-8°CHam's F12, 1X, with 1.00 mM L-glutamine ● SH30026.01 500 mL 2-8°C

● SH30026.FS 6 x 500 mL 2-8°C● SH30026.02 1000 mL 2-8°C

Ham's F12, Kaighn's Modification ● SH30526.01 500 mL 2-8°C○ SH30526.02 1000 mL 2-8°C

Insect MediaGrace's Unsupplemented (liquid) ○ SH30610.01 500 mL 2-8°C

TNM-FH (liquid) ● SH30280.02 500 mL 2-8°C● SH30280.03 1000 mL 2-8°C

Iscove's Modified Dulbecco's Medium (IMDM)IMDMwith L-glutamine, HEPES, without Alpha-Thioglycerol ● SH30228.01 500 mL 2-8°C

● SH30228.FS 6 x 500 mL 2-8°C● SH30228.02 1000 mL 2-8°C

IMDMwithout L-glutamine ● SH30259.01 500 mL 2-8°C● SH30259.02 1000 mL 2-8°C

Leibovitz MediumLeibovitz L-15 Medium, with 2.05 mM L-glutamine ● SH30525.01 500 mL 2-8°C

● SH30525.02 1000 mL 2-8°CMedium 199M199 with Earle's Balanced Salt Solution (EBSS), L-glutamine ● SH30253.01 500 mL 2-8°C

○ SH30253.FS 6 x 500 mL 2-8°C● SH30253.02 1000 mL 2-8°C

M199 with Hank's Balanced Salt Solution (HBSS), L-glutamine ○ SH30233.01 100 mL 2-8°C○ SH30233.02 500 mL 2-8°C

M199 with Hank's Balanced Salt Solution (HBSS), L-glutamine,L-Amino Acids

○ SH30330.01 500 mL 2-8°C○ SH30330.02 1000 mL 2-8°C

McCoy's MediumMcCoy's 5A with L-glutamine ● SH30200.01 500 mL 2-8°C

● SH30200.FS 6 x 500 mL 2-8°C● SH30200.02 1000 mL 2-8°C

McCoy's 5A with L-glutamine, without Phenol Red ● SH30270.01 500 mL 2-8°C○ SH30270.02 1000 mL 2-8°C

McCoy's 5A with L-glutamine, HEPES ○ SH30602.01 500 mL 2-8°C○ SH30602.02 1000 mL 2-8°C○ SH30602.03 100 mL 2-8°C

○ SH30610.02 1000 mL 2-8°C



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Classical Liquid Media (continued)

Description Part Number Packaging Storage TempMinimum Essential Medium (MEM)MEMwith Earle's Balanced Salt Solution (EBSS), with 2.0 mM L-glutamine ● SH30024.01 500 mL 2-8°C


MEMwith Earle's Balanced Salt Solution (EBSS), Suspension Modification, with L-glutamine ○ SH30235.01 500 mL 2-8°C○ SH30235.02 1000 mL 2-8°C

MEMwith Earle's Balanced Salt Solution (EBSS), without L-glutamine ● SH30244.01 500 mL 2-8°C● SH30244.FS 6 x 500 mL 2-8°C● SH30244.02 1000 mL 2-8°C○ SH30244.LS 6 x 1000 mL 2-8°C

MEM Alpha Modification, with L-glutamine, Ribo- and Deoxyribonucleosides ● SH30265.01 500 mL 2-8°C●

SH30265.02 1000 mL2-8°C

●SH30265.FS 6 x 500 mL

2-8°CMEM/Alpha Modification without L-glutamine, Ribo- and Deoxyribonucleosides ● SH30568.01 500 mL 2-8°C

○ SH30568.FS 6 x 500 mL 2-8°C○ SH30568.02 1000 L 2-8°C

MEM (Richter's Modification), without Phenol Red ○ SH30600.01 500 mL 2-8°C○ SH30600.02 1000 mL 2-8°C

MEM (Richter's Modification) with L-glutamine and Phenol Red ● SH30601.01 500 mL 2-8°C○ SH30601.02 1000 mL 2-8°C

MEM for Suspension Cultures, without L-glutamine, Calcium Chloride, or Magnesium ○ SH30603.01 500 mL 2-8°C○ SH30603.02 1000 mL 2-8°C

RPMI 1640 MediumRPMI 1640, 1X, with 2.05 mM L-glutamine (Same as SH30091) ● SH30027.01 500 mL 2-8°C


RPMI 1640, 1X, with 2.05 mM L-glutamine (Same as SH30027) ○ SH30091.01 20 L Bag 2-8°C○ SH30091.02 10 L Bag 2-8°C

RPMI 1640, without L-glutamine ● SH30096.01 500 mL 2-8°C● SH30096.FS 6 x 500 mL 2-8°C● SH30096.02 1000 mL 2-8°C○ SH30096.LS 6 x 1000 mL 2-8°C○ SH30096.04 10 L Bag 2-8°C○ SH30096.05 20 L Bag 2-8°C

RPMI 1640, without L-glutamine, with HEPES ○ SH30203.05 500 mL 2-8°C○ SH30203.06 1000 mL 2-8°C

RPMI 1640 Medium, with 25 mM HEPES, with L-glutamine ● SH30255.01 500 mL 2-8°C● SH30255.FS 6 x 500 mL 2-8°C● SH30255.02 1000 mL 2-8°C

RPMI 1640, without L-glutamine or Phenol Red ● SH30605.01 500 mL 2-8°C○ SH30605.02 1000 mL 2-8°C



20


HyQPAK Convenience ProductsThe following Thermo Scientific HyClone Classical Media products are provided in convenient HyQPAK 6-Packs. Formulations forpublished, non-proprietary products begin on Page 168

HyQPAK Convenience ProductsDescription Part Number Packaging Storage TempHyQPAK Classical MediaDMEMwith High Glucose, 4.0 mM L-glutamine, Sodium Pyruvate ● SH30243.FS 6 x 500 mL 2-8°C

● SH30243.LS 6 x 1000 mL 2-8°CDMEMwith High Glucose, 4.0 mM L-glutamine, without Sodium Pyruvate ● SH30022.FS 6 x 500 mL 2-8°C

● SH30022.LS 6 x 1000 mL 2-8°CDMEMwith High Glucose, without L-glutamine and Sodium Pyruvate ● SH30081.FS 6 x 500 mL 2-8°C

● SH30081.LS 6 x 1000 mL 2-8°CDMEMwith High Glucose, without L-glutamine, with Sodium Pyruvate ● SH30285.FS 6 x 500 mL 2-8°C

● SH30285.LS 6 x 1000 mL 2-8°CDMEMwith Low Glucose, 4.0 mM L-glutamine, 110 mg/Sodium Pyruvate ● SH30021.FS 6 x 500 mL 2-8°CDMEM/F12 1:1, with 2.50 mM L-glutamine, 15 mM HEPES ● SH30023.FS 6 x 500 mL 2-8°CDMEM/F12 1:1, with L-glutamine, without HEPES ○ SH30271.FS 6 x 500 mL 2-8°CDMEM/F12 1:1, without L-glutamine, with HEPES ● SH30126.FS 6 x 500 mL 2-8°CHam's F12, 1X, with 1.00 mM L-glutamine ● SH30026.FS 6 x 500 mL 2-8°CIMDMwith L-glutamine, HEPES, without Alpha-Thioglycerol ● SH30228.FS 6 x 500 mL 2-8°CM199 with Earle's Balanced Salt Solution (EBSS), L-glutamine ○ SH30253.FS 6 x 500 mL 2-8°CMcCoy's 5A with L-glutamine ● SH30200.FS 6 x 500 mL 2-8°CMEM Alpha Modification, with L-glutamine, Ribo- and Deoxyribonucleosides ● SH30265.FS 6 x 500 mL 2-8°CMEMwith Earle's Balanced Salt Solution (EBSS), 2.0 mM L-glutamine ● SH30024.FS 6 x 500 mL 2-8°C

● SH30024.LS 6 x 1000 mL 2-8°CMEMwith Earle's Balanced Salt Solution (EBSS), without L-glutamine ● SH30244.FS 6 x 500 mL 2-8°C

MEM/AlphaModification without L-glutamine, Ribo- and Deoxyribonucleosides ○ SH30568.FS 6 x 500 mL 2-8°CRPMI 1640 Medium, with 25 mM HEPES, L-glutamine ● SH30255.FS 6 x 500 mL 2-8°CRPMI 1640, 1X, with 2.05 mM L-glutamine (Same as SH30091) ● SH30027.FS 6 x 500 mL 2-8°C

● SH30027.LS 6 x 1000 mL 2-8°CRPMI 1640, without L-glutamine ● SH30096.FS 6 x 500 mL 2-8°C

○ SH30096.LS 6 x 1000 mL 2-8°CHyQPAK Serum-Free MediaADCF-MAb (liquid) ○ SH30349.LS 6 x 1000 mL 2-8°CADCF-MAb without L-glutamine (liquid) ○ SH30547.LS 6 x 1000 mL 2-8°CCDM4CHO with L-glutamine (liquid) ○ SH30557.LS 6 x 1000 mL 2-8°CCDM4CHO without L-glutamine (liquid) ○ SH30558.LS 6 x 1000 mL 2-8°CCDM4MAb with L-glutamine (liquid) ○ SH30801.LS 6 x 1000 mL 2-8°CCDM4MAb without L-glutamine (liquid) ○ SH30802.LS 6 x 1000 mL 2-8°CCDM4NS0 without L-glutamine (liquid) ○ SH30579.LS 6 x 1000 mL 2-8°CPF CHO LS with L-glutamine (liquid) ○ SH30359.LS 6 x 1000 mL 2-8°CSFM4CHO without L-glutamine (liquid) ○ SH30548.LS 6 x 1000 mL 2-8°CSFM4CHO-Utility with L-glutamine (liquid) ● SH30516.LS 6 x 1000 mL 2-8°C

● Item in stock.

○ SH30244.LS 6 x 1000 mL 2-8°C

SFM4CHO-A without L-glutamine NNEEWW ○ SH30820.LS 6 x 1000 mL 2-8°CSFM4CHO-A without L-glutamine NNEEWW ○ SH30821.LS 6 x 1000 mL 2-8°CCDM4HEK293 without L-glutamine NNEEWW ○ SH30858.LS 6 x 1000 mL 2-8°CSFM4TRANSfx-293 without L-glutamine NNEEWW ● SH30860.LS 6 x 1000 mL 2-8°CCDM4PERMAb without L-glutamine NNEEWW ○ SH30871.LS 6 x 1000 mL 2-8°C


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HyQPAK Convenience Products (continued)

● Item in stock.

Description Part Number Packaging Storage TempSFM4HEK293 without L-glutamine (liquid) ○ SH30521.LS 6 x 1000 mL 2-8°CSFM4MAb with L-glutamine (liquid) ○ SH30513.LS 6 x 1000 mL 2-8°CSFM4MAb-Utility with L-glutamine (liquid) ○ SH30382.LS 6 x 1000 mL 2-8°CSFM4MegaVir without L-glutamine (liquid) ○ SH30552.LS 6 x 1000 mL 2-8°CSFX-Insect (liquid) ● SH30278.LS 6 x 1000 mL 2-8°CSFM4CHO (liquid) ○ SH30549.LS 6 x 1000 mL 2-8°CHyQPAK WaterWater, Cell Culture Grade, Endotoxin-free, (P 0.005 EU/mL),Deionized, Distilled, 0.1 Rm Sterile Filtered

● SH30529.FS 6 x 500 mL 2-30°C● SH30529.LS 6 x 1000 mL 2-30°C

Water, Molecular Biology Grade, DNase, RNase, Protease free (no activity withinassay detectability limits), Deionized, Distilled, 0.1 Rm Sterile Filtered


Water, WFI Quality, Meets current USP monograph criteria for WFI packaged in bulkfor commercial use elsewhere. Not for Diagnostic or Therapeutic Use.

● SH30221.LS 6 x 1000 mL 2-30°C

HyQPAK Balanced Salt SolutionsDulbecco's Phosphate Buffered Saline (DPBS), 1X, with Calcium, Magnesium,without Phenol Red

● SH30264.FS 6 x 500 mL 15-30°C

Dulbecco's Phosphate Buffered Saline (DPBS), 1X, without Calcium,Magnesium, Phenol Red


Hank's Balanced Salt Solution (HBSS), 1X, with Calcium, Magnesium,without Phenol Red

● SH30268.LS 6 x 1000 mL 15-30°C

Hank's Balanced Salt Solution (HBSS), 1X, without Calcium, Magnesium,with Phenol Red

● SH30031.FS 6 x 500 mL 15-30°C

Phosphate Buffered Saline (PBS), 1X, 0.0067M PO4, without Calcium,Magnesium, Phenol Red



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Classical Dry Powdered Media

Thermo Scienitifc HyClone Dry PowderedMedia are manufactured from the highestgrade raw materials and in a uniquely designed,state-of-the-art facility. Available in standard orcustom formulations, our Powdered Media arequality products that provide flexibility to cellbiologists for research and industrial cellculture applications.

Exacting manufacturing processes ensurethe highest consistency both in media basepowders produced for onsite manufacture oflarge volume liquids and in finished packagedpowders delivered directly to the customer.Intrinsic to Thermo Scientific HyClone ClassicalDry Powder Media Products are:• Highest quality raw materials of USP ormulticompendial grade where available

• Manufactured under cGMP guidelines andISO 9001:2000 and ISO 13485:2003-certifiedprocesses

• Full traceability and documented origin of allformula ingredients

• Validated and consistent productionprocesses

• Integrated weight verification• Scaleable batch sizes from 0.5 to 6,500 kg• Complete QA/QC testing and release

Thermo Scientific HyClone Dry PowderedMedia includes:• Basal Medium Eagle (BME)• Dulbecco's Modified Eagle's Medium(DMEM)

• Dulbecco's Modified Eagle's Medium/Ham'sNutrient Mixture F12 (DMEM/F12) 1:1

• Ham's Nutrient Mixtures F10 and F12• Iscove's Modified Dulbecco's Medium(IMDM)

• Leibovitz L-15• McCoy's 5A• Medium 199 w/Earle's Balanced Salts(M199/EBSS)

• Minimum Essential Medium (MEM)• RPMI 1640• William's E Medium

Media formulations are listed in the formulation section.

Classical Dry Powdered Media

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Description Part Number Packaging Storage TempBasal Medium Eagle with Earle's Balanced SaltsBME/EBSS with L-glutamine P SH30159.02 2 x 5 L 2-8°C

P SH30159.03 1 x 10 L 2-8°CP SH30159.04 1 x 50 L 2-8°C

Dulbecco's Modified Eagle's Medium (DMEM)DMEM/HIGH with L-glutamine, without Sodium Pyruvate Q SH30003.02 2 x 5 L 2-8°C

Q SH30003.03 1 x 10 L 2-8°CQ SH30003.04 1 x 50 L 2-8°C

DMEM/HIGH with L-glutamine, Sodium Pyruvate P SH30045.02 2 x 5 L 2-8°CP SH30045.03 1 x 10 L 2-8°CP SH30045.04 1 x 50 L 2-8°C

DMEM/HIGH without L-glutamine, Sodium Pyruvate P SH30053.02 2 x 5 L 2-8°CP SH30053.03 1 x 10 L 2-8°CP SH30053.04 1 x 50 L 2-8°C

DMEM/HIGH with L-glutamine, without Sodium Pyruvate, Phenol Red P SH30211.05 2 x 5 L 2-8°CP SH30211.01 1 x 10 L 2-8°CP SH30211.03 1 x 50 L 2-8°C

DMEM/HIGH without L-glutamine, Sodium Pyruvate, Calcium, Magnesium P SH30346.01 2 x 5 L 2-8°CP SH30346.02 1 x 10 L 2-8°CP SH30346.03 1 x 50 L 2-8°C

Q Item in stock. P Item is made to order. Lead times and minimum order quantities apply.


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Classical Dry Powdered Media (continued)Description Part Number Packaging Storage TempDulbecco's Modified Eagle's Medium (DMEM)DMEM/LOWwith L-glutamine, Sodium Pyruvate Q SH30002.02 2 x 5 L 2-8°C

Q SH30002.03 1 x 10 L 2-8°CQ SH30002.04 1 x 50 L 2-8°C

DMEM/LOWwith L-glutamine, Sodium Pyruvate, without Phenol Red P SH30044.02 2 x 5 L 2-8°CP SH30044.03 1 x 10 L 2-8°CP SH30044.04 1 x 50 L 2-8°C

DMEM/F12 1:1 with L-glutamine, HEPES P SH30004.02 2 x 5 L 2-8°CP SH30004.03 1 x 10 L 2-8°CQ SH30004.04 1 x 50 L 2-8°C

DMEM/F12 1:1 with L-glutamine, without HEPES P SH30069.02 2 x 5 L 2-8°CP SH30069.03 1 x 10 L 2-8°CP SH30069.04 1 x 50 L 2-8°C

Ham's Nutrient MixtureHam's F10, with L-glutamine P SH30009.02 2 x 5 L 2-8°C

P SH30009.03 1 x 10 L 2-8°CP SH30009.04 1 x 50 L 2-8°C

Ham's F12, with L-glutamine Q SH30010.02 2 x 5 L 2-8°CQ SH30010.03 1 x 10 L 2-8°CQ SH30010.04 1 x 50 L 2-8°C

Ham's F12, without L-glutamine P SH30056.02 2 x 5 L 2-8°CP SH30056.03 1 x 10 L 2-8°CP SH30056.04 1 x 50 L 2-8°C

Iscove's Modified Dulbecco's Medium (IMDM)IMDMwith L-glutamine, HEPES, without Alpha-Thioglycerol Q SH30005.02 2 x 5 L 2-8°C

P SH30005.03 1 x 10 L 2-8°CP SH30005.04 1 x 50 L 2-8°C

IMDMwithout L-glutamine, with HEPES P SH30380.02 2 x 5 L 2-8°CP SH30380.03 1 x 10 L 2-8°CP SH30380.04 1 x 50 L 2-8°C

Leibovitz MediumLeibovitz L-15 with L-glutamine P SH30048.02 2 x 5 L 2-8°C

P SH30048.03 1 x 10 L 2-8°CP SH30048.04 1 x 50 L 2-8°C

McCoy's MediumMcCoy's 5A Iwakata and Grace Modification, with L-glutamine P SH30049.02 2 x 5 L 2-8°C

P SH30049.03 1 x 10 L 2-8°CP SH30049.04 1 x 50 L 2-8°C

Medium 199M199/EBSS with L-glutamine, L-Amino Acids P SH30297.02 2 x 5 L 2-8°C

P SH30297.03 1 x 10 L 2-8°CP SH30297.04 1 x 50 L 2-8°C

M199/EBSS Filter Friendly, with L-glutamine P SH30351.01 1 x 10 L 2-8°CP SH30351.02 1 x 50 L 2-8°C

M199/EBSS with L-glutamine, without Phenol Red P SH30254.01 2 x 5 L 2-8°CP SH30254.02 1 x 10 L 2-8°CP SH30254.03 1 x 50 L 2-8°C



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Classical Dry Powdered Media (continued)Description Part Number Packaging Storage TempMinimum Essential Medium (MEM)MEM/EBSS with L-glutamine Q SH30008.02 2 x 5 L 2-8°C

Q SH30008.03 1 x 10 L 2-8°CQ SH30008.04 1 x 50 L 2-8°C

MEM/EBSS without L-glutamine P SH30054.02 2 x 5 L 2-8°CP SH30054.03 1 x 10 L 2-8°CP SH30054.04 1 x 50 L 2-8°C

MEM/EBSS with L-glutamine, without Phenol Red P SH30171.02 2 x 5 L 2-8°CP SH30171.03 1 x 10 L 2-8°CP SH30171.04 1 x 50 L 2-8°C

MEM/EBSS (Autoclavable) P SH30327.01 1 x 10 L 2-8°CP SH30327.02 1 x 50 L 2-8°CQ SH30050.02 2 x 5 L 2-8°CQ SH30050.03 1 x 10 L 2-8°CQ SH30050.04 1 x 50 L 2-8°C

MEM Alpha Modification with L-glutamine, Ribo- and Deoxyribonucleosides P SH30007.02 2 x 5 L 2-8°CP SH30007.03 1 x 10 L 2-8°CP SH30007.04 1 x 50 L 2-8°C

MEM Alpha Modification with L-glutamine, without Ribo- and Deoxyribonucleosides P SH30219.01 1 x 10 L 2-8°CP SH30219.02 1 x 50 L 2-8°C

MEM Alpha Modification without L-glutamine, Ribo- and Deoxyribonucleosides P SH30205.02 2 x 5 L 2-8°CP SH30205.03 1 x 10 L 2-8°CP SH30205.04 1 x 50 L 2-8°C

MEM GlasgowModification with L-glutamine P SH30269.01 1 x 10 L 2-8°CP SH30269.02 1 x 50 L 2-8°C

MEM/HBSS with L-glutamine P SH30193.02 2 x 5 L 2-8°CP SH30193.03 1 x 10 L 2-8°CP SH30193.04 1 x 50 L 2-8°C

RPMI 1640 MediumRPMI 1640, with L-glutamine Q SH30011.02 2 x 5 L 2-8°C

Q SH30011.03 1 x 10 L 2-8°CQ SH30011.04 1 x 50 L 2-8°C

RPMI 1640, Bakers X-Tra Soluble with L-glutamine P SH30012.02 2 x 5 L 2-8°CP SH30012.03 1 x 10 L 2-8°CP SH30012.04 1 x 50 L 2-8°C

RPMI 1640, without L-glutamine P SH30057.02 2 x 5 L 2-8°CP SH30057.03 1 x 10 L 2-8°CP SH30057.04 1 x 50 L 2-8°C

RPMI 1640, with L-glutamine, without Phenol Red P SH30197.02 2 x 5 L 2-8°CP SH30197.03 1 x 10 L 2-8°CP SH30197.04 1 x 50 L 2-8°C

William's E MediumWilliam's E Medium with L-glutamine P SH30094.02 2 x 5 L 2-8°C

P SH30094.03 1 x 10 L 2-8°CP SH30094.04 1 x 50 L 2-8°C

MEM/EBSS with L-glutamine, Non-Essential Amino Acids



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HyQ-RS Reduced Serum MediaDMEM-RSq, MEM-RSq, and Ham's F12-RSqwhich are standard basal formulations thathave been nutritionally enhanced to allowmaintained performance while reducing serum(or serum replacement) supplementation by upto 80 percent.

Thermo Scientific HyClone HyQ-RS productsare provided as a convenient 1X liquid in both500 and 1000 mL sizes.

Product Applications

HyQ-RS products are suitable for a wide varietyof cell types normally used with standardformulations. As with the introduction of anynew cell culture media, we recommend thatthe reduced serum media be performancetested on a sampling of customer cells.

Cell types for Ham's F12-RS include, but arenot limited to:• CHO-K1 cells

Cell types for DMEM-RS include, but are notlimited to:• VERO• MRC-5• MDBK• COS-7• A549

Cell types for MEM-RS include, but are notlimited to:• WI-38• VERO• PK-15• MDCK• HEp-2

As with all of Thermo Scientific HyCloneClassical Media products, all HyQ-RS areSystem Tested with Thermo ScientificHyClone serum products. Shelf life for all RSproducts is 12 months. Reduction in theamount of serum used will vary with the cell

line and basal medium used. The recommendedconcentration of serum (or serum replacement)is 2 to 4 percent. Reduction in serum usingHyQ-RS media results in the followingkey benefits:• maximize individual lots of serum• minimize lot-to-lot serum variability• reduce costs• maintain consistent cell culture results

HyQ-RS MediaDescription Part Number Packaging Storage TempMEM-RS with L-glutamine Q SH30564.01 500 mL 2-8°CMEM/EBSS-RS with L-glutamine, Complete Powder P SH30629.01 5 L Bottle 2-8°C

P SH30629.02 10 L Bottle 2-8°CP SH30629.03 50 L Bottle 2-8°CP SH30629.04 100 L Bottle 2-8°CP SH30629.05 500 L Bottle 2-8°C

DMEM-RS with L-glutamine Q SH30565.01 500 mL 2-8°CQ SH30565.02 1000 mL 2-8°C

DMEM-RS with L-glutamine, Complete Powder P SH30628.01 5 L Bottle 2-8°CP SH30628.02 10 L Bottle 2-8°CP SH30628.03 50 L Bottle 2-8°CP SH30628.04 100 L Bottle 2-8°CP SH30628.05 500 L Bottle 2-8°C

Ham's F12-RS with L-glutamine P SH30623.01 500 mL 2-8°CP SH30623.02 1000 mL 2-8°C



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AdvanceSTEM Cell Culture ProductsAdvancing Stem Cell Research

The number of studies involving stem cells hasincreased considerably over the past fewdecades, with a corresponding increase in thedemand for sera and media designedspecifically for stem cell applications. As stemcell research advances through clinical trialsand into the therapeutic arena, we are uniquelypositioned to serve your cell culture needs.

The Unique Properties of Stem Cells

Our cell culture experts understand the uniquefeatures of stem cells and their special needs.Stem cells are defined by two common criteria:their ability for prolonged undifferentiatedproliferation, and their ability to differentiateinto more than one type of specialized celltype. There are two broad categories of stemcells: pluripotent and multipotent. The mostcommonly studied pluripotent stem cell is theembryonic stem cell (ESCs), which is derivedfrom the inner cell mass of a blastocyststage embryo.

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Multipotent, or adult, stem cells have beenidentified and isolated from most of the majororgans and tissues, including adipose tissueand bone marrow.

ESCs are unique cells that retain their capacity in culture for both self-renewal anddifferentiation. Publications dating from 1998on the isolation and growth of human ESCshave intrigued scientists and laymen alike, andgenerated the hope of learning how to controland direct the differentiation of these cells. Inturn, this research would help elucidate theearliest stages of human development, howspecialized tissues and organs are generated,and how we might be able to use these cells todevelop therapies that might be able to treatcomplex degenerative diseases that currenttherapies cannot adequately address.

Murine, or mouse, ESCs have been used by biologists to study mammalian developmentand model human diseases for the past 25years. Additionally, they have become standardresearch tools. ESCs are sensitive to cultureconditions and can easily differentiate and lose pluripotency if not properly maintained.

We set the standard for high-quality fetalbovine serum used for the culture of embryonicstem cells and provides lot tested batches thathave been carefully analyzed for their ability tosupport the growth of mESCs.

As part of continuing our efforts to address the research and development needs for thehighest-quality stem cell culture reagents, wehave built on our expertise in sera and cell culture nutrition and added new media andreagents that have been carefully formulatedto support the culture of mESCs.

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HyClone_026-027:Media_015-020 3/26/2010 2:05 PM Page 26

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AdvanceSTEM Murine Embryonic Stem CellCulture ProductsProviding optimal culture conditions can be oneof the greatest challenges in stem cell research,particularly in keeping cells in an undifferentiatedstate or directing differentiation when desired.

The Thermo Scientific HyClone AdvanceSTEMline of stem cell culture products can help youmeet those challenges.

ES-Qualified Stem Cell Products

Classic culture conditions for 129-derivedmurine embryonic stem cells (mESCs) includeculturing in medium containing DMEM, FBS,LIF (Protocol on Page 140) and co-culturingwith primary mouse embryonic fibroblasts(MEFs). Pluripotent mESCs have a very distinctmorphology when cultured under theseconditions; growing as tightly clusteredcolonies with smooth phase bright borders.Murine ESCs grow quickly and requiredaily maintenance.

Pluripotent B6-ESCs have a very distinctmorphology when cultured under certainconditions, growing as tightly clusteredcolonies with smooth phase bright borders. ThePRX- B6N ESCs grow quickly and require dailymaintenance. When co-cultured in the mediumdescribed in with mouse embryonic fibroblasts(MEFs), these B6 ESCs are very robust. Fora complete listing of the Thermo ScientificHyClone AdvanceSTEM products availablefor these conditions, reference the orderinginformation table below.

Serum Replacement

AdvanceSTEM Serum Replacement has beenspecifically developed to maintain murineembryonic stem cells (mESC) in culture.• No requirement for fetal bovine serum• Does not contain serum• Does not contain leukemia inhibitory factory(LIF); supplementation with LIF is requiredfor murine embryonic stem cell culture

• Concentrations of 15 to 20% SerumReplacement supplementation are suggested

• Not recommended for plating mouseembryonic feeder cells (MEFs)

Low Osmo DMEM

This basal medium has an optimized osmolalitywhich approximates that of murine embryonictissue. The formulation specifically supportsthe growth and maintenance of murine ESCs inculture. Some murine embryonic stem celllines have been observed to double morerapidly in AdvanceSTEM Low Osmo DMEMthan classical DMEM formulations. In keepingwith good cell culture techniques, cells shouldbe microscopically observed daily tomonitor status.

• Not recommended for plating mouseembryonic feeder cells (MEFs)

• Formulation does not contain serum.Concentrations of 15 to 20% SerumReplacement supplementation are suggested

• Do not use serum replacement atconcentrations (30%

• Does not contain leukemia inhibitory factory(LIF); supplementation with LIF is requiredfor murine embryonic stem cell culture

NOTE: Some murine embryonic stem cell lineshave been observed to double more rapidly inAdvanceSTEM Low Osmo DMEM thanclassical DMEM formulations. Following goodcell culture techniques, cells should bemicroscopically observed daily tomonitor status.

Embryonic Stem (ES) Cell Screened FetalBovine Serum, U.S. Origin

For research involving embryonic stem cells(ESCs), it is critical to maintain ESCs in theirundifferentiated state. Historically, researcherswere required to screen several lots of serumto find one suitable for ESC research. ThermoScientific HyClone ES Screened FBS has beenscreened for the ability to promote the rapidgrowth of ES cells while retaining theirpluripotent state, eliminating the need forcustomers to prescreen serum lots. For moreinformation, see our ES Screened FBSon Page 79.

Murine Embryonic Stem Cell ProductsDescription Part Number Packaging Storage TempAdvanceSTEM IMDM4SC without L-glutamine Q SH30822.01 500 mL 2-8°C

P SH30822.02 1000 mL 2-8°CAdvanceSTEM DMEM4SC with 4.5 mg/L Glucose, without L-glutamineand Sodium Pyruvate

Q SH30824.01 500 mL 2-8°CP SH30824.02 1000 mL 2-8°C

AdvanceSTEM ES Qualified Dulbecco's Phosphate Buffered Saline (DPBS)without Calcium or Magnesium

P SH30850.02 500 mL 15-30°CP SH30850.03 1000 mL 15-30°C

AdvanceSTEM ES Qualified HEPES (1 M) Q SH30851.01 100 mL 2-8°CAdvanceSTEM ES Qualified L-glutamine 200 mM Q SH30852.01 100 mL FrozenAdvanceSTEM ES Qualified Non-Essential Amino Acids (100X) Q SH30853.01 100 mL 2-8°CAdvanceSTEM Low Osmo DMEMwithout L-glutamine Q SH30870.01 500 mL 2-8°C

Q SH30870.02 1000 mL 2-8°CAdvanceSTEM Serum Replacement Q SH30874.01 50 mL Frozen

Q SH30874.02 100 mL FrozenQ SH30874.03 500 mL Frozen

Thermo Scientific HyClone ES Screened FBS, U.S. Origin Q SH30070.02E 100 mL -20°CQ SH30070.03E 500 mL -20°C

Q Item in stock.

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Primogenix Cells

Primogenix Murine Embryonic Stem Cells

First isolated in 1981, murine embryonic stemcells, or "mESCs," are used to produce mousemodels of human disease that aid in theunderstanding of and in the development oftherapies for genetic diseases in humans. Thediscovery and use of murine ES cells andassociated technology that allows the precisemodification of individual genes has had sucha major impact on biomedical research that thethree scientists who laid the ground work forthe use of mouse ES cells and the associatedgene targeting technology were awarded the2007 Nobel Prize in Medicine.

In October of 2007, Thermo Fisher Scientificannounced its agreement with Primogenix, Inc.of Laurie, Mo., which covers the marketing ofmurine embryonic stem cells and media usedto grow these cells. This partnership wasformed in response to growing demand amongresearchers for high-quality murine embryonicstem cells.

Primogenix was founded with the purpose ofproviding high quality, robust murine embryonicstem cell lines to researchers around the worldin order to enhance their research efforts andallow for more efficient production of mousemodels of human diseases. The company isdedicated to deriving and validating novelmouse embryonic stem cell lines that are usedto understand mammalian gene function.Leveraging nearly two decades of experiencein embryonic stem cell technology, Primogenixis applying proven expertise to assist researchersin achieving their project goals faster and atlower cost. The company provides high qualitymouse embryonic stem cells, de novo derivationof mouse embryonic stem cell lines, andconsulting services that accelerateresearch programs.

Mouse embryonic stem cells, commonlyabbreviated as "mESCs," are used in manybiomedical research laboratories and for manyscientists have become a standard laboratorytool for evaluating gene function in the contextof complex mammalian physiology via theproduction of gene targeted or knockout mice.

We are pleased to introduce two mouse ESClines derived from two of the most commonlyused mouse strains for generating knockoutmice:129/X1 and B6N.

129-derived mESC lines tend to be stable whencultured under classical conditions in mediumcontaining DMEM, FBS, and LIF. 129-derivedlines are often the workhorse of the genetargeting laboratory and have been used tomake literally thousands of mouse knockouts.

In recent years, however, there has beengrowing interest in producing mouse modelsin the C57BL/6 (B6)-derived ESC lines, thusavoiding the lengthy and expensive backcrossinginto the B6 strain, which is preferred for somephysiological studies.

These two highly robust mESC lines have beencarefully tested and shown to be germlinecompetent, karyotyped by G-banding, and arefree from common mouse pathogens andmycoplasma as teste by PCR assay. These lineshave been derived and validated using highquality Thermo Scientific HyClone ClassicalBasal Media, in the presence of ThermoScientific HyClone ES-Qualified FetalBovine Serum.

Primogenix PRX-129/X1MurineEmbryonic Stem Cells (mESCs)

Primogenix PRX-129/X1 have been derivedfrom the inner cell mass of a 129/X1 blastocyststage embryo. They are karyotypically normalmale: 40XY, IMPACT tested (pathogen free),and germline competent. Germline chimerasobtained when 8-10 cells injected into B6mouse blastocysts.

Primogenix PRX-B6NMurine EmbryonicStem Cells (mESCs)

Primogenix PRX-B6N mESCs are derived fromthe inner cell mass of a B6N blastocyst stageembryo. They are karyotypically normal male:40XY, IMPACT tested, and germline competent.Germline chimeras obtained when 10-14 cellsare injected into BALB/c, C57BL/6, or C57BL/6tyr2cJ blastocysts.

Description Part Number Packaging Storage TempPrimogenix PRX-B6NMouse EmbryonicStem Cells (mESCs)

Q SV30109.01 Single vial containing at least2 x 106 cells at passage 10 or 11

-140°C in liquid nitrogen

Primogenix PRX-129/X1 Mouse Embryonic StemCells (mESCs)

Q SV30098.01 Single vial containing at least2 x 106 cells at passage 10 or 11

-140°C in liquid nitrogen

Q Item in stock.

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PPRRXX--112299//XX11 MMuurriinnee EEmmbbrryyoonniicc SStteemm CCeellllss((mmEESSCCss))


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AdvanceSTEM Mesenchymal Stem Cell Culture Platform

Multipotent stem cells, sometimes called adultstem cells (ASCs), have been found to persist intheir undifferentiated state in both embryonicand adult tissues. These cells can be derivedfrom most tissues and, depending on theirorigin, have different properties and capacityfor generating a variety of progeny. However,unlike embryonic stem cells, they have a finitelife span in culture and are not totipotent.

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Mesenchymal stem cells represent an interestingand widely used stem cell type, possessing theability to differentiate into a variety of celltypes, including adipocytes, chondrocytes, osteocytes, cardiomyocytes, neurons, andmany others.

The AdvanceSTEM line of stem cell culture products has been developed specifically forapplications in stem cell research.

The line comprises numerous products designedto support a wide range of applications such asthe expansion and maintenance of humanadult mesenchymal stem cells, to the directeddifferentiation of these cells into such celltypes as neurons, adipocytes, chondrocytes,and osteocytes.

Recently, Thermo Fisher Scientific entered into an agreement with Cellular EngineeringTechnologies (CET) to provide a wide array ofhuman non-embryonic stem cells, growthmedia and differentiation media.

This agreement covers the production and marketing of human non-embryonic stem cells,growth media used to grow these cells and differentiation media used to convert the stemcells into other type of human cells, whichcould be used in the future to treat conditionssuch as diabetes, cardiovascular failure andParkinson's disease.

Cellular Engineering Technologies, Inc., is abiotechnology tissue engineering companywhich combines engineering expertise with lifescience for preclinical drug discovery. CET specializes in stem cells obtained from humannon-embryonic sources, such as fat collectedduring liposuction procedures, umbilical cords,umbilical cord blood, and placentas collectedafter full-term deliveries. The company wasfounded by Dr. Alan Moy, CEO, in 2000 and isIowa's premier non-embryonic stem cell company.


Mesenchymal Stem Cell Expansion Kit

The AdvanceSTEMMesenchymal Stem CellExpansion Kit has been developed to supportthe expansion and maintenance ofundifferentiated Human Mesenchymal StemCells (hMSCs), including HumanWharton'sJelly Stem Cells (HWJSCs), Human Adipose-derived Mesenchymal Stem Cells (HAMSCs),Human Amniotic Mesenchymal Stem Cells,Multipotent Cord Blood Unrestricted SomaticStem Cells (MCBUSSCs) and bone marrow-derived hMSCs.

Each kit includes 1000 mL of AdvanceSTEMMesenchymal Stem Cell Basal Medium and100 mL of AdvanceSTEM Growth Supplement.The medium and growth supplement should beused together; however, components are alsoavailable individually.

Amniotic Epithelial Expansion Kit

The AdvanceSTEM Amniotic EpithelialExpansion Kit has been developed to supportthe expansion of undifferentiated HumanAmniotic Epithelial Stem Cells.

The expansion kit comprises of proprietaryAmniotic Epithelial expansion medium (450 mL)and AdvanceSTEMGrowth Supplement (50 mL).The two products combined produce thecomplete medium used for Amniotic Epithelialexpansion.

Adipogenic Differentiation Kit

Post expansion using the AdvanceSTEMMesenchymal Expansion Kit, differentiation ofMSCs into adipocytes can be accomplishedusing the AdvanceSTEM AdipogenicDifferentiation Kit. This kit has been developedto support adipogenic differentiation fromHuman Adipose-derived Mesenchymal StemCells (HAMSCs), and Human Bone MarrowMesenchymal Stem Cells (HBMSCs).

The differentiation kit comprises a proprietarybasal Adipogenic Differentiation Medium(450 mL), to which an aliquot of AdvanceSTEMGrowth Supplement (50 mL) is added at thetime of use. Basal Adipogenic DifferentiationMedium and Growth Supplement should beused together, but are available as separatecomponents.

Chondrogenic Differentiation Medium

The AdvanceSTEM Chondrogenic DifferentiationMedium has been developed to supportdifferentiation of a variety of hMSCs intochondrocytes. After expansion of hMSCs usingtheAdvanceSTEMMesenchymal Expansion kit,chondrogenic differentiation of Human BoneMarrowMesenchymal Stem Cells (HBMSCs)can be accomplished using this kit.

Osteogenic Differentiation Kit

After expanding hMSCswith the AdvanceSTEMMesenchymal Expansion Kit, AdvanceSTEMOsteogenic Differentiation Kit has been developedto support the differentiation of a variety ofHumanMesenchymal Stem Cells—includingHuman Adipose-derivedMesenchymal StemCells (HAMSCs), Human BoneMarrowMesenchymal Stem Cells (HBMSCs) and HumanMultipotent Cord Blood Unrestricted SomaticStem Cells (MCBUSSCs) .

The differentiation kit comprises a proprietarybasal Osteogenic Differentiation Medium(450 mL), to which an aliquot of AdvanceSTEMGrowth Supplement (50 mL) is added at thetime of use. Basal Osteogenic DifferentiationMedium and Growth Supplement should beused together, but are available as separatecomponents.

Neural Differentiation Kit

The AdvanceSTEM Neural Differentiation Kithas been developed to facilitate the optimalneural differentiation of Adipose-DerivedMesenchymal Stem Cells, Bone MarrowMesenchymal Stem Cells, and MultipotentCord Blood Unrestricted Somatic Stem Cells(MCBUSSCs).

The differentiation kit comprises a proprietarybasal Neural Differentiation Medium (450 mL),to which an aliquot of AdvanceSTEM GrowthSupplement (50 mL) is added at the time of use.Basal Neural DifferentiationMedium and GrowthSupplement line should be used together, but areavailable as separate components.

Cryopreservation Medium

The AdvanceSTEM Cryopreservation Mediumhas been formulated to support the cryopreser-vation of a variety of cell types, includingHuman Mesenchymal Stem Cells, multipotentunrestricted stem cells of Umbilical Cord Bloodand Human Amniotic Epithelial Stem Cells.

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Mesenchymal Stem Cell Products

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Description Part Number Packaging Storage TempMesenchymal Stem Cell KitsMesenchymal Stem Cell Expansion Kit Q SH30875.KT 1 KitAdipogenic Differentiation Kit Q SH30876.KT 1 KitOsteogenic Differentiation Kit Q SH30877.KT 1 KitNeural Differentiation Kit Q SH30892.KT 1 Kit

Growth SupplementsGrowth Supplement Q SH30878.02 50 mL -10° C

Q SH30878.01 100 mL -10° CBasal and Cryopreservation MediaMesenchymal Stem Cell Basal Medium Q SH30879.01 500 mL 2-8°C

Q SH30879.02 1000 mL 2-8°CAdipogenic Differentiation Medium Q SH30886.02 450 mL 2-8°CChondrogenic Differentiation Medium Q SH30889.02 450 mL 2-8°COsteogenic Differentiation Medium Q SH30881.02 450 mL 2-8°CNeural Differentiation Medium Q SH30893.02 450 mL 2-8°C

Cryopreservation Medium Q SH30894.01 100 mL 2-8°C

Amniotic Epithelial Expansion Kit Q SH30904.KT 1 Kit

Amniotic Epithelial Expansion Medium Q SH30900.02 450 mL 2-8°C

SerumUS Human Mesenchymal Stem Cell Screened FBS Q SH30070.02M 100 mL -10°C or lower

Q SH30070.03M 500 mL -10°C or lower


Human Bone Marrow Mesenchymal Stem Cellsare isolated from human red bone marrowcollected during a bone marrow aspirationprocedure.

Human bone marrow mesenchymal stem cellsmay be differentiated into Adipogenic,chondrogenic, Osteogenic, neuron like cells andother cell types.

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Human Wharton's Jelly Stem Cells (HWJSC)are isolated and expanded from the innerportion of the human umbilical cord.The umbilical cord is longitudinally sectioned(length wise), stripped of its blood vessels,digested with enzymes and then the cellsare harvested.

Like other mesenchymal stem cells, these cellshave multilineage differentiation potential andimmunomodulatory functions in animal models.

CET Human Wharton's JellyMesenchymal Stem Cells

Human Adipose-Derived Mesenchymal StemCells (HAMSC) are isolated and expanded fromhuman lipoaspirate using enzymatic treatment.

HAMSC are easily isolated and capable ofbeing grown in large numbers. In addition,these cells also have multilineagedifferentiation potential and immunomodulatoryfunctions in animal models.

Tissue differentiation applications includeconversion into cell types such as neurons andcell types such as osteocytes.

CET Human Adipose-DerivedMesenchymal Stem Cells

Human Amniotic Mesenchymal Stem Cells areenzymatically isolated from the AmnioticMembrane of Placentas, after epithelial cellsare first removed.

Like other mesenchymal stem cells, these cellshave multilineage differentiation potential andimmunomodulatory functions in animal models.

CET Human Amniotic MesenchymalStem Cells

CET Mesenchymal Stem CellsDescription Part Number Packaging Storage TempCET Human Wharton's Jelly Mesenchymal Stem Cells ≥ 100,000 Q SV30101.01 2.0 mL vial -196°C in liquid nitrogenCET Human Wharton's Jelly Mesenchymal Stem Cells ≥ 500,000 Q SV30101.02 2.0 mL vial -196°C in liquid nitrogenCET Human Adipose-Derived Mesenchymal Stem Cells ≥ 100,000 Q SV30102.01 2.0 mL vial -196°C in liquid nitrogenCET Human Adipose-Derived Mesenchymal Stem Cells ≥ 500,000 Q SV30102.02 2.0 mL vial -196°C in liquid nitrogenCET Human Amniotic Mesenchymal Stem Cells ≥ 100,000 Q SV30103.01 2.0 mL vial -196°C in liquid nitrogenCET Human Amniotic Mesenchymal Stem Cells ≥ 500,000 Q SV30103.02 2.0 mL vial -196°C in liquid nitrogenCET Human Bone Marrow Mesenchymal Stem Cells ≥ 100,000 Q SV30110.01 2.0 mL vial -196°C in liquid nitrogenCET Human Bone Marrow Mesenchymal Stem Cells ≥ 500,000 Q SV30110.02 2.0 mL vial -196°C in liquid nitrogen

CET Mesenchymal Stem Cells

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CET Human Bone Marrow MesenchymalStem Cells

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Unlike Mesenchymal Stem Cells, HCBHSC aremainly used in animal models to study theimmune system and cardiovascular orcirculatory defects. Tissue differentiationapplications include conversion intoendothelial progenitor cells.

Validation for CD34+ and CD133+ are both,growth in hematopoietic stem cell media.Molecular Validation (Flow Cytometry ofProtein Cell Surface Markers) CD34+ andCD133+ Surface Antigen.

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Human Amniotic Epithelial Cells (HAEC) areisolated from the amniotic membrane of freshplacentas using an enzyme-based treatment.The cells retain embryonic stem cellcharacteristics and are useful for tissuedifferentiation of all three germ layers,the ectoderm-endoderm and the mesoderm.

Tissue differentiation applications includeconversion into pancreatic islet cells, liverhepatocytes, cardiomyocytes and neurons.Cells are enzymatically isolated from theamniotic membrane of a human placenta.Cells are differentially plated and isolatedusing their morphology.

CET Somatic Stem Cells

Multipotent Cord Blood Unrestricted SomaticStem Cells are isolated from human umbilicalcord blood using differential plating techniquesand a proprietary media formulation.

These cells may be differentiated into celltypes such as neuron-like cells andosteoblasts.

Three passages, using tissue culture plasticadhesion, are conducted to achieve ahomogenous population.

CETMultipotent Cord Blood UnrestrictedSomatic Stem Cells

CET Somatic Stem Cells

CET Human Amniotic Epithelial Stem Cells CET Human Cord Blood CD34+Stem Cells

CET Human Cord Blood CD133+Stem Cells

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Human CD34+ or CD133+ Hematopoietic StemCells (HCBHSC) are isolated robotically fromfresh umbilical cord blood using a magneticallycoupled antibody, which is specific for eitherthe CD34+ or C133+ antigen. This is donerobotically to maximize purity and yield whileminimizing human handling and chances ofcontamination.

HCBHSC are important in a variety ofapplications including reconstitution of theimmune system, synthesis of endothelium orbasement membrane and revascularization ofdamaged blood vessels. HCBHSC are alsobeing used to study aberrant stem cell functionin the progression of immune related cancers.

Description Part Number Packaging Storage TempCET Human Amniotic Epithelial Stem Cells ( 100,000 Q SV30104.01 2.0 mL vial -196°C in liquid nitrogenCET Human Amniotic Epithelial Stem Cells ( 500,000 Q SV30104.02 2.0 mL vial -196°C in liquid nitrogenCET Human Multipotent Cord Blood Unrestricted Somatic Stem Cells ( 100,000 Q SV30105.01 2.0 mL vial -196°C in liquid nitrogenCET Human Multipotent Cord Blood Unrestricted Somatic Stem Cells ( 500,000 Q SV30105.02 2.0 mL vial -196°C in liquid nitrogenCET Human Cord Blood CD34+ Stem Cells ( 100,000 Q SV30106.01 2.0 mL vial -196°C in liquid nitrogenCET Human Cord Blood CD34+ Stem Cells ( 500,000 Q SV30106.02 2.0 mL vial -196°C in liquid nitrogenCET Human Cord Blood CD133+ Stem Cells ( 100,000 Q SV30107.01 2.0 mL vial -196°C in liquid nitrogenCET Human Cord Blood CD133+ Stem Cells ( 500,000 Q SV30107.02 2.0 mL vial -196°C in liquid nitrogen


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Providing optimal culture conditions can beone of the greatest challenges in stem cellresearch. Aside from optimal nutrientrequirements, an optimal substrate is alsoimportant. There are several key considerationsto take into account when choosing theappropriate substrate on which to expand anddifferentiate these very important cells.

Whether your requirements mandate ananimal component-free system or yourresearch requires the ability to incorporategrowth factors and peptides into your stem cellculture system, there is a HyStem Hydrogel kitto meet your needs.

Thermo Scientific HyClone HyStemHydrogels

HyStem Hydrogel ProductsDescription Part Number Packaging Storage TempHyStem Kit 2.5 mL Q SV30138.01 Kit -10°C or lowerHyStem Kit 7.5 mL Q SV30138.02 Kit -10°C or lowerHyStem-C Kit 2.5 mL Q SV30139.01 Kit -10°C or lowerHyStem-C Kit 7.5 mL Q SV30139.02 Kit -10°C or lowerHyStem-HP Kit 2.5 mL Q SV30140.01 Kit -10°C or lowerHyStem-HP Kit 7.5 mL Q SV30140.02 Kit -10°C or lower

HyStem Hydrogels

HyStemHydrogels

Charactistics Key Features Key Benefits

HyStemChemically modifiedhyaluronan crosslinkedwith PEGDA

1. Hyaluronan-based

2. Just add water and go

3. Can change formulation orstiffness before gelation

1. Recommended forapplications requiringattachment factor optimization

1. Animal-free

2. Synthetic

3. Customizable before gelation

4. Injectable

5. Ease in changing stiffness

6. Batch to batch consistency

7. Non-toxic

8. Customizable before gelation

9. ECM protein incorpation

10. 2-D plating

11. 3-D encapsulation

HyStem-C HyStem but with chemicallymodified gelatin added

1. Just add water and go

2. Gelatin (or denatured collagen)is an excellent additive to startwith when optimizing your ownmatrix/surface

1. Starting point for optimizingyour own surface or matrix

HyStem-HP HyStem-C but withchemically modified heparin

1. Contains heparin which actsby ionically binding to growthfactors

1. Slow release of growth factors

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Thermo Scientific HyClone Serum-FreeMedia (SFM)

Our versatile Serum Free Media productsare developed through Metabolic PathwayDesign to support superior performancein multiple cell culture platforms. Thisdevelopmental approach produces SFMtargeted to increasing process yields for eachrespective cell platform. Current SFMapplications include hybridoma, NS0, insect,CHO, PER.C6, HEK293, MDCK, MDBK,COS-7, and Vero cell cultures. The use ofSFM provides many time- and cost-savingadvantages, including:• eliminating the need to pre-screen

serum lots• simplified regulatory documentation• consistent media performance• reduced downstream purification challenges

Applications for serum-free media can befound in many biotechnology fields includingthe production of human and animalbiopharmaceuticals, diagnostics reagents andbioagricultural products. Due to the increasingattention to producing biologicals representativeof their native form, our Serum-Free Mediahave been focused on supporting mammalianand invertebrate cell culture platforms. Someof the critical cell culture platforms forbiotechnology include Chinese Hamster Ovary(CHO) cells, hybridomas and myelomas, insectcells, cells for the production of gene therapyviral vectors, and cells for the production ofviral vaccines. These cell culture platformsconstitute the majority of applications forbiopharmaceutical research, development andmanufacture employing eukaryotic cell culture.

Metabolic Pathway Design

High performance with our serum-free mediafor specific cell culture platforms is achievedthrough Metabolic Pathway Design technology.This approach to media formulationdevelopment ensures cell productivity duringgrowth and production phases of cell life.Extensive work in media development hasresulted in products that provide a consistentnutrient supply in a form acceptable tocultured cells.

The Metabolic Pathway Design approachbalances nutrient supply against metabolicwaste accumulation, determines the effectivedose of nutrients critical to the production ofrecombinant proteins, and provides complexedlipids and phospholipids that facilitate deliverythrough the cell membrane. The objectivefor developing serum-free media throughMetabolic Pathway Design is to provide theintended cell line with the nutrients needed topromote cell growth and stimulate productivity.

Serum-Free Media PlatformsCHO, Hybridoma, Myeloma, Gene Therapy, Insect, and Viral Vaccine


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The CHO cell culture platform has led thebiopharmaceutical industry in its use ofserum-free media products (SFM). ThermoScientific HyClone SFM products lead theindustry with continued development on aselection of superior media to meet clients'needs for CHO cell culture. A selection of SFMfor the unique needs of CHO cells is available.

CDM4CHO

CDM4CHO is a chemically defined mediumcontaining no animal derived components.This regulatory-friendly medium is developedthrough Metabolic Pathway Design to increaseprocess yields in the manufacture ofrecombinant proteins using a variety of CHOcell clones. CDM4CHO has been successfullytested in a variety of culture systems, includingT-flasks, shaker flasks and bioreactors,including fed-batch and perfusion.

CDM4CHO contains Pluronice F68 andL-glutamine, and does not contain phenol red.It is also available without L-glutamine tosupport the GS gene expression system.

SFM4CHO

SFM4CHO is a high performance cell culturemedium developed through Metabolic PathwayDesign to increase process yields for theindustrial manufacturing of recombinantproteins for therapeutic use in a variety ofCHO cells.

SFM4CHO is a protein-free formulation thatcontains no components of bovine origin andhas been designed for high performance ina variety of culture vessels, includingbioreactors. It is formulated using ourproprietary lipid complexing process forenhanced stability and growth promotion ofvarious CHO cell lines. SFM4CHO containsL-glutamine and Pluronic F68, and does notcontain phenol red. It has been designed tosupport the DHFR selection/amplificationsystem, and is also available withoutL-glutamine to support the GS geneexpression system.

SFM4CHO-Utility

SFM4CHO-Utility is a versatile protein-free cellculture medium developed through MetabolicPathway Design to support the growth ofmultiple CHO cell clones and production of avariety of recombinant proteins. It enablessuperior growth of many CHO cells withminimal adaptation.

SFM4CHO-Utility is ideally suited for thecost-effective manufacturing of recombinantproteins for academic and industrial research,genomics and proteomics, drug targetscreening and validation, and manufacturingof preclinical lots. It is formulated using ourproprietary lipid complexing process forenhanced stability and broad-spectrumgrowth promotion.

SFM4CHO-Utility contains Pluronic F68,and does not contain phenol red. It has beendesigned to support the DHFR selection/amplification system. This protein-free mediumhas been successfully tested in a variety ofculture systems including T-flasks, spinner andshaker flasks, roller bottles and bioreactors.

SFM4CHO-A

SFM4CHO-A is a protein-free mediumdeveloped through the Metabolic PathwayDesign to support the growth of multiple CHOcell clones and the production of a variety ofrecombinant proteins in adherent cultures. Thismedium’s formulation equals the robustnessof serum-based media in adherence, spreadingand growth using standard tissue-culturesurfaces – without concerns associatedwith animal-derived products. It has beensuccessfully tested in a range of culturesystems including T-flasks, and microcarriercultures. SFM4CHO-A does not containphenol red and is available with or withoutL-glutamine.

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Serum-Free Media

CHO Cell Culture Platform


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CHO Cell Culture Platform MediaDescription Part Number Packaging L-glutamine Phenol Red HEPES Sodium

BicarbonateStorage Temp

CDM4CHO with L-glutamine (liquid) Q SH30557.01 500 mL 4 mM no no 2.2 g/L 2-8°CQ SH30557.02 1000 mLP SH30557.LS 6 x 1000 mLP SH30557.03 5 L BPCP SH30557.04 10 L BPCP SH30557.05 20 L BPCP SH30557.06 50 L BPCP SH30557.07 100 L BPCP SH30557.08 200 L BPCP SH30557.09 500 L BPC

CDM4CHO without L-glutamine (liquid) Q SH30558.01 500 mL no no no 2.2 g/L 2-8°CQ SH30558.02 1000 mLP SH30558.LS 6 x 1000 mLP SH30558.03 5 L BPCP SH30558.04 10 L BPCP SH30558.05 20 L BPCP SH30558.06 50 L BPCP SH30558.07 100 L BPCP SH30558.08 200 L BPCP SH30558.09 500 L BPC

CDM4CHO (powder) Q SH30556.01 1 x 5 L no no no no 2-8°CQ SH30556.02 1 x 10 LP SH30556.03 1 x 50 LQ SH30556.04 1 x 100 LP SH30556.05 1 x 500 LP 1 x 1000 L

SFM4CHO (liquid) Q SH30549.01 500 mL 4 mM no no 2.2 g/L 2-8°CQ SH30549.02 1000 mLP SH30549.LS 6 x 1000 mLP SH30549.03 5 L BPCP SH30549.04 10 L BPCP SH30549.05 20 L BPCP SH30549.06 50 L BPCP SH30549.07 100 L BPCP SH30549.08 200 L BPCP SH30549.09 500 L BPC

SFM4CHO without L-glutamine (liquid) Q SH30548.01 500 mL no no 2.2 g/L 2-8°CQ SH30548.02 1000 mLP SH30548.LS 6 x 1000 mLP SH30548.03 5 L BPCP SH30548.04 10 L BPCP SH30548.05 20 L BPCP SH30548.06 50 L BPCP SH30548.07 100 L BPCP SH30548.08 200 L BPCP SH30548.09 500 L BPC


In addition to the aforementioned products, wecontinue to offer early generation serum-freeCHO media. PF CHOq and PF CHO LSq havebeen successfully tested in a variety ofapplications and accepted in numerousresearch and biopharmaceutical settings. Ourfirst two products for the CHO cell cultureplatform, CCM5q and SFX-CHOq have beendesigned for anchorage-dependent andsuspension CHO cells, respectively.

CHO Cell Culture products available:

• CDM4CHO– Chemically Defined, ADCF– Designed for Suspension Cultures– Support Large-Scale Culture Applications

• SFM4CHO– Protein-Free– Designed for Suspension Cultures– Support High Density Bioreactor Culture

• SFM4CHO-A– Protein-Free, ADCF– Developed for Adherent CHO Cells

– Support Multiple CHO Cell Lines andCulture Environment

• SFM4CHO-Utility– Protein-Free– Designed for Suspension Cultures– Supports Multiple CHO Cell Lines andCulture Environments

• PF-CHO LS• PF-CHO• SFX-CHO• CCM5

SH30556.06

no


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CHO Cell Culture Platform Media (continued)Description Part Number Packaging L-glutamine Phenol Red HEPES Sodium


SFM4CHO (powder) Q SH30518.01 1 x 5 L no no no no 2-8°CQ SH30518.02 1 x 10 LQ SH30518.03 1 x 50 LP SH30518.04 1 x 100 LP SH30518.05 1 x 500 LQ SH30518.06 1 x 1000 L

SFM4CHO-Utility with L-glutamine(liquid)

Q SH30516.01 500 mL 4 mM no no 2.2 g/L 2-8°CQ SH30516.02 1000 mLQ SH30516.LS 6 x 1000 mLP SH30516.04 10 L BPCP SH30516.03 5 L BPCP SH30516.05 20 L BPCP SH30516.06 50 L BPCP SH30516.07 100 L BPCP SH30516.08 200 L BPCP SH30516.09 500 L BPC

SFM4CHO-Utility (powder) Q SH30517.01 1 x 5 L no no no no 2-8°CQ SH30517.02 1 x 10 LP SH30517.03 1 x 50 LP SH30517.04 1 x 100 LP SH30517.05 1 x 500 LP SH30517.06 1 x 1000 L

PF CHO LS with L-glutamine (liquid) P SH30359.01 500 mL 4 mM no no 2.2 g/L 2-8°CQ SH30359.02 1000 mLP SH30359.LS 6 x 1000 mLP SH30359.03 20 L BPCP SH30359.04 50 L BPCP SH30359.05 100 L BPCP SH30359.06 200 L BPCP SH30359.07 500 L BPC

PF-CHO (powder) Q SH30333.01 1 x 5 L no no no no 2-8°CQ SH30333.02 1 x 10 LP SH30333.03 1 x 50 LQ SH30333.04 1 x 100 LP SH30333.05 1 x 1000 L

PF-CHO (liquid) P SH30220.02 500 mL 2 mM yes no 2.0 g/L 2-8°CQ SH30220.01 1000 mLP SH30220.04 20 L BPCP SH30220.03 50 L BPCP SH30220.05 100 L BPCP SH30220.06 200 L BPCP SH30220.07 20 L BPC Q/C

SFX-CHO (liquid) P SH30187.01 500 mL no yes 1.8 g/L 1.6 g/L 2-8°CQ SH30187.02 1000 mLP SH30187.03 20 L BPCP SH30187.04 50 L BPCP SH30187.05 100 L BPC

CCM5 (liquid) P SH30100.02 500 mL 2.4 mM yes no 1.7 g/L 2-8°CQ SH30100.03 1000 mLP SH30100.04 10 L BPCP SH30100.05 20 L BPCP SH30100.06 200 L BPC


SFM4CHO-A With L-glutamine,Liquid

Q SH30821.01 500 mL 10 mM no no 1.2g/L 2-8°CQ SH30821.02 1000 mLP SH30821.LS 6 x 1000 mLP SH30821.03 5 L BPCP SH30821.04 10 L BPCP SH30821.05 20 L BPCP SH30821.06 50 L BPCP SH30821.07 100 L BPCP SH30821.08 200 L BPCP SH30821.09 500 L BPCP SH30821.10 900 L BPC


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The application of serum-free media in theproduction of monoclonal antibodies (MAbs)from hybridoma and recombinant myeloma celllines for research and diagnostic purposes israpidly growing. Typically, serum-containingmedia are used to culture these cell lines;however, growing concerns over serum supplyand the potential for exogenous serumcontamination have driven the transition toserum-free media.

Since each hybridoma or recombinant myelomacell line is unique, it is necessary that theserum-free medium of choice offer nutrientsfor a variety of applications. In addition to this,unique requirements of some cell lines requirespecific media supplementation to be effective(e.g., cholesterol auxotrophic cell line, NS0).

CDM4NS0

CDM4NS0 is a chemically defined mediumcontaining no animal derived components.This regulatory-friendly medium is developedthrough Metabolic Pathway Designq toincrease process yields in the manufacturer ofmonoclonal antibodies (MAbs) using a varietyof NS0 cell clones. CDM4NS0 requires nocholesterol or GS supplementation, as it hassufficient amounts to support NS0 cell culture

already in the formulation. It has beensuccessfully tested in a variety of culturesytems,including T-flasks, shaker flasks and biore-actors using batch and fed-batch strategies.

CDM4NS0 contains Pluronic F68, and does notcontain L-glutamine and Phenol Red.

CDM4MAb

CDM4MAb is a chemically defined mediumcontaining no animal dervied components.This regulatory-friendly medium is developedthrough our Metabolic Pathway Designqapproach to increase process yields for themanufacture of monoclonal antibodies fortherapeutic use in a variety of engineeredhybridoma and recombinant myeloma cell lines.

CDM4MAb contains Pluronic F68 and does notcontain Phenol Red. It is available with andwithout L-glutamine.

SFM4MAb

SFM4MAb is a high performance cell culturemedium developed throughMetabolic PathwayDesign to increase the process yields for theindustrial manufacture of human and humanizedrecombinant antibodies and antibody fragmentsfor therapeutic use in a variety of engineeredhybridoma and recombinant myeloma cell lines.

SFM4MAb is a low protein formulation and isoptimized for downstream purification usingProtein A, Protein G and other matrices tofacilitate product recovery. It is formulatedusing a proprietary lipid and phospholipidcomplexing process for enhanced stabilityand growth promotion of various cell types.

SFM4MAb contains synthetic cholesterol,L-glutamine and Pluronic F68, and doesnot contain phenol red. It is also availablewithout L-glutamine.

SFM4MAb-Utility

SFM4MAb-Utility is a versatile serum-free cellculture medium developed through MetabolicPathway Design to support the growth ofmultiple hybridoma cell types and productionof a variety of immunoglobulins. It enablessuperior growth of many hybridomas andrecombinant myelomas with minimal adaptation.

SFM4MAb-Utility is ideally suited for thecost-effective manufacture of monoclonal

antibodies for academic and industrialresearch, genomics and proteomics, in vitrodiagnostics, drug target screening andvalidation, and manufacturing of preclinicallots. It is formulated using our proprietarylipid and phospholipids complexing processfor enhanced stability and broad-spectrumgrowth promotion.

Serum-Free Media

Hybridoma and Myeloma Cell Culture Platform

TThheerrmmoo SScciieennttiifificc HHyyCClloonnee SSeerruumm--FFrreeee MMeeddiiaapprroodduuccttss ffoorr HHyybbrriiddoommaa aanndd MMyyeelloommaa cceellllss

PPrroodduuccttiioonn ooff IIggGG11 uussiinngg aa pprroopprriieettaarryy SSpp22//00--ddeerriivveedd hhyybbrriiddoommaa ccuullttuurreedd iinnSSFFMM44MMAAbb uussiinngg aa 1100 LL ssttiirrrreedd ttaannkk bbiioorreeaaccttoorr.. CCuullttuurree pprroocceessss:: bbaattcchh mmooddee ((00--9966hh));; mmeeddiiuumm eexxcchhaannggee ((9966hh));; ppeerrffuussiioonnmmooddee ((114444--226644hh));; ddiiaafifillttrraattiioonn ((226644--331122hh)).. PPeerr--ffuussiioonn//ddiiaafifillttrraattiioonn rraatteess iinnddiiccaatteedd oonn ggrraapphh iinnwwoorrkkiinngg vvoolluummeess ppeerr ddaayy ((wwvv//dd)).. IInnsseerrtt:: IIggGG11 ppuurriifificcaattiioonn uussiinngg aa pprree--ppaacckkeeddHHiiTTrraapp rrPPrrootteeiinn GG HHPP ccoolluummnn ooppeerraatteeddtthhrroouugghh AAKKTTAAffppllccqq..

PPrroodduuccttiioonn ooff IIggGG uussiinngg aa pprrooppiieettaarryy GGSS--NNSS00cceellll lliinnee ccuullttuurreedd iinn CCDDMM44NNSS00 dduurriinngg 110000 mmLLsshhaakkeerr ccuullttuurreess.. FFeedd--BBaattcchh ccuullttuurree eemmppllooyyeeddLLSS11000000 ((SSHH3300555544)) aanndd GGSS MMaaxx ((SSHH3300558866)) aatt 7722hhoouurrss..

PPrroodduuccttiioonn ooff IIggGG22aa uussiinngg aa PP33--ddeerriivveedd hhyybbrriiddoommaa ccuullttuurreedd iinn SSFFMM44MMAAbb--UUttiilliittyy dduurriinngg aa 5500 mmLL bbaattcchhssppiinnnneerr ccuullttuurree.. IInnsseerrtt:: IIggGG22aa ppuurriifificcaattiioonn uussiinngg aa pprreeppaacckkeeddHHiiTTrraapp rrPPrrootteeiinn GG HHPP ccoolluummnn ooppeerraatteeddtthhrroouugghh AAKKTTAAffppllcc((ttmm))..

PPrroodduuccttiioonn ooff IIggGG uussiinngg aann SSpp22//00--ddeerriivveedd hhyybbrriiddoommaa cceellll lliinnee ccuullttuurreedd iinn CCDDMM44MMAAbb dduurriinngg aa sshhaakkeerr flflaasskk ccuullttuurree..


2-8°Cnono

40

Hybridoma and Myeloma Cell Culture Platform Media

SFM4MAb-Utility contains cholesterol, anddoes not contain phenol red or Pluronic F68.This serum-free medium has been successfullytested in a variety of culture systems includingT-flasks, spinners, roller bottles and variousbioreactors, including hollow fiber.

ADCF-MAb

ADCF-MAb is a protein-free medium containingno animal derived components. This regulatory-friendly medium is developed throughMetabolic Pathway Design to increase theprocess yields for the manufacture of antibodiesand antibody fragments for therapeutic use in avariety of engineered hybridoma andrecombinant myeloma cell lines. ADCF-MAbcontains L-glutamine and Pluronic F68, anddoes not contain phenol red. It is also availablewithout L-glutamine to support the GS geneexpression system.

LS250

LS250 is a chemically defined, ADCF lipidsupplement specifically developed throughMetabolic Pathway Design to stimulate cellgrowth and monoclonal antibody (MAb)production in cholesterol auxotrophic NS0 celllines. This 250X concentrate lipid supplementhas been formulated using a proprietary lipidcomplexing process for enhanced stability andfilterability. LS250 has been designed tosupplement a variety of Thermo ScientificHyClone serum-free media, and may be used inthe preparation of liquid media from drypowdered media.

LS1000

LS1000 is a chemically defined, ADCF,cholesterol supplement specifically designed to beadded as a fed-batch supplement or to sterile liq-uidmedia at the time of use. This 1000Xconcentrated supplement has been formulatedusing a proprietary complexing process forenhanced cholesterol delivery.

In addition to these recently developedserum-free media, support continues for earlygeneration Thermo Scientific HyCloneProducts, such as, CCM1, PF-MAb and SFX-MAb. CCM1, a serum-free medium fora variety of hybridoma and myeloma cell lines,has been shown to work in a variety ofalternative cell lines, including lymphocytesand epithelial cells. SFX-MAb is a serum-freemedium for hybridoma and myeloma cell lines.

PF-MAb is a 100X concentrate designed for usewith RPMI 1640 and other basal formulationsto reduce or eliminate serum dependency.

Thermo Scientific HyClone Media available forHybridoma and Myeloma Cell Culture include:• CD4MNS0– Chemically Defined, ADCF

• CDM4MAb– Chemically Defined, Designed for NS0cells, ADCF

– Designed for Suspension Cultures– Support Large-Scale Culture Applications

• SFM4MAb– Serum-Free– Ultra-Low Protein– Designed for Suspension Culture– Supports High Density Bioreactor Cultures

• SFM4MAb-Utility– Serum-Free– Low Protein– Supports Multiple Hybridoma/MyelomaCell lines in a variety of culture systems

• ADCF-MAb– Animal Derived Component Free– Protein-Free

• LS250– Chemically Defined, ADCF

• LS1000– Chemically Defined, ADCF– Contains 2.5 mg/mL Cholesterol

• SFX-MAb• CCM1• PF-MAb

BBaattcchh pprroodduuccttiioonn ooff IIggGG iinn AADDCCFF--MMAAbb uussiinnggaann PP33--ddeerriivveedd hhyybbrriiddoommaa ccuullttuurreedd iinn aa 110000 mmLLsshhaakkeerr flflaasskk..

Description Part Number Packaging L-glutamine Phenol Red HEPES Sodium Bicarbonate

Storage Conditions

CDM4NS0 (liquid) P SH30579.01 500 mL no no no 3.2 g/L 2-8°CQ SH30579.02 1000 mL P SH30579.LS 6 x 1000 mLP SH30579.03 5 L BPCP SH30579.04 10 L BPCP SH30579.05 20 L BP CP SH30579.06 50 L BPCP SH30579.07 100 L BPCP SH30579.08 200 L BPCP SH30579.09 500 L BPC

CDM4NS0 (powder) P SH30578.01 1 x 5 L no noQ SH30578.02 1 x 10 LP SH30578.03 1 x 50 LQ SH30578.04 1 x 100 LP SH30578.05 1 x 500 LP SH30578.06 1 x 1000 L



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Hybridoma and Myeloma Cell Culture Platform Media (continued)


Description Part Number Packaging L-glutamine Phenol Red HEPES SodiumBicarbonate

StorageTemp

CDM4MAb with L-glutamine (liquid) Q SH30801.01 500 mL 6 mM no no 3.2 g/L 2-8°CQ SH30801.02 1000 mLP SH30801.LS 6 x 1000 mLP SH30801.03 5 L BPCP SH30801.04 10 L BPCP SH30801.05 20 L BP CP SH30801.06 50 L BPCP SH30801.07 100 L BPCP SH30801.08 200 L BPCP SH30801.09 500 L BPC

CDM4MAb without L-glutamine (liquid) Q SH30802.01 500 mL no no no 3.2 g/L 2-8°CQ SH30802.02 1000 mLP SH30802.LS 6 x 1000 mLP SH30802.03 5 L BPCP SH30802.04 10 L BPCP SH30802.05 20 L BP CP SH30802.06 50 L BPCP SH30802.07 100 L BPCP SH30802.08 200 L BPCP SH30802.09 500 L BPC

CDM4MAb (powder) P SH30800.01 1 x 5 L no no no no 2-8°CP SH30800.02 1 x 10 LP SH30800.03 1 x 50 LP SH30800.04 1 x 100 LP SH30800.05 1 x 500 LP SH30800.06 1 x 1000 L

SFM4MAb with L-glutamine (liquid) Q SH30513.01 500 mL 6 mM no no 3.2 g/L 2-8°CQ SH30513.02 1000 mLP SH30513.LS 6 x 1000 mLP SH30513.03 5 L BPCP SH30513.04 10 L BPCP SH30513.05 20 L BP CP SH30513.06 50 L BPCP SH30513.07 100 L BPCP SH30513.08 200 L BPCP SH30513.09 500 L BPC

SFM4MAb without L-glutamine (liquid) P SH30391.01 500 mL no no no 3.2 g/L 2-8°CP SH30391.02 1000 mLP SH30391.LS 6 x 1000 mLP SH30391.03 5 L BPCP SH30391.04 10 L BPCP SH30391.05 20 L BP CP SH30391.06 50 L BPCP SH30391.07 100 L BPCP SH30391.08 200 L BPCP SH30391.09 500 L BPC

SFM4MAb (powder) P SH30535.02 1 x 5L no no no no 2-8°CP SH30535.03 1 x 10 LP SH30535.04 1 x 50 LP SH30535.05 1 x 100 LP SH30535.06 1 x 500 LP SH30535.07 1 x 1000 L

SFM4MAb-Utility with L-glutamine (liquid) Q SH30382.01 500 mL 6 mM no 3.5 g/L 2.3 g/L 2-8°CQ SH30382.02 1000 mLP SH30382.LS 6 x 1000 mLP SH30382.03 5 L BPCP SH30382.04 10 L BPCP SH30382.05 20 L BP CP SH30382.06 50 L BPCP SH30382.07 100 L BPCP SH30382.08 200 L BPC

SFM4MAb-Utility (powder) Q SH30550.01 1 x 5 L no no 3.5 g/L no 2-8°CQ SH30550.02 1 x 10 LP SH30550.03 1 x 50 LP SH30550.04 1 x 100 LP SH30550.05 1 x 500 LP SH30550.06 1 x 1000 L


42

Hybridoma and Myeloma Cell Culture Platform Media (continued)

** Storage temperatures: Long-term: -20°C (24 months); Short term: 2–8°C (12 months).

Description PartNumber

Packaging L-glutamine PhenolRed

HEPES SodiumBicarbonate

Storage Temp

ADCF-MAb (liquid) Q SH30349.01 500 mL 4.5 mM no 3.6 g/L 1.6 g/L 2-8°CQ SH30349.02 1000 mLP SH30349.LS 6 x 1000 mLP SH30349.03 5 L BPCP SH30349.04 10 L BPCP SH30349.05 20 L BP CP SH30349.06 50 L BPCP SH30349.07 100 L BPCP SH30349.08 200 L BPC

ADCF-MAb without L-glutamine (liquid) P SH30547.01 500 mL no no 3.6 g/L 1.6 g/L 2-8°CQ SH30547.02 1000 mLP SH30547.LS 6 x 1000 mLP SH30547.03 5 L BPCP SH30547.04 10 L BPCP SH30547.05 20 L BP CP SH30547.06 50 L BPCP SH30547.07 100 L BPCP SH30547.08 200 L BPC

ADCF-MAbq (powder) P SH30635.01 1 x 5 L no no 3.6 g/L no 2-8°CP SH30635.02 1 x 10 LP SH30635.03 1 x 50 LP SH30635.04 1 x 100 LP SH30635.05 1 x 500 LP SH30635.06 1 x 1000 L

SFX-MAb (liquid) P SH30206.03 500 mL no yes 3.6 g/L 1.6 g/L 2-8°CP SH30206.01 1000 mLP SH30206.02 20 L BPC

CCM1 (liquid) P SH30043.02 500 mL 2.5 mM yes 3.6 g/L 2.0 g/L **Q SH30043.03 1000 mL

CCM1 (powder) P SH30058.01 10 x 1 L 2.5 mM yes 3.6 g/L no 2-8°CP SH30058.02 2 x 5 LP SH30058.03 1 x 10 LP SH30058.04 1 x 50 LP SH30058.05 1 x 100 L

CCM1 (powder) P SH30059.01 10 x 1 L 2.5 mM no 3.6 g/L no 2-8°CP SH30059.02 2 x 5 LP SH30059.03 1 x 10 LP SH30059.04 1 x 50 LP SH30059.05 1 x 100 L

LS1000 Lipid Supplement, ADCF (liquid) P SH30554.01 50 mL no no no no 2-8°CQ SH30554.02 100 mLP SH30554.03 500 mL

LS250 Lipid Supplement, ADCF (liquid) Q SH30555.01 100 mL no no no no 2-8°CP SH30555.02 500 mLP SH30555.03 1000 mL

PF-MAb (liquid) P SH30138.01 100 mL no no no no 15-30°CP SH30138.05 500 mL


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Serum-Free Media

HEK 293 and PER.C6 Cell Culture Platform

As biotherapeutics are desired more closelyrepresentative of their native form, theapplication of human-derived cell lines forrecombinant protein, monoclonal antibodiesand gene therapy viral vectors has rapidlyemerged.

Thermo Scientific HyClone products includehigh performing serum-free media capable ofproviding optimal cell culture conditions forHEK 293 and PER.C6 cells in theseapplications. These media have beendeveloped through the Metabolic PathwayDesign approach to ensure that the needs ofhuman-derived cell lines are met to enable thehighest level of productivity.

Products available for HEK 293 and PER.C6cells include:

CDM4HEK293• Chemically defined, ADCF• Designed for high performance suspensionHEK 293 cultures

SFM4Transfx-293• Protein-free, ADCF• Designed to promote transfection inHEK 293 cultures

SFM4HEK293• Protein-free, ADCF• Designed for suspension HEK 293 culturesfor a variety of applications

CDM4Retino• Chemically defined, ADCF• Designed for high adenoviral vectorproduction in suspension PER.C6 cultures

CDM4PERMAb• Chemically defined, ADCF• Designed for high MAb expression inPER.C6 culturesTThheerrmmoo SScciieennttiifificc HHyyCClloonnee PPrroodduucctt ffoorr

HHEEKK229933 aanndd PPEERR..CC66 CCeellll CCuullttuurreess

HyClone_042-043:0 3/30/2010 4:44 PM Page 43

CDM4HEK293 is a chemically defined, animalderived component free and protein-free cellculture medium designed to support the growthof HEK 293 cultures, and promoteadenovirus and recombinant proteinproduction. This regulatory-friendly mediumwas developed to support high cell densityand specific cell productivity insuspension cultures.

CDM4HEK293

SFMTransfx-293 is a serum-free, animal derivedcomponent free medium designed to supportthe growth of HEK 293 cultures and promotetransfection using lipofection or similarmethods. This regulatory-friendly mediumwas developed to support high transfectionefficiency, productivity and cell density insuspension cultures.

SFM4Transfx-293

This versatile cell culture medium wasdeveloped to support the production ofadenoviral vectors and recombinant proteinsin HEK 293 cultures. SFM4HEK293 is aprotein-free medium containing no animalderived components. It has been formulated forsuperior yields, and has been tested in a varietyof culture systems including T-flasks, shakerflasks and bioreactors.

SFM4HEK293

CDM4Retino is a chemically defined mediumcontaining no animal derived components.Thishigh performance cell culture medium has beendeveloped to increase process yields in theproduction of adenoviral vectors andrecombinant proteins using PER.C6 cells. It hasbeen successfully tested in a variety ofapplications, including perfusion bioreactors.

CDM4Retino

CDM4PERMAb is a chemically defined mediumcontaining no animal derived components.This high performance cell culture medium hasbeen developed to increase process yields inthe production of human antibodies andrecombinant proteins using PER.C6technology. It has been successfully tested ina variety of applications, including fed-batchbioreactors.

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CDM4PERMAb

PPrroodduuccttiioonn ooff pprroopprriieettaarryy AAdd55 uussiinngg HHEEKK229933cceellllss ccuullttuurreedd iinn SSFFMM44HHEEKK229933 dduurriinngg aa 1100 LLbbaattcchh bbiioorreeaaccttoorr ccuullttuurree.. TThhee ccuullttuurree wwaass iinnffeecctteedd oonnccee tthhee cceellll ddeennssiittyy rreeaacchheedd >>11..00 xx 110066 cceellllss//mmLL..

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2-8°CnonoSH30871.01Q

HEK293 and PER.C6 Cell Culture Platform MediaDescription Part Number Packaging L-glutamine Phenol Red HEPES Sodium

BicarbonateStorageTemp

CDM4PERMAb withoutL-glutamine (liquid)

500 mL no 3.2 g/LQ SH30871.02 1000 mLP SH30871.LS 6 x 1000 mLP SH30871.03 1 L BPCP SH30871.04 5 L BPCP SH30871.05 10 L BPCP SH30871.06 20 L BPCP SH30871.07 50 L BPCP SH30871.08 100 L BPCP SH30871.09 200 L BPCP SH30871.10 500 L BPC

CDM4PERMAb withoutL-glutamine (powder)

P SH30872.01 1 x 5 L no no no no 2-8°CP SH30872.02 1 x 10 LP SH30872.03 1 x 50 LP SH30872.04 1 x 100 LP SH30872.05 1 x 500 LP SH30872.06 1 x 1000 L

CDM4HEK293 withoutL-glutamine (liquid)

Q SH30858.01 500 mL no no no 2.0 g/L 2-8°CQ SH30858.02 1000 mLP SH30858.LS 6 x 1000 mLP SH30858.03 1 L BPCP SH30858.04 5 L BPCP SH30858.05 10 L BPCP SH30858.06 20 L BPCP SH30858.07 50 L BPCP SH30858.08 100 L BPCP SH30858.09 200 L BPCP SH30858.10 500 L BPC

CDM4HEK293 withoutL-glutamine (powder)

P SH30859.01 1 x 5 L no no no no 2-8°CP SH30859.02 1 x 10 LP SH30859.03 1 x 50 LP SH30859.04 1 x 100 LP SH30859.05 1 x 500 LP SH30859.06 1 x 1000 L

SFM4Transfx-293 withoutL-glutamine (liquid)

Q SH30860.01 500 mL no no no 2.0 g/L 2-8°CQ SH30860.02 1000 mLQ SH30860.LS 6 x 1000 mLP SH30860.03 1 L BPCP SH30860.04 5 L BPCP SH30860.05 10 L BPCP SH30860.06 20 L BPCP SH30860.07 50 L BPCP SH30860.08 100 L BPCP SH30860.09 200 L BPCP SH30860.10 500 L BPC

SFM4Transfx-293 withoutL-glutamine (powder)

Q SH30861.01 1 x 5 L no no no no 2-8°CQ SH30861.02 1 x 10 LP SH30861.03 1 x 50 LP SH30861.04 1 x 100 LP SH30861.05 1 x 500 LP SH30861.06 1 x 1000 L


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2-8°Cno4 mMSH30521.01Q

HEK293 and PER.C6 Cell Culture Platform Media (contd.)

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Description Part Number Packaging L-glutamine Phenol Red HEPES SodiumBicarbonate

StorageTemp

SFM4HEK293 with L-glutamine (liquid) 500 mL no 2.0 g/LQ SH30521.02 1000 mL

P SH30521.03 5 L BPCP SH30521.04 10 L BPCP SH30521.05 20 L BPCP SH30521.06 50 L BPCP SH30521.07 100 L BPCP SH30521.08 200 L BPCP SH30521.09 500 L BPC

SFM4HEK293 (powder) P SH30522.01 1 x 5 L no no no no 2-8°CP SH30522.02 1 x 10 LP SH30522.03 1 x 50 LP SH30522.04 1 x 100 LP SH30522.05 1 x 500 LP SH30522.06 1 x 1000 L

CDM4Retino (liquid) P SH30520.01 500 mL 4 mM no no 2.0 g/L 2-8°CP SH30520.02 1000 mLP SH30520.03 5 L BPCP SH30520.04 10 L BPCP SH30520.05 20 L BPCP SH30520.06 50 L BPCP SH30520.07 100 L BPCP SH30520.08 200 L BPCP SH30520.09 500 L BPC

CDM4Retino (powder) P SH30519.01 1 x 5 L no no no no 2-8°CP SH30519.02 1 x 10 LP SH30519.03 1 x 50 LP SH30519.04 1 x 100 LP SH30519.05 1 x 500 LP SH30519.06 1 x 1000 L

PF-293 (liquid) P SH30356.01 500 mL no no no 2.0 g/L 2-8°CP SH30356.02 1000 mLP SH30356.03 20 L BPCP SH30356.04 50 L BPCP SH30356.05 100 L BPCP SH30356.06 200 L BPC

PF-293 MPS (powder) P SH30355.06 1 x 1 L no no no no 2-8°CP SH30355.01 1 x 5 LP SH30355.02 1 x 10 LP SH30355.03 1 x 50 LP SH30355.04 1 x 100 LP SH30355.05 1 x 1000 L


P SH30521.LS 6 x 1000 mL


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Insect Cell Culture Platform

The development of insect cell cultureproduction systems has produced opportunitiesfor the expression of recombinant proteins forresearch and therapeutic applications in anon-mammalian cell culture environment.

Many different insect cell lines have beendeveloped for use with the BaculovirusExpression vector System (BEVS). Additionally,the development of stable transfectedDrosophila and lepidopteran cell lines hasoffered a non-lytic culture for the productionof recombinant proteins from insect cells.

SFX-Insect

This versatile protein-free cell culturemedium is developed through MetabolicPathway Design to support the growth ofmultiple insect cell lines and production of avariety of recombinant proteins. It providessuperior growth of many key insect celllines,including Sf9, Sf21, High Five, andD.mel (2), requiring minimal adaptation.

SFX-Insect is ideally suited for the costeffective manufacture of recombinantproteins for academic and industrialresearch, genomics and proteomics, drugtarget screening and validation, andmanufacturing for therapeutic applications.

SFX-Insect is formulated using our proprietarylipid and phospholipid complexing processfor enhanced stability and performance.SFX-Insect contains Pluronice F68 and hasbeen developed for superior performance intested in a variety of culture systems rangingfrom T-flasks, spinners, shaker flasks(including Fernbach), and various bioreactorsincluding hollow fiber.

Serum-Free Media

Insect Cell Culture Platform

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FFiigguurree 11.. GGrroowwtthh kkiinneettiiccss ooff bbaattcchh ccuullttuurreess ooffSSff99 cceellllss ccoommppaarriinngg tthhee nneeww SSFFMM44IInnsseecctt((ggrreeeenn)) ttoo oouurr ttrraaddiittiioonnaall sseerruumm--ffrreeee ffoorrmmuullaattiioonn SSFFXX--IInnsseecctt ((ppuurrppllee))..

SFM4Insect

Thermo Scientific HyClone SFM4Insect™ isan animal-derived component free cell culturemedium developed through Metabolic Path-way Design to support growth of many keyinsect cell lines and production of a varietyof recombinant proteins. Insect cells needonly minimal adaptation to SFM4Insect toprovide growth performance superior to for-mulations that contain animal origin compo-nents. The medium has been successfullytested in a variety of culture systems includingtraditional and disposable bioreactors for production of recombinant proteins using the Baculovirus Expression Vector System.

In addition to component traceability, regulatory friendly ADCF and protein-freecharacteristics, cGMP and ISO compliant facility manufacturing have also been maintained to provide cell culture and bioprocessing customers a quality product.SFM4Insect is available in liquid form in 500 mL and 1 L bottles, and in 1 L to 200 LBioProcess Containers or custom packaging.The medium is also available as a powderfor research through production applications.

In addition to SFX-Insect and SFM4Insect,other Thermo Scientific HyClone Insect Culture Products include TNM-FH, IPL-41,CCM3™ and the BEVS PlaKit™. TNM-FH is amodification of the original Grace's mediumto contain yeast extract and lactalbumin hydrolysate. When supplemented with 10 percent Insect Screened FBS, TNM-FH becomes a complete insect cell culturemedium which requires minimal adaptation.Grace's Medium (unsupplemented) is alsoavailable. Thermo Scientific HyClone IPL-41is a basal medium, frequently used forserum-free media development/optimization.CCM3 is our first serum-free medium for Sf9 cells.

The Thermo Scientific HyClone BEVS PlaKitprovides the necessary reagents for a reproducible and accurate baculovirus plaque assay kit.

FFiigguurree 22.. DDoouubblliinngg ttiimmeess ffrroomm bbaattcchh ccuullttuurreessooff SSff99 cceellllss ccoommppaarriinngg tthhee nneeww SSFF44IInnsseecctt ttoo oouurr ttrraaddiittiioonnaall sseerruumm--ffrreeee ffoorrmmuullaattiioonn,, SSFFXX--IInnsseecctt..


48

Insect Cell Culture Platform MediaDescription Part Number Packaging L-Glutamine Phenol Red HEPES Sodium


SFX-Insect (liquid) Q SH30278.01 500 mL PETE 10 mM no no 0.35 g/L 2-8°CQ SH30278.02 1000 mL PETEQ SH30278.LS 6 x 1000 mLP SH30278.05 5 L BPCP SH30278.06 10 L BPCP SH30278.03 20 L BPCP SH30278.04 50 L BPCP SH30278.07 100 L BPC

SFX-Insect (powder) P SH30350.04 1 x 1 L 10 mM no no no 2-8°CQ SH30350.02 1 x 5 LQ SH30350.03 1 x 10 L

Grace's Unsupplemented (liquid) P SH30610.01 500 mL 4 mM no no 0.35 g/L 2-8°CP SH30610.02 1000 mL PETE

TNM-FH (liquid) Q SH30280.02 500 mL 4 mM no no no 2-8°CQ SH30280.03 1000 mL

IPL-41 (powder) P SH30282.01 1 x 5 L 7 mM no no no 2-8°CP SH30282.02 1 x 10 LP SH30282.03 1 x 50 LP SH30282.04 1 x 100 L

BEVS PlaKit P SH30340 1 Kit n/a no n/a n/a 2-8°CCCM3 (liquid) Q SH30065.01 500 mL PETE 7mM no no 0.35g/L 2-8°C

Q SH30065.02 1000 mL PETEP SH30065.03 50 L BPCP SH30065.04 10 L BPC

CCM3 (DPM) P SH30061.01 10 x 1 L 7mM no no no 2-8°CP SH30061.02 2 x 5 LP SH30061.03 1 x 10 LP SH30061.04 1 x 50 LP SH30061.05 1 x 100 L


SFM4Insect with L-glutamine,liquid

Q SH30913.01 500 mL PETE 10mM no no 0.35g/L 2-8°CQ SH30913.02 1000 mL PETEP SH30913.LS 6 x 1000 mL PETEP SH30913.03 1 L BPCP SH30913.04 5 L BPCP SH30913.05 10 L BPCP SH30913.06 20 L BPCP SH30913.07 50 L BPCP SH30913.08 100 L BPCP SH30913.09 200 L BPC

SFM4Insect with L-glutamine,powder

P SH30912.01 5 L HDPE 10mM no no no 2-8°CP SH30912.02 10 L HDPEP SH30912.03 50 L HDPEP SH30912.04 100 L Polybag/pailP SH30912.05 500 L Polybag/pailP SH30912.06 1000 L Polybag/pail


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Q SH30552.01 no 2-8°Cno

HEPES

Serum-Free Media

Viral Vaccine Cell Culture Platform

The application of serum-free media in the

production of virus vaccines has been largely

driven by concerns over serum supply and

exogenous contaminants. Key cell lines in this

industry are the African green monkey kidney

cell, Vero, and the canine kidney and bovine

kidney epithelial cells, MDCK and MDBK.

SFM4MegaVir

SFM4MegaVir is a protein-free mediumcontaining no animal derived components.This regulatory-friendly medium is developedthrough Metabolic Pathway Design toincrease process yields in the manufacture ofviral vaccines in a variety of cell lines includingVero, COS-7, MDCK, and MDBK.

SFM4MegaVir has been successfully tested ina variety of culture systems for cell growthand virus production including T-flasks andsuspension microcarrier cultures. SFM4MegaVirdoes not contain Pluronic F68 or L-glutamine.It does contain Phenol Red.

Alongside SFM4MegaVir, a protein-freemedium designed specifically for Vero cells,PF-Veroq is also available. This medium hasdemonstrated superior performance in multipleculture environments, and was the foundationfor developing SFM4MegaVir.

Thermo Scientific HyClone Viral Vaccine CellCulture Media include:• SFM4MegaVir– Protein-Free– Animal Derived Component Free– Developed for multi-cultureenvironments, including microcarrierbioreactor cultures

• PF-Vero

Viral Vaccine Cell Culture Platform Media

SSHH3300555522..0011,, SSHH3300558877..0022


Description L-glutamine Sodium Bicarbonate

StorageTemp

SFM4MegaVir without L-glutamine (liquid)

500 mL yes 2.95 g/LQ SH30552.02 1000 mLP SH30552.LS 6 x 1000 mLP SH30552.03 5 L BPCP SH30552.04 10 L BPCP SH30552.05 20 L BPCP SH30552.06 50 L BPCP SH30552.07 100 L BPCP SH30552.08 200 L BPCP SH30552.09 500 L BPC

SFM4MegaVir (powder) P SH30587.01 1 x 5 L yes no no 2-8°CP SH30587.02 1 x 10 LP SH30587.03 1 x 50 LP SH30587.04 1 x 100 LP SH30587.05 1 x 500 LP SH30587.06 1 x 1000 L

IInnflfluueennzzaa AA pprroodduuccttiioonn uussiinngg MMDDCCKK cceellllss((MMOOII==11 00)) aanndd PPoolliioovviirruuss pprroodduuccttiioonn uussiinngg VVeerroo cceellllss ((MMOOII==00..11)) ccuullttuurreedd iinnSSFFMM44MMeeggaaVViirr aanndd DDMMEEMM ++55%%FFBBSS..

no

Part Number Packaging Phenol Red


50

Process Supplements Platform

The fed-batch version of stirred-tank culturehas become most popular at large scales. Theprimary driver of this trend is based on addingnutrients to a batch culture in mid-run toincrease the quantity of product harvested.

The prevalence of fed-batch over other modesis due to many practical factors, such asreliability, ease of scalability andapplication latitude.

The Thermo Scientific HyClone ProcessSupplements platform has been designed tosupport demand in the area of fed-batch cellculture and media customization. Eachsupplement is developed through theMetabolic Pathway Design approach toprovide specific nutrient combinations for avariety of applications, and have beensuccessfully tested in CHO, Hybridoma, NS0,HEK 293, PER.C6 and other cell lines.

The six Thermo Scientific HyClone Cell Boostsupplements have been designed to providesuch nutrients as amino acids, vitamins, lipids,cholesterol, glucose and/or growth factors innovel combinations for multiple mammaliancell types. Our Cell Boost supplements arechemically defined and ADCF. In addition to theCell Boost line, we offer chemically defined,ADCF concentrated liquid supplements

designed to provide lipids and cholesterol tosuch cell lines as NS0.

Each Cell Boost supplement is designed toprovide nutrients for fed-batch cultureapplications or media customization. Thesupplements each contain a unique blend ofkey cell culture nutrients. To facilitateselecting the appropriate Cell Boost ProcessSupplement, the Thermo Scientific HyCloneCell Boost Process Supplement kit contains a100 g bottle of each formulation. Each CellBoost Process Supplement has been tested ina variety of cell culture systems, includingshaker flasks and bioreactors in CHO, HEK 293,PER.C6, hybridoma and myeloma, and othercell lines.

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Product Name Amino Acids Vitamins Glucose Trace Element Growth Factor(s) Hypoxanthine/ Lipids SyntheticThymidine Cholesterol

Cell Boost 1 (R06.2) X X XCell Boost 2 (R15.4) X X XCell Boost 3 (JM3.5) X X X X XCell Boost 4 (PS307) X X X X X X XCell Boost 5 (CN-F) X X X X X X X XCell Boost 6 (CN-T) X X X X X X X XLS1000 XLS250 X X


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Cell Boost 1 is a supplement designed toprovide nutrients such as amino acids, vitaminsand glucose. It is designed to improveproductivity through a multi-vitamin feedapproach. Cell Boost 1 has been tested on avariety of cell lines including CHO andHEK 293 cells.

Cell Boost 1

Cell Boost 2 is a supplement designed toprovide nutrients such as amino acids andglucose. It is also designed to improveproductivity through a single-vitamin feedapproach. Cell Boost 2 has been tested on avariety of cell lines including CHO and PER.C6.

Cell Boost 2

Cell Boost 3 is a supplement designed toprovide nutrients such as glucose, amino acids,vitamins and trace elements. Cell Boost 3 hasbeen tested on a variety of cell lines including,hybridomas and myelomas.

Cell Boost 3

Cell Boost 4 is a supplement designed toprovide nutrients such as amino acids,vitamins, glucose, trace elements and growthfactors. Cell Boost 4 has been tested on avariety of cell lines including CHO.

Cell Boost 4

Cell Boost 5 is a supplement designed toprovide nutrients such as lipids, amino acids,vitamins and growth factors. Cell Boost 5 hasbeen tested in a variety of cell lines including,CHO, Hybridoma, NS0 and HEK 293.

Cell Boost 5

Cell Boost 6 is a supplement designed toprovide nutrients such as lipids, amino acids,vitamins and growth factors. Cell Boost 6 hasbeen tested in a variety of cell lines including,CHO, Hybridoma, NS0, HEK 293 and T-cells.

Cell Boost 6

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PPrroodduuccttiioonn ooff MMoonnoocclloonnaall AAnnttiibbooddyy uussiinngg aarrMMAAbb CCHHOO cceellll lliinnee ccuullttuurreedd iinn CCDDMM44CCHHOO dduurr--iinngg aa 33 LL ssttiirrrreedd ttaannkk bbiioorreeaaccttoorr ccuullttuurree.. FFeedd--bbaattcchh ccuullttuurree eemmppllooyyeedd oonn ddaayyss 00,, 33,, 55 aanndd 77uussiinngg CCeellll BBoooosstt 22 ((RR1155..44))..

PPrroodduuccttiioonn ooff IIggGG11 uussiinngg pprroopprriieettaarryy SSpp22//00--ddeerriivveedd hhyybbrriiddoommaa ccuullttuurreedd iinn CCDDMM44MMAAbb.. FFeedd--bbaattcchh ccuullttuurree eemmppllooyyeedd oonn ddaayyss 44 aanndd 66 uussiinngg CCeellll BBoooosstt 33 ((JJMM33..55))..

PPrroodduuccttiioonn ooff rrPPrrootteeiinn uussiinngg CCHHOO cceellll lliinnee ccuullttuurreedd iinn CCDDMM44CCHHOO dduurriinngg aa 33 LL ssttiirrrreedd--ttaannkk bbiioorreeaaccttoorr ccuullttuurree.. FFeedd--bbaattcchh ccuullttuurree eemmppllooyyeedd oonn ddaayyss 33,, 55 aanndd 77 uussiinngg TThheerrmmooSScciieennttiifificc HHyyCClloonnee CCeellll BBoooosstt 44 ((PPSS330077))..

PPrroodduuccttiioonn ooff CCDD4466 aannttiibbooddyy iinn PPEERR..CC66 cceellllss ccuullttuurreedd iinn CCDDMM44PPEERRMMAAbb.. FFeedd--bbaattcchh ccuullttuurreeeemmppllooyyeedd ddaaiillyy ffeeeeddiinnggss ooff CCeellll BBoooosstt 55 ((CCNN--FF))..

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52

Description Part Number Packaging Storage TempCell Boost Process Supplement Kit Q SH30890 1 Kit 2-8°CCell Boost 1 (R05.2) Q SH30584.02 100 g 2-8°C

P SH30584.03 500 g 2-8°CP SH30584.04 1000 g 2-8°CP SH30584.05 5000 g 2-8°C

Cell Boost 2 (R15.4) Q SH30596.01 100 g 2-8°CP SH30596.02 500 g 2-8°CP SH30596.03 1000 g 2-8°CP SH30596.04 5000 g 2-8°C

Cell Boost 3 (JM3.5) Q SH30825.01 100 g 2-8°CP SH30825.02 500 g 2-8°CP SH30825.03 1000 g 2-8°CP SH30825.04 5000 g 2-8°C

Cell Boost 4 (PS307) Q SH30857.01 100 g 2-8°CP SH30857.02 500 g 2-8°CP SH30857.03 1000 g 2-8°CP SH30857.04 5000 g 2-8°C

Cell Boost 5 (CN-F) Q SH30865.01 100 g 2-8°CP SH30865.02 500 g 2-8°CP SH30865.03 1000 g 2-8°CP SH30865.04 5000 g 2-8°C

Cell Boost 6 (CN-T) Q SH30866.01 100 g 2-8°CP SH30866.02 500 g 2-8°CP SH30866.03 1000 g 2-8°CP SH30866.04 5000 g 2-8°C

LS1000 Lipid Supplement, ADCF (liquid) P SH30554.01 50 mL 2-8°CQ SH30554.02 100 mL 2-8°CP SH30554.03 500 mL 2-8°C

LS250 Lipid Supplement, ADCF (liquid) Q SH30555.01 100 mL 2-8°CP SH30555.02 500 mL 2-8°CP SH30555.03 1000 mL 2-8°C

LS1000 is a chemically defined, animal derivedcomponent free cholesterol supplementdesigned to be added as a fed-batch supplement.This 1000X concentrated supplement has beenformulated using a proprietary complexingprocess for enhanced cholesterol delivery. Ithas been successfully tested in a variety ofserum-free media cultures, including ThermoScientific HyClone CDM4NS0 and CDM4MAb.LS1000 delivers 2.5 mg of cholesterol per mL.

LS1000

LS250 is a chemically defined, animal derivedcomponent free lipid supplement developed tostimulate cell growth and MAb production inNS0 cell cultures using traditional hybridomaserum-free media. It has been successfullytested in a variety of media, including ThermoScientific CDM4MAb and SFM4MAb.

LS250

GS-Max is a chemically defined, animalderived component free glutamine synthetase(GS) cell culture supplement. Its developmenthas built upon traditional GS supplementformulations to provide the additional nutrientsneeded for high productivity in applicable CHOand NS0 cell lines. It has been successfullytested as both a media preparation supplement,and as a fed-batch supplement.

GS-Max


Process Supplements

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PPrroodduuccttiioonn ooff MMAAbb uussiinngg pprroopprriieettaarryy GGSS--NNSSOOcceellll lliinnee ccuullttuurreedd iinn SSFFMM44MMAAbb wwiitthh GGSS--MMaaxxssuupppplleemmeenntt.. LLSS225500 wwaass aaddddeedd ttoo bbootthh ccuull--ttuurreess ttoo ssttiimmuullaattee pprroodduuccttiivviittyy.. LLSS11000000 wwaassaaddddeedd ttoo tthhee ffeedd--bbaattcchh ccuullttuurree aass aa 11000000XXccoonncceennttrraattee bbeeggiinnnniinngg aatt 4488 hhoouurrss..PPrroodduuccttiioonn ooff MMoonnoocclloonnaall AAnnttiibbooddyy uussiinngg

aa pprroopprriieettaarryy NNSSOO cceellll lliinnee ccuullttuurreedd iinnCCDDMM44NNSSOO dduurriinngg aa 5500 LL SSiinnggllee UUSSee BBiioorreeaaccttoorr ccuullttuurree.. FFeedd--bbaattcchh ccuullttuurree eemmppllooyyeedd oonn ddaayyss 11,, 33 aanndd 66 uussiinngg LLSS11000000 ttoooovveerrccoommee ttrraaddiittiioonnaall cchhoolleesstteerrooll aaddssoorrppttiioonncchhaalllleennggeess iinn ddiissppoossaabbllee cceellll ccuullttuurree ssyysstteemmsswwiitthh cchhoolleesstteerrooll aauuxxoottrroopphhiicc cceellll lliinneess..

GS-MAX Q SH30586.01 100 mL 2-8°CP SH30586.02 500 mL 2-8°CP SH30586.03 1000 mL 2-8°C


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Nutrient Media Supplements

Living cells in vivo obtain the nutrients theyrequire directly from the blood and biologicaltissues that surround them. Cell culture mediaare composed of inorganic salts, sugars(energy sources), vitamins, amino acids andother components which are intended to mimicas nearly as possible these natural biological

fluids. Duplicating nature's miracle withexactness is, of course, impossible. Accordingly,animal derived serum is a popular mediasupplement (FBS in particular). FBS is a naturalbiological fluid, is readily available, providesnot only the necessary nutrients, but containsall the other biological and environmentalfactors that cells in vitro are accustomed to.We are recognized as the world leader inquality and supply.

Serum may present a challenge for somebiopharmaceutical and biologics manufacturersin that it contains potentially undesired proteinsthat need to be purified from the end-product.

Regulatory pressures to remove any animalderived components from products intendedfor human therapeutic use may compoundthis challenge.

Accordingly, many biopharmaceuticalmanufacturers are developing or purchasingserum-free and protein-free media formulationsor supplements. We address this need both byresearching and developing a high-performanceline of serum-free media platforms and byassembling a portfolio of nutritional andgrowth supplements.

Nutrient Media Supplements

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Description Part Number Packaging Storage TempBovine Serum Albumin (BSA), Cell Culture Grade, pH 7.0, Lyophilized Powder Q SH30574.01 10 g 2-8°C

Q SH30574.02 100 g 2-8°CP SH30574.03 500 g 2-8°C

Calcium Chloride Solution, 1 M Solution P SH30289.01 1000 mL 15-30°CD-Glucose (powder) P SH30371.01 5 kg 15-30°C

P SH30371.02 10 kg 15-30°CP SH30371.03 100 kg 15-30°CP SH30371.04 1 kg 15-30°C

HEPES 1 M Solution Q SH30237.01 100 mL 2-8°CHEPES Solution, 50 mM, 0.15 M NaCl, 20 mM EDTA, pH 8.0 P SH30291.01 1000 mL 15-30°CHypoxanthine/Thymidine (HT) Solution, 50X P SH30614.01 100 mL < 0°CLS250 Lipid Supplement, ADCF (liquid) Q SH30555.01 100 mL 2-8°C

P SH30555.02 500 mL 2-8°CP SH30555.03 1000 mL 2-8°C

LS1000 Lipid Supplement, ADCF (liquid) P SH30554.01 50 mL 2-8°CQ SH30554.02 100 mL 2-8°CP SH30554.03 500 mL 2-8°C

SG-200, (Stable dipeptide of L-AlanyL-glutamine) 200 mM, in 0.85% NaCl Solution Q SH30590.01 100 mL < 0°CSoy Hydrolysate UF (25%) (liquid) P SH30357.01 500 mL 2-8°C

P SH30357.02 1000 mL 2-8°C


Lactalbumin Hydrolysate (LAH) growth enhancement supplement for serum-free or serum-reduced cell culture systems (powder)

P SH30322.01 100 g 15-30°CP SH30322.02 500 g 15-30°CP SH30322.03 1 kg 15-30°CP SH30322.04 5 kg 15-30°C

L-glutamine, 200 mM Solution 29.2 mg/mL-L-glutamine in 0.85% NaCl Q SH30034.01 100 mL -20°CQ SH30034.02 500 mL -20°CP SH30034.03 1000 mL -20°C

L-glutamine (powder) P SH30336.01 25 g 15-30°CP SH30336.02 100 g 15-30°CP SH30336.03 500 g 15-30°C

MEM Amino Acid Solution, 50X Q SH30598.01 100 mL 2-8°CMEM Vitamin Solution, 100X Q SH30599.01 100 mL <0°CNon-Essential Amino Acid, (NEAA) Solution, 100X Q SH30238.01 100 mL 2-8°CPluronic 10% Solution P SH30594.01 100 mL 15-30°CPluronic (powder) P SH30612.01 100 g 15-30°CSodium Pyruvate 100 mM Solution Q SH30239.01 100 mL 2-8°CTryptose Phosphate Broth (TPB) (powder) P SH30394.01 5 kg 15-30°C

P SH30394.02 10 kg 15-30°C


54


Reagents and Cell Dissociation Products

To further support the needs of cell culturescientists, we offer an array of ancillaryproducts used in various aspects of culturingcells. Product descriptions are given below.See product table at end of section for specificproducts and ordering information.

Dissociation Reagents

Dissociation reagents such as Trypsin are usedto detach and disaggregate adherent cellsfrom the surface or substrate to which they areattached as well as for dissociating tissuesfor primary cell culture. Our product offeringincludes two commonly used concentrationsof gamma-irradiated porcine Trypsin (1:250)as well as a new non-mammalian Trypsinalternative called Thermo Scientific HyQTaseSee table below for specific productdescriptions.

HyQTase is an ultra-filtered solution ofproteolytic and collagenolytic enzymescombined to achieve rapid dissociation,while being gentle to cells. Its non-mammalianformulation makes it ideal for serum-freeapplications and eliminates the need forneutralizing or enzyme inhibitors.

Treatment results in rapid cell detachment,single cell suspension and high cell viabilitywith minimal passage-to-passage variability.

Reagents and Cell Dissociation Products

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Description Part Number Packaging Storage TempHyQTase Cell Detachment Solution, in DPBS with EDTA (non-mammalian) Q SV30030.01 100 mL < 0°CTrypsin, 0.05% 1X, with 0.5 g porcine Trypsin (1:250/L gamma irradiated)in HBSS with 0.2 g/L EDTA, without calcium, magnesium (liquid)

Q SH30236.01 100 mL < 0°CQ SH30236.02 500 mL < 0°C

Trypsin, 0.25% 1X, with 2.5 g porcine Trypsin (1:250/L gamma irradiated) in HBSS with 0.2 g/L EDTA, without calcium, magnesium (liquid)

Q SH30042.01 100 mL < 0°CQ SH30042.02 500 mL < 0°C

Trypsin 0.25% 1X Solution, with 2.5 g Porcine Trypsin (1:250/L) in HBSS, without Calcium and Magnesium, with 0.1% EDTA

Q SV30031.01 100 mL < 0°CP SV30031.02 500 mL < 0°C

Trypsin 2.5% 10X Solution, with 25.0 g Porcine Trypsin (1:250/L) in HBSS, without Calcium and Magnesium, without EDTA or Phenol Red

Q SV30037.01 100 mL < 0°C

Trypan Blue Solution, 0.4% in Phosphate Buffered Saline Q SV30084.01 100 mL 15-30°C


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Process Liquids, Buffers and Salt Solutions

Today's rapid advances in biotechnology havecreated a growing need for ultra-pure processliquids in large volumes. From our Water ForInjection (WFI) Quality water to wash/suspension solutions and purification buffers,we are the premier supplier of research andindustry scale process liquids.

For every liter of cell culture medium feedinga bioreactor, 5 to 20 L of sterile-filtered processliquids are required for downstream processingof desired end products. Downstreamprocesses include suspension, separation,and purification.

Whether large volumes of WFI Quality Water,washing and rinsing solutions, or other processliquids such as buffers for chromatography arerequired, we have the resources and expertiseto ensure that our customers have the processliquids they need, when they need them, andpackaged in convenient delivery systems.

Thermo Scientific HyClone Process Liquids aremanufactured under cGMP, ISO 9001:2000and ISO 13485:2003 guidelines. We utilizeUSP grade components when available.Multicompendial grade chemicals areavailable upon request.

All of our Process Liquids are fully QC testedand packaged in convenient ready-to-usedelivery systems. Standard product sizes rangefrom PETE/G bottles in 100 mL, 500 mL, and1000 mL sizes to Thermo Scientific HyCloneBioProcess Containers in 10, 20, 50, 100, 200,and 500 L sizes. Process liquids and packagingcan be produced on a customized basis to meetcustomer requirements.

Thermo Scientific HyClone Standard ProcessLiquid products include:• WFI Quality Water• Cell Culture Grade Water• Molecular Biology Grade Water• Balanced Salts Solutions• Buffers

Water is one of the major components used bythe pharmaceutical industry, widely used asa cleaning or rinsing agent, as an excipientin media and buffer preparation, and inreconstitution of products in both therapeuticand diagnostic applications. Furthermore, thereare various applications for uniquely qualifiedtypes of water. For example, in cell cultureapplications, the water used must be free ofcontaminating endotoxin. It is crucial in manymolecular biology applications that water befree of nucleases (i.e., DNase and RNase)and proteases. To meet these exactingrequirements, we offer the following lineof specialty water products.

WFI Quality Water

Installation of a WFI system represents a majorcapital expenditure for any biopharmaceuticalcompany. As a result, many leadingbiopharmaceutical and contract manufacturersoutsource their large volume demands for WFIQuality Water. Even companies with WFIsystems in place recognize the value of havinga qualified second source for their large volumeWFI Quality Water demands. Consider some ofthe advantages of outsourcing:• Eliminates large, up-front investments inland, buildings and equipment

• Eliminates the need for WFI systemsoperation, maintenance, and validation

• Eliminates the costs associated within-house testing facilities and procedures

• Reduces costs associated with down timesand equipment failure

• Provides back-up security when productiondemands exceed internal capacity

Thermo Scientific HyClone WFI Quality Waterundergoes an extensive purification processthat includes multimedia filtration, mineraland hardness reduction, reverse osmosis,continuous deionization, ultraviolet lighttreatment, and distillation with continuous hot

loop circulation. Our WFI Quality WaterSolutions meets current USP production andmonograph test requirements for Water ForInjection packaged in bulk for commercial useelsewhere. It is available in a variety ofvolumes and container options suitable forboth research and manufacturing.

Cell Culture GradeWater (Endotoxin-free)

Thermo Scientific HyClone Cell Culture GradeWater is not just "endotoxin screened," it isendotoxin-free (to the maximum detectionlimit of P0.005 EU/mL by turbidimetric assay).Endotoxin interferes with normal cellproliferation and growth. Its effects are notcompletely predictable and it can actuallyartificially enhance cell culture growth. Incultures used to produce human or animalproducts, its presence can elicit a febrileresponse in the patient.

Accordingly, it is vital to keep the cultures freeof contaminating endotoxins. The assurancethat water is free of endotoxin not onlyquells concerns about the reagent's physicalproperties, but it also assures that the reagentwas prepared under the best of processesand procedures. Use of endotoxin-free waterremoves one more potential doubt or problem.The purification process for our Cell CultureGrade Water includes deionization, reverseosmosis, distillation and 0.1 Rm filtration.Applications include:• Media preparation• Buffer preparation• LAL reagent testing

Cell Culture Grade Water is available in avariety of volumes and container optionssuitable for both research and manufacturingprocesses.

Molecular Biology GradeWater

In order to work with nucleotides such asRNA and DNA and to prevent subsequentdegradation due to enzymatic activity, it isnecessary to use reagents, including water,that are devoid of all nucleases. Nucleasesare naturally occurring enzymes whose role is

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5656 Q Item in stock. P Item is made to order. Lead times and minimum order quantities apply.

specifically to cleave nucleotide polymericmolecules such as DNA or RNA at variouspoints. Theoretically, just one molecule of anuclease could systematically facilitate thedestruction of an entire DNA or RNA sampleover time.

Thermo Scientific HyClone Molecular BiologyGrade Water is purified, tested and certified tohave no detectable contaminating activity inDNase, RNase, and Protease assays. It is verylow in endotoxin and contains no DEPC or anyother carcinogenic chemicals. Our purificationprocess includes continuous deionization,reverse osmosis, ultra-violet light treatment,multi-effect distillation, and 0.1 Rm filtration.

Applications include:• Buffer preparation• DNA/RNA purification• DNA sequencing and libraries• Gene mapping• Recombinant DNA and cloning

• PCR analysis• Northern Blot, Southern Blot, and other assays

Molecular Biology Grade Water is available ina variety of volumes and container optionssuitable for both research and manufacturingprocesses.

Balanced Salt Solutions and Buffers

Our commitment to being a full service supplierto the bioresearch and biopharmaceuticalcommunity includes offering a complementary

line of the most commonly used buffers andbalanced salts. Most products are available inboth powder and liquid forms in lot sizes thatfacilitate scale-up and manufacturingoperations.

All formulations incorporate a rigorousspecification and approval process thatincludes a meticulous screening of incomingraw materials and suppliers to ensureconformance to cGMP, ISO 9001:2000, ISO13485:2003 and our own quality standards.

Dry buffers are formulated using eitherconventional ball milling technologies or ournew continuous micronization process with lotsizes up to 6500 kg. Large volume liquid buffersin either standard or custom formulations areavailable in unique Thermo Scientific HyCloneBioProcess Containers in lot sizes up to 10,000L and volumes of up to 500 L per container.

Process Liquids, Buffers and Salts

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Description Part Number Packaging Storage TempBalanced Salt Solutions, Buffers and SaltsDulbecco's Phosphate Buffered Saline (DPBS), without Calcium, Magnesium (powder)

Q SH30013.02 2 x 5 L 2-8°CQ SH30013.03 1 x 10 L 2-8°CQ SH30013.04 1 x 50 L 2-8°C

Dulbecco's Phosphate Buffered Saline (DPBS), 1X, without Calcium, Magnesium, Phenol Red

Q SH30028.01 100 mL 15-30°CQ SH30028.02 500 mL 15-30°CQ SH30028.FS 6 x 500 mL 15-30°CQ SH30028.03 1000 mL 15-30°CQ SH30028.LS 6 x 1000 mL 15-30°CQ SH30028.04 20 L Bag 15-30°CP SH30028.08 10 L Bag 15-30°CP SH30028.09 5 L Bag 15-30°C

Dulbecco's Phosphate Buffered Saline (DPBS), 1X, with Calcium, Magnesium, without Phenol Red

Q SH30264.01 500 mL 15-30°CQ SH30264.FS 6 x 500 mL 15-30°CQ SH30264.02 1000 mL 15-30°C

Dulbecco's Phosphate Buffered Saline (DPBS), 10X, without Calcium, Magnesium, Phenol Red

P SH30378.01 100 mL 15-30°CQ SH30378.02 500 mL 15-30°C

Dulbecco's Phosphate Buffered Saline (DPBS), 10X, with Calcium, Magnesium

P SH30597.01 500 mL 15-30°C

Earle's Balanced Salt Solution (EBSS), with Calcium, Magnesium, Phenol Red (powder)

P SH30014.03 2 x 5 L 2-8°CP SH30014.03 1 x 10 L 2-8°CP SH30014.04 1 x 50 L 2-8°C

Earle's Balanced Salt Solution (EBSS), 1X, with Calcium, Magnesium, Phenol Red

Q SH30029.01 100 mL 15-30°CQ SH30029.02 500 mL 15-30°CP SH30029.03 1000 mL 15-30°C

Hank's Balanced Salt Solution (HBSS), with Calcium, Magnesium, Phenol Red (powder)

Q SH30015.02 2 x 5 L 2-8°CQ SH30015.03 1 x 10 L 2-8°CP SH30015.04 1 x 50 L 2-8°C

Hank's Balanced Salt Solution (HBSS), without Calcium, Magnesium, with Phenol Red (powder)

Q SH30016.02 2 x 5 L 2-8°CP SH30016.03 1 x 10 L 2-8°CP SH30016.04 1 x 50 L 2-8°C

Hank's Balanced Salt Solution (HBSS), 1X, with Calcium, Magnesium, Phenol Red

Q SH30030.02 500 mL 15-30°CQ SH30030.03 1000 mL 15-30°C

Hank's Balanced Salt Solution (HBSS), 1X, without Calcium, Magnesium, with Phenol Red

Q SH30031.01 100 mL 15-30°CQ SH30031.02 500 mL 15-30°CQ SH30031.FS 6 x 500 mL 15-30°CQ SH30031.03 1000 mL 15-30°C


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Process Liquids, Buffers, and Salts (continued)Description Part Number Packaging Storage TempHank's Balanced Salt Solution (HBSS), without Calcium,Magnesium, Phenol Red (powder)

P SH30107.02 2 x 5 L 2-8°CP SH30107.03 1 x 10 L 2-8°CP SH30107.04 1 x 50 L 2-8°C

Hank's Balanced Salt Solution (HBSS), 1X, with Calcium,Magnesium, without Phenol Red

Q SH30268.01 500 mL 15-30°CQ SH30268.02 1000 mL 15-30°CQ SH30268.LS 6 x 1000 mL 15-30°C

Hank's Balanced Salt Solution (HBSS), 1X, without Calcium,Magnesium, Phenol Red

Q SH30588.01 500 mL 15-30°CQ SH30588.02 1000 mL 15-30°C

HEPES 1M Solution Q SH30237.01 100 mL 2-8°CHEPES Solution, 50 mM, 0.15 M NaCl, 20 mM EDTA, pH 8.0 P SH30291.01 1000 mL 15-30°CHEPES Buffer (powder) P SH30337.01 25 g 15-30°C

P SH30337.02 50 g 15-30°CP SH30337.03 100 g 15-30°C

Phosphate Buffered Saline (PBS), 1X, 0.0067 M PO4,without Calcium, Magnesium, Phenol Red

Q SH30256.01 500 mL 15-30°CQ SH30256.FS 6 x 500 mL 15-30°CQ SH30256.02 1000 mL 15-30°CQ SH30256.LS 6 x 1000 mL 15-30°C

Phosphate Buffered Saline (PBS) 10X, 0.067 M PO4,without Calcium, Magnesium, Phenol Red

Q SH30258.01 500 mL 15-30°CQ SH30258.02 1000 mL 15-30°C

Sodium Bicarbonate Solution, with 75.0 g/L Sodium Bicarbonate Q SH30033.01 100 mL 15-30°CSodium Bicarbonate (powder) P SH30173.02 105 g 15-30°C

P SH30173.04 120 g 15-30°CP SH30173.06 2.2 kg 15-30°CP SH30173.08 220 g 15-30°CP SH30173.09 1 kg 15-30°C

Specialty WaterWater, Cell Culture Grade, Endotoxin-free,(P 0.005 EU/mL), Deionized, Distilled, 0.1 Rm Sterile Filtered

Q SH30529.01 100 mL 2-30°CQ SH30529.02 500 mL 2-30°CQ SH30529.FS 6 x 500 mL 2-30°CQ SH30529.03 1000 mL 2-30°CQ SH30529.LS 6 x 1000 mL 2-30°C

Water, Molecular Biology Grade, DNase, RNase,Protease free (no activity within assay detectability limits),Deionized, Distilled, 0.1 Rm Sterile Filtered

Q SH30538.01 100 mL 2-30°CQ SH30538.02 500 mL 2-30°CQ SH30538.FS 6 x 500 mL 2-30°CQ SH30538.03 1000 mL 2-30°CQ SH30538.LS 6 x 1000 mL 2-30°C

Water, WFI Quality, Meets current USP monograph criteriafor WFI packaged in bulk for commercial use elsewhere.Not for Diagnostic or Therapeutic Use.

Q SH30221.10 1000 mL 2-30°CQ SH30221.LS 6 x 1000 mL 2-30°CP SH30221.12 10 L Bag 2-30°CQ SH30221.17 500 mL 2-30°CP SH30221.18 100 mL 2-30°CQ SH30221.24 10 L Bag 2-30°CQ SH30221.25 20 L 2-30°CQ SH30221.26 50 L 2-30°CQ SH30221.27 100 L 2-30°CQ SH30221.28 200 L 2-30°CP SH30221.29 500 L 2-30°C


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Antibiotics and Selection AgentsWe offer a selection of quality antibiotics tocontrol bacterial, yeast, fungi, and mycoplasmacontaminations, as well as premium selectionantibiotics.

Note: Antibiotics/antimycotics should notbe used as an alternative to proper aseptictechnique.

The introduction of laminar-flow hoods coupledwith good aseptic techniques have greatlyreduced the necessity to add antibiotics inmost cell culture protocols. However,antibiotics are still routinely added to cellculture media.

Improving aseptic techniques and limitingantibiotic use is desirable, as overuse ofantibiotics can encourage the developmentof antibiotic-resistant organisms, and hide thepresence of a low-level contamination that canbecome fully operative if:• the antibiotics are removed• the culture conditions change• resistant strains develop

They can also have anti-metabolic effects thatcould cross-react with mammalian cells.

We recommend that the use of antibioticsbe limited and removed from the culture ifpossible. When antibiotics are used for longperiods of time, parallel cultures should bemaintained free of antibiotics.

The following general information about thevarious antibiotics, antimycotics, and selectionagents is provided for information only. Dosagemay vary for each individual application.

Penicillin/Streptomycin is a broad spectrumbacteriostatic with activity against gramnegative and gram positive organisms. It isused as an antibacterial or antimycobacterial.Penicillin interferes with the final stage ofsynthesis of the bacterial cell wall.Streptomycin, through protein synthesis,inhibits elongation at transpeptidation step.Binds to 30S subunit causing misreading.

Common working concentration for penicillin is100 U/mL; for Streptomycin: 100 Rg/mL.

Gentamicin Solution is a broad spectrum cellculture antibiotic that is non-toxic to virusesand mammalian cells at antibacterial levels. Itis active against gram positive and gram nega-tive bacteria and Mycoplasma, and is useful forlong-term virus and tissue culture studies. It isused as an antibacterial or antimycobacterial.Gentamicin interferes with bacterial proteinsynthesis by binding to L6 protein of 50S ribo-somal subunit. Common working concentrationis 5 µg/mL. It is cytotoxic at >300 µg/mL.

G418 Solution is an aminoglycoside used asa selective agent of transfected mammalian,yeast, plant and bacteria cells. G418 is relatedto Gentamicin. G418 interferes with the func-tion of 80S ribosomes and protein synthesisin eukaryotic cells. Resistance is conferredby the bacterial gene for aminoglycoside-3'-phophotransferase that can be expressed ineukaryotic cells.

Common working concentration for G418 is300–1000 µg/mL.

Hygromycin B is an aminoglycosidicantibiotic that kills bacteria, fungi and highereukaryotic cells by inhibiting protein synthesis.The resistance gene codes for a kinase thatinactivates Hygromycin B throughphosphorylation. Cloning of the resistancegene and fusion with eukaryotic promotershas resulted in the development of vectors thatpermit selection for resistance to Hygromycin Bin both prokaryotic and eukaryotic cells. Itis useful as an antifungal or antibacterial.Hygromycin B inhibits protein synthesis bydisrupting translocation and promotingmistranslation of the 80S ribosome. It isused for the selection and maintenance ofprokaryotic and eukaryotic cells transfectedwith the hygromycin resistance gene, hph.

Working concentration for mammalian cellselection is normally between 50 Rg/mL and1 mg/mL; for plant cells: 20–200 Rg/mL, forbacteria: 20–200 Rg/mL, and for fungi:200 Rg–1 mg/mL.

Puromycin is an antibiotic used to select cellsmodified by genetic engineering. Puromycinallows the selection and maintenance of cellsexpressing the puromycin-resistance gene. Itinhibits the growth of a wide range ofprokaryotic and eukaryotic cells by interferingwith protein synthesis. Its common workingconcentration is 0.1–30 µg/mL.

Amphotericin B (Fungizone) is an antibioticagent that is active against fungi. It is used asan antifungal, anti-yeast, and anti-mold agent.It interferes with the permeability of the cellmembrane of sensitive fungi by binding sterols.The common working concentration is2.5 Rg/mL, and it is cytotoxic at 30 µg/mL.

Antibiotic/Antimycotic Solution(Pen/Strep/Fungizone) contains 10,000 unitsof Penicillin, 10,000 Rg of streptomycin, and25 Rg of Amphotericin B per mL. This solutionhas a wide spectrum of activity against gramnegative and gram positive bacteria as well asactivity against fungi and yeast. It is commonlyused as an anti-fungal, anti-yeast, anti-mold,or antibacterial. Its common working dose is10 mL/L of 100X solution.

Note: These products are offered for ResearchUse and are not intended for Diagnostic orTherapeutic Use.


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Antibiotics and Selection AgentsDescription Part Number Packaging Storage TempAmphotericin B (Fungizone) Solution, 250 Rg/mL Q SV30078.01 50 mL < 0°CAntibiotic Antimycotic Solution (Pen/Strep/Fungizone),100X, 10,000 U/mL Penicillin G, 10,000 Rg/mL Streptomycin,25 Rg/mL Amphotericin B

Q SV30079.01 100 mL < 0°C

G418 Sulfate Solution, 50 mg/mL Q SV30069.01 20 mL 2-8°CG418 Sulfate, ) 90% Purity P SV30068.01 1 g 2-8°C

Q SV30068.02 5 g 2-8°CGentamicin Solution, 50 mg/mL Q SV30080.01 10 x 10 mL 15-30°CHygromycin B Solution, 50 mg/mL Q SV30070.01 20 mL 15-30°CPen/Strep/Glutamine Solution Penicillin/Streptomycin/Glutamine Solution Q SV30082.01 100 mL -20°CPenicillin-Streptomycin Solution 10,000 U/mL Penicillin,10,000 Rg/mL Streptomycin in 0.85% NaCl

Q SV30010 100 mL -20°C

Puromycin 2 HCl, Ultra Pure Grade Q SV30075.01 100 mg < 0°C

Q Item in stock.


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MicrocarriersWe offer two types of microcarriers:Thermo Scientific HyQSpheres and CultiSphersMicrocarriers are tiny beads or particles with asurface chemistry that facilitates attachmentand growth of anchorage-dependent cells incell culture processes. There are numeroustypes of microcarriers, widely varying in theircomposition, size, shape and density.

Microcarriers typically range in size from70–350 Rm in diameter and have a specificgravity such that they can be maintained insuspension with gentle stirring. They may becomposed of animal-derived or syntheticmaterials, including collagen, dextran, andplastic. Most are spherical in shape and maybe either solid or porous.

Surface treatments with biological proteins,synthetic compounds, and/or cationic chargesmay be added to further enhance cellattachment and propagation. All thesedifferences present unique advantagesdepending on the intended application.

A primary benefit of microcarriers is that theyprovide increased surface area to volumeratios, allowing large-scale culture inside arelatively small footprint. For example, athree-liter microcarrier culture can potentiallyreplace up to 26 typical 850 cm2 roller bottles.They can be maintained in a simple spinner

flask or in a highly controlled stirred-tankbioreactor. For the large-scale cultivation ofanchorage dependent cells, the incorporationof microcarrier technology in bioreactorsystems provides numerous additional benefitsversus roller bottles or other flat surfaceformats, including:• Scalability—Easy sub-passaging of cells• Significant cost reduction• Simpler harvesting, downstream processing• Reduction in required technical labor• Precise control of cell growth conditions• Increase in titers

HyQSpheres are available from Thermo FisherScientific, a company that has extensiveexperience in both serum-containing andserum-free cell culture. Combined with theprotocol development and process optimizationcapabilities of SoloHill, customers haveunparalleled technical support over the fullline of products.

A Tradition of Research, Innovationand Quality

Ongoing R&D efforts have resulted in theindustry's most diverse product offerings,including microcarriers that are free of animalderived components. We are the leading globalsupplier of quality solutions to challengesfaced by the biopharmaceutical industry and

Cell TypeCGEN 102-L P102-L P Plus 102-L FACT 102-L HLX II-170 Pro-F 102-LSV30044 SV30056 SV30052 SV30040 SV30090 SV30048

Vero MonkeyKidney (VERO)

X X X X X

Chick EmbryoFibroblast (CEF)

X X

Swine Testes (ST) XBovine Turbinate (BT) XRabbit SkinFibroblast (P4RSF)

X

Human DipolidFibroblast (MRC-5)

X

Human EpithelialCarcinoma (KB)

X

Human ForeskinFibroblast (HFF)

X X X X X

Human SquamousCarcinoma (UMSCC)

X X X X X

Madin-Darby BovineKidney (MDBK)

X X X X X

Chinese HamsterOvary (CHO)

X

Madin-Darby CanineKidney (MDCK)

X X X X X

animal cell culture based researchers. We haveaccomplished this by applying our expertisein animal cell nutrition as it influences cellfunction and product yield. We have combinedour expertise to provide not just individualproducts, but system solutions that incorporatemicrocarrier technology. Working together withour customers, we develop cell culturesystems and protocols that utilize animalprotein-free components throughout the entiremanufacturing process.

HyQSpheres are manufactured utilizing thesame core material (cross-linked polystyrene)as T-flasks, roller bottles, and similar plasticcultureware, thus facilitating the attachmentprocess. This rigid substrate also providesoptimal durability (no fragmentation) and doesnot allow the extra-cellular matrix to grow intothe bead.

Our scientists, in conjunction with academicand other researchers, have demonstratedcompatibility of the cell types in the followingtable with HyQSpheres. Successful cell cultureon microcarriers depends on many factors:serum, media, microcarrier type, cell status, stirspeed, etc. The data in the table to the left arenot comprehensive; however, in principle, anyanchorage-dependent cell should be able toattach and grow on microcarriers.

HyQSpheres are available in both collagen-coated and ADCF formats. This facilitatesadaptation from other microcarriers thatutilize animal-derived components as well astransitioning into ADCF cell culture systems.

Type of HyQSphere


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HyQSpheres

HyQSpheres incorporate various surfacemodifications, including cationic charges, andanimal-based proteins such as collagen orproprietary recombinant proteins, to furtherenhance the attachment process and tofacilitate their use with a wide array of celllines and applications.

Choosing a bead one type at a time can becostly. The HyQSphere Microcarrier Starter Kitallows you to devise an experimental matrix fortesting a variety of microcarriers to determinethe bead that will work best with yourapplication.

This HyQSphere Microcarrier Starter Kitcontains 5 g bottles of the followingHyQSpheres:• SV30040.06 FACT 102-L• SV30044.06 CGEN 102-L• SV30048.06 Pro-F 102-L• SV30052.06 PPlus 102-L• SV30056.06 P102-L• SV30090.06 HLX II-170

Description Part Number Packaging Storage TempHyQSphere Microcarriers, FACT 102-L Collagen-coated plasticwith surface charge. General purpose bead.

P SV30040.01 10 g AmbientP SV30040.02 50 g AmbientP SV30040.03 100 g AmbientP SV30040.04 500 g AmbientP SV30040.05 1000 g Ambient

HyQSphere Microcarriers, CGEN 102-L Same as fact bead without surface charge. P SV30044.01 10 g AmbientP SV30044.02 50 g AmbientP SV30044.03 100 g AmbientP SV30044.04 500 g AmbientP SV30044.05 1000 g Ambient

HyQSphere Microcarriers, Pro-F 102-L ADCF Plastic bead coated with ProNectine F,a recombinant protein. Superior performance bead facilitatesattachment of fastidious cell lines in ADCF conditions


HyQSphere Microcarriers, plus 102-L ADCF Plastic bead with surfacecharge. General purpose bead for use with serum-containing media culturesor other non-fastidious cell lines.


HyQSphere Microcarriers, P 102-L P SV30056.01 10 g AmbientP SV30056.02 50 g AmbientP SV30056.03 100 g AmbientP SV30056.04 500 g AmbientP SV30056.05 1000 g Ambient

HyQSphere Microcarriers, HLX II-170 Polystyrene microcarrier coated withcationic trimethyl ammonium with a nominal specific gravity of 1.11 andbead diameter of 160–180 Rm. This bead has a surface charge.


HyQSphere Microcarrier Starter Kit Contains 5 Grams Each of Pro-F 102-L, P 102-L,P Plus 102-L, FACT 102-L, CGEN 102-L, and HLX II-170 Microcarriers.

P SV30092.01 6 x 5 g Ambient



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products intended for implantation. Use of themicrocarrier in these cellular therapeuticproducts has been shown to enhance thesurvival of the implanted cells.

CultiSphers

In addition to Thermo Scientific HyCloneHyQSpheres, the CultiSpher line ofmicrocarriers is also offered.

CultiSphers are unique protein-basedmacroporous microcarriers, manufactured frompharmaceutical grade porcine gelatin via aprocess which yields a highly cross-linkedgelatin matrix with high mechanical andthermal stability. When used in cell cultures,cells can attach to both the external and theinternal surfaces of the matrix. The increasedsurface area of the matrix together with theprotection from stress afforded to the cells inthe interior of the matrix results in enhancedcell production capabilities. An additionaladvantage of the product is that the matrix canbe dissolved with proteolytic enzymes resultingin the harvesting of cells with almost 100%viability.

CultiSphers may also be utilized as anexcipient component in preparations of cellular

Technical Specifications of CultiSpher GMicrocarriersParticle diameter (Rm*) 130-380Volume (mL/g dry*) 12-18Density (g/mL*) 1.04Approx. no. of microcarriers ( /g dry) 1,000,000Average pore diameter (Rm*) 20* in PBS

CultiSpher microcarriers are made of gelatin.Gelatin is a product derived from the partialhydrolysis of collagen and have large regions ofidentical structure. The majority of primarycells will attach to the gelatin structure. Sincethe macroporous microcarrier structure createsa three-dimensional framework it mimics thecooperative conformation demonstrated intissue.

The density is around 1.02–1.04 g/mL. Whenthe beads are filled with cells their weight willsignificantly increase the density. This is one ofthe reasons for increasing agitation rate duringlater parts of the culture.

Cultures of cells grown on CultiSphermicrocarriers can be scaled-up in steps of 50times; for instance, the cells harvested from a1 liter fermentor are sufficient to inoculate a50 liter fermentor. This is possible due to themacroporous structure and the digestiblegelatin matrix. The macroporous structureallows the cells to increase from 10–20 to2000–3000 on each bead. As the matrix iseasily digested with tissue culture gradeTrypsin, all these cells can be recovered andused for scale-up purposes.

Description Part Number Packaging Storage TempCultiSpher G Microcarriers, Macroporous gelatin bead P SV30008.02 10 g 20-26°C

P SV30008.03 100 g 20-26°CP SV30008.04 500 g 20-26°C

CultiSpher GL Microcarriers, Macroporous gelatin bead P SV30006.02 10 g 20-26°CP SV30006.03 100 g 20-26°CP SV30006.04 500 g 20-26°C

CultiSpher S Microcarriers, Macroporous gelatin bead P SV30009.02 10 g 20-26°CP SV30009.03 100 g 20-26°CP SV30009.04 500 g 20-26°C

P Item is made to order. Lead times and minimum order quantities apply.


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Antifoam

An easy-to-use antifoaming agent Thermo

Scientific HyClone Antifoam 10% Irradiated

Solution HyClone Antifoam solutions are

developed to deliver 3% active Simethicone in

sterile Thermo Scientific HyClone BioProcess

Container (BPC) Systems using a validated

gamma irradiation process. The Antifoam

Irradiated BPCs are designed with tubing lines

and fittings to enable easy connection and

delivery to any cell culture system.

Antifoam Irradiated has been shown to

effectively prevent foam from occurring in cell

culture systems, as well as quickly eliminate

foam already formed. Two types of Antifoam

Solutions are available (See ordering information),

with the HyClone ADCF Antifoam Irradiated

containing no components of animal origin.

Application Information

Due to the normal characteristics of

Simethicone emulsions, some product

separation may occur during shipping and

storage. Shake Antifoam Irradiated well

before use.

HyClone Antifoam Irradiated and ADCF Antifoam

Irradiated have been demonstrated to directly

replace autoclaved Simethicone solutions.

As individual systems may vary, optimal

concentrations and conditions may need to be

determined prior to use. Aseptic techniques and

processing is required to delivery the antifoam

solution to cell culture systems and media as it

is not filterable.

SStteerriilliizzaattiioonn IInnffoorrmmaattiioonn

HyClone Antifoam Irradiated and ADCF Antifoam

Irradiated deliver a 3% active Simethicone

concentration. The 30% Simethicone emulsion

is diluted in Water for Injection (WFI) quality

water. Gamma irradiation sterilization of these

products has been validated to a sterility

assurance level (SAL) of 10-6. The gamma

radiation process is based on the Association for

the Advancement of Medical Instrumentation

(AAMI) technical information report number

33:2005 standard (title: Sterilization of Health

Care Products - Radiation - Substantiation of a

Selected Sterilization Dose - Method VDmax).

A sub-lethal sterilization dose was determined

according to AAMI/ANSI/ISO11137-2006 part

2 - method VDmax25. This process validates a

radiation dose based on the determination of

the product bioburden and comparison of that

bioburden to a model population having a

defined resistance to radiation.

Diagram 1: 500 mL Antifoam in 1 L BPC

Size Material Tubing Size Fittings

Line 1 16” (41 cm) Medial-Grade PVC 1/8” ID x 1/16” Wall Female Luer Lock

Line 2 36” (91 cm) C-Flex 1/8” ID x 1/16” Wall Female Luer Lock

Diagram 1


QQ

QQ

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Diagram 2: 2.5 L Antifoam in 5 L BPC

Size Material Tubing Size Fittings

Line 1 10.5” (27 cm) Medial-Grade PVC 3/8” ID x 1/16” Wall Quick Connect Body

Line 2 36” (91 cm) C-Flex 3/8” ID x 1/8” Wall Quick Connect Body

Line 3 36” (91cm) C-Flex 1/8” ID x 1/16” Wall Female Luer Lock

Diagram 2

Ordering Information

Name Part Number/Unit Size Raw Material ADCF Active Simethicone ConcentrationAntifoam Irradiated SH30896.01 (500 mL)

SH30896.02 (2.5 L)Dow Corning 7-9245, 30%Simethicone Emulsion USP

No Manufactured to 3%

Name Part Number/Unit Size Raw Material ADCF Active Simethicone ConcentrationAntifoam Irradiated SH30897.01 (500 mL)

SH30897.02 (2.5 L)Dow Corning Q7-2587, 30%Simethicone Emulsion USP

Yes Manufactured to 3%

Q Item in stock.


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Custom Media Products

Custom Media, Buffers, Reagents,and Process Liquids

We recognize that many industrial andresearch customers have proprietary mediaformulations. In addition to standard mediaproducts, we offer an extensive customizationprogram whereby proprietary formulation ismanufactured and packaged in a deliverysystem to meet customer applications. Customcell culture media, buffers and reagents can bemanufactured in both liquid and powderedform. Liquids can be packaged for conveniencein bottles and Thermo Scientific HyCloneBioProcess Containers (BPCs).

By understanding that many cell cultureapplications are unique, we have become aleader in formulation and packagingcustomization.

As a leading BPC manufacturer, we have theability to produce single-use, disposablecustomized delivery systems ranging in sizefrom 50 mL to 1,000 L. Standard PETE/PETGbottles are also available in 100, 250, 500, and

1,000 mL. Custom liquid production lot sizesrange from 50 to 10,000 L, while dry powderedmedia and buffers are available in lot sizesfrom 1 to 6,300 kg.

To ensure that requirements for customproducts are met, we have developed anISO 9001:2000- compliant Contract ReviewProcedure. This procedure was implementedto facilitate the development of productspecifications that are mutually agreed uponby our organization and our customer.

The process begins with the completion of aProduct Brief that defines a product's particularrequirements. Typical requirements defined inthe Product Brief include:• product name and description• packaging requirements and delivery

systems• product formulation• release criteria and Certificate of Analysis

reporting• storage and shipping conditions• expiration information

Once the Product Brief is submitted, a qualifieddesign development team conducts anextensive review of the specified requirementsto ensure that the product requirements canbe met. Our staff is trained to assess eachcustom request to confirm that all productrequirements are adequately understoodand defined.

Upon acceptance of the Product Brief byour development team—which consistsof delegates from Product Management,Manufacturing, R&PD, Quality Control, andEngineering—the requirements are translatedinto a Product Specification, which is assignedits own unique part number. The specificationis then presented to the customer for reviewand approval.

Once approved by the customer and returned tous, the Product Specification undergoes finalapproval in our Document Control System.Upon receipt of a purchase order, the product isready for production. The flow diagram belowrepresents the custom product developmentprocess.


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Rapid Response Production (RRP) services areoffered to those customers in need of quick-turnThermo Scientific HyClone Cell Culture mediaand Reagents to facilitate research andproduct/process development. Requests aretypically manufactured within seven days.

Customized and optimized prototypes forindustrial applications must be evaluatedquickly to facilitate final product selectionand implementation. In research settings,minor modifications of existing media andreagent formulations are necessary toaccommodate specific goals and objectives.

RRP is a media manufacturing service designedfor the production of client-specific media andreagents with the shortest turnaround times.RRP facility is a non-cGMP media and reagentsmanufacturing suite capable of preparing upto 200 L liquid lot sizes and up to 10 kg drypowdered lot sizes. Liquid lots are packagedin 100, 500, and 1,000 mL bottles, or in ThermoScientific HyClone BioProcess Containers(100 L maximum). Dry powdered lots arepackaged in volume specific bottles or inbulk containers.

Our RRP facility offers the highest qualityproducts by following consistent andreproducible manufacturing practices.Documentation on media and reagents isdesigned to provide component-specifictracking, as well as initiate technical transferto our cGMP manufacturing facility whenappropriate.

Quality ControlAvailable quality control tests for RRP liquidlots:• Sterility• pH• Osmolality• Endotoxin

Available quality control tests for RRPdry powdered lots:• Solubility• Moisture• Particle size• pH• Osmolality

Delivery SystemsA wide range of standard packaging sizes anddelivery systems to meet your specific productrequirements are offered. Custom packagingis also available.

Facilities and PersonnelRapid Response Production is operated in afacility with stringent access and operationalrestrictions. Rapid Response Productionpersonnel are trained in accordance withestablished manufacturing practices.Additional features include:• Liquid media lots are 0.2 Rm sterile filtered

(unless otherwise specified) in a certifiedlaminar flow hood;

• Single-contact, ADCF liquid mediamanufacturing is available upon request; and

• Rapid Response Production personnelfollow established gowning and sterilizationprocedures

RawMaterialsThe components used in RRP are the samequality components used in our cGMPmanufacturing facility. These components havepassed our Quality Control testing and vendorlot traceability. Should your RRP lot requirecomponents not presently qualified, they willbe sourced from one of our approved vendorswhenever possible.

Rapid Response Production RequestsFor more information about RRP, or otherThermo Scientific HyClone Products, contactyour Account Manager, or visitwww.thermo.com/hyclone.

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Format Lot Size Delivery SystemLiquid 10-20 L 100 mL Plastic BottleLiquid 10-100 L 500 mL Plastic BottleLiquid 10-200 L 1000 mL Plastic BottleLiquid 20-200 L 5, 10, 20 L BPCsDry Powder 500 g-20 kg By volume (L)Dry Powder 500 g-20 kg Bulk Powder


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About HyClone Sera Products

Serum Origin

Serum origin refers to the country in which theraw blood was collected. This is not to beconfused with the country in which the rawserum was sterile filtered, having originatedelsewhere. With Thermo Scientific HyCloneSerum, the country of origin is not a secret.The documentation you receive will clearlystate the country or countries of origin. Thisinformation is on the Certificate of Analysisand the Certificate of Origin.

Importance of Origin

The United States Department of Agriculture(USDA) considers certain countries to be freeof bovine spongiform encephalopathy (BSE)and foot and mouth disease (FMD). Some ofthese countries are New Zealand, Australia,and all countries in Central America whereour serum is collected. However, the USDApermits only serum of New Zealandorigin to be imported into the U.S. withoutsafety testing. Serum of Australian,Mexican, and Central American origin mustbe quarantined, sampled, and tested by theUSDA for the presence of exotic strains ofBluetongue virus and, in the case of Australianserum, Akabane virus as well. Once thequarantined serum has successfully completedsafety testing, it can be sold and used in theU.S. without restriction.

The results of the safety testing are availableto you with the purchase of ThermoScientific HyClone Serum. There are manyareas of the world which are prohibited fromexporting serum into the US. You can becertain that Thermo Scientific HyClone Serumconforms to USDA regulations. Verification oforigin is even more critical for serum userswho are producing products requiringregulatory approval through the United StatesFood and Drug Administration (FDA), the USDA,or other regulatory agencies.

Traceability

Traceability is the ability to provide a verifiabledocument trail from the serum you receive tothe geographic origin of the raw blood usedin its production. It is critical to the safe useof serum. Issues related to BSE and FMD, forexample, can only be controlled by use ofserum from locations where FMD does notexist, or where BSE does not exist or has beendetected at a very low level. We are dedicatedto proving complete traceability on all serumproducts.

Method of Collection

Controlling the initial collection of bovine bloodis at least as important to serum quality assterile filtration and aseptic packaging. Aclosed-system collection method (cardiacor venipuncture) and rapid processing areessential to a quality finished serum product.Low levels of endotoxin and hemoglobin are anexcellent measure of the care with whichcollection and processing were done.

Some blood collectors use blood collectionvessels, which are open to the environment ofthe packing house. This is especially true forcalf blood collection where little care may betaken to avoid contaminants including stomachcontents. In addition, collection of fetal bloodby expressing it from the umbilicus into opencontainers is still practiced elsewhere.We do not use these practices in bloodcollection.

Collectors

Trained collectors are very important to ourquality serum products. As with any importantprocedure, skill and training are as essentialin harvesting blood as in any step of its productpreparation. We control every step of thecollection process by careful training andsupervision of personnel to offer customersa superior product.

Serum Brokers

Common practice in the serum supply chain isthe use of companies or individuals who dealin the trading of raw serum for supply to thosewho perform sterile filtration. Some brokersacquire raw blood either by collecting the bloodthemselves and processing it into raw serum orbuying the raw serum from other brokers.Where possible, we do not use serum brokers.

Age at Time of Collection

Blood sourced from an older animal will havedifferent characteristics than blood sourcedfrom a younger animal. Also, the type of feedwill make a difference (i.e. 16 to 22 week-oldformula fed veal animals vs. range fedanimals). Some differences will be seen whencomparing IgG, total protein, and albuminlevels (to name a few).

Lot Size

Serum is a natural product resulting inlot-to-lot variability. Large lot sizes tend tominimize this variability. Additionally, if youutilize large quantities of serum, the cost perliter of lot QC testing prior to purchase isreduced with large lots. Even if you use onlysmall quantities, a serum supplier whose lotsizes are large (2,000 to 3,000 L) willprovide the best opportunity of providingconsistency order after order. We produce thelargest lot sizes possible in this industry.


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Filtration Method

Understanding the types of filtration processesthat serum suppliers use is critical. Serum isfiltered using a variety of filter types. Commonfinal filters used in serum processing havepore-size ratings ranging from 100 nm to450 nm and may be used as a single filter orin a series. We recommend that yourspecifications require use of a minimum of100 nm pore-size rated filters and that threefilters in a series are used. This has becomean industry standard. Forty nm pore-size ratedfilters are the most retentive filters used toprocess commercial-sized lots. This method isused to produce Thermo Scientific HyCloneDefined Fetal Bovine Serum.

"True Pool" Processing

True pool processing means that the serumis pooled after filtration and before finalpackaging. If serum is pooled, filtered, andbottled directly after filtration, the serum willlikely have increased variability frombottle-to-bottle. This is due to changes infiltration properties as hundreds of liters passthrough the filters.

With true pool processing, customers areensured of more consistent quality from thefirst bottle filled to the last. Variability inserum components is natural and is the result ofa variety of factors (age of the animal, origin,season collected, etc.). Large lots processedusing true pool technology increase consistencyand minimize variability within a lot.

Required Testing

This is dependent on each customer's needs.Most serum suppliers do perform a variety oftests. We consider the following tests to befundamental in offering a high quality serumproduct:• Endotoxin (LAL or EP)• Hemoglobin• Sterility (by current USP and EP methods)Bacteria and Fungi

• Virus Testing (Full 9CFR113.53 or per EMEA)BluetongueBovine AdenovirusBovine ParvovirusBovine Respiratory Syncytial VirusBovine Viral Diarrhea VirusRabiesReovirusCytopathogenic Agents (IBR)Hemadsorbing Agents (P13)

• Mycoplasma (Points to Consider orcurrent EP)

• Biochemical Assay panel• Electrophoretic profile

Additional tests can also be done on acustom basis.

Post-Filtration Treatments

The most commonly used post-filtrationtreatment is exposure to gamma irradiation.This is a powerful tool to further reduce the riskof adventitious viruses present in serum. The ir-radiation process used should bevalidated to deliver a minimum dose of 25 kGy.If you are manufacturing products for use inhumans or in animals, irradiation should be aroutine requirement.

Heat inactivation by exposing the serum to atemperature of 56° C for 30 minutes is also acommon post-filtration process. It may beeffective in reducing some adventitious agentsand may be used in addition to, but neversubstituted for, gamma irradiation. Bothservices are available by request.

Benefits• Supplier's reputation• Degree of vertical integration• Quality of technical assistance• cGMP• Clear, thorough documentation, verifiedby audit

• Quality of customer service• Quality of technician training• Variety of products and origins offered• State-of-the-art filtration options• ISO 9001:2000 and ISO 13485:2003certifications

• Availability of Certificates of Suitability• Adequate supply of serum• Blood collection techniques used• Origin clearly stated and certified• Validated post-filtration processes


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Defined FBS is the highest quality FBSavailable and is widely used by those cellculturists who have a concern for viralcontaminants and require an extensivebiochemical profile. Defined FBS is filteredthrough serial 40 nm (0.04 Rm) pore-size ratedfilters, which are the most retentive filters usedin commercial FBS production. This type offiltration is a practical, cost competitive methodof viral load reduction. Data shows that 40 nmfiltration will remove as many as eight logs ofviral challenge. This is a dramatic improvementover the current industrystandard of using filters with mean poresizes of 100 nm. Studies demonstrate thatthis filtration regimen has no adverse effecton cell growth. Finished Defined FBS has over50 components analyzed and the results areincluded on the Certificate of Analysis and theBiochemical Assay.

Characterized FBS is comparable to the highestquality FBS from other suppliers. It is lower inprice than Defined FBS, but meets therequirements of most discriminating FBSusers. Characterized FBS is filtered throughthree sequential 100 nm (0.1 Rm) pore-sizerated filters. US and Canadian sourced.

Test SpecificationEndotoxin(Limulus AmebocyteLysate—Gel Clot)

,25 EU/mL

Hemoglobin(Spectrophotometric)

,25 mg/dl

SterilityTesting(Current USP and EP)Bacteria & Fungi No GrowthVirusTesting (9 CFR 113.53)FluorescentAntibodyBluetongue Not DetectedBovine Adenovirus Not DetectedBovine Parvovirus Not DetectedBovine Respiratory Syncytial Virus Not DetectedBovine Viral Diarrhea Virus Not DetectedRabies Not DetectedReovirus Not DetectedCytopathogenic Agents—e.g., IBR Not DetectedHemadsorbing Agents—e.g., PI3 Not DetectedMycoplasmaLarge Volume, Direct Culture Not DetectedHoechst DNA Stain Not DetectedCertificate of Suitability Included

Additional assays performed (subject to change withoutnotice): Gamma Globulin, Alkaline Phosphatase, LactateDehydrogenasse, Glutamic Pyruvic Transaminase (SGPT),Glutamic Oxaloacetic Transaminase (SGOT), pH, TotalProtein, Albumin, Blood Urea Nitrogen, Creatinine, TotalBilirubin, Sodium, Potassium, Calcium, Chloride, InorganicPhosphorus, Osmolality, Iron, Total Iron Binding Capacity(TIBC), Percent Saturation, Glucose, and IgG.

Characterized Fetal Bovine SerumTest Specifications,10 EU/mL


,10 mg/dl

SterilityTesting(Current USP and EP)Bacteria & Fungi No GrowthVirusTesting (9 CFR 113.53)FluorescentAntibodyBluetongue Not DetectedBovine Adenovirus Not DetectedBovine Parvovirus Not Detected

Bovine Respiratory Syncytial Virus Not Detected

Bovine Viral Diarrhea Virus Not DetectedRabies Not DetectedReovirus Not DetectedCytopathogenic Agents—e.g., IBR Not DetectedHemadsorbing Agents—e.g., PI3 Not DetectedMycoplasmaLarge Volume,Direct Culture Not DetectedHoechst DNA Stain Not DetectedCertificate of Suitability Included

Additional assays performed (subject to change withoutnotice): Progesterone, Insulin, Cholesterol, High DensityLipo-Protein Cholesterol, Low Density Lipo-ProteinCholesterol, Triglycerides, Gamma Globulin, Phospholipids,Alkaline Phosphatase, Lactate Dehydrogenase, GlutamicPyruvic Transaminase (SGPT), Glutamic OxaloaceticTransaminase (SGOT), Thiamine (B1), Riboflavin (B2),Pyridoxine (B6), Nicotinic Acid (Niacin), Vitamin H (Biotin),Vitamin A (Retinol), Carotene, Total Vitamin E (Tocopherol),Cyanocobalamine (B12), Pteroylglutamic Acid (Folate),Vitamin C, 1,25 Dihydroxy Vitamin D, pH, Total Protein,Albumin, Blood Urea Nitrogen, Creatinine, Total Bilirubin,Inorganic Phosphorus, Osmolality, Iron, Selenium, IgG,Sodium, Potassium, Calcium, Chloride, Magnesium, Glucose,Copper, Total Iron Binding Capacity (TIBC), and PercentSaturation.

Defined Fetal Bovine Serum, US Origin

Fetal Bovine Serum (FBS)

Endotoxin(Limulus AmebocyteLysate—Gel Clot


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This quality cell culture grade FBS is used byprice-conscious cell culturists. Standard FBSis filtered through three sequential 100 nm(0.1 Rm) pore-size rated filters and is procuredin the same manner as Defined andCharacterized FBS US and Canadian sourced.


For informationonly


For informationonly

SterilityTesting(Current USP and EP)Bacteria & Fungi No GrowthVirusTesting (9 CFR 113.53)FluorescentAntibodyBluetongue Not DetectedBovine Adenovirus Not DetectedBovine Parvovirus Not DetectedBovine Respiratory Syncytial Virus Not DetectedBovine Viral Diarrhea Virus F10Rabies Not DetectedReovirus Not DetectedCytopathogenic Agents—e.g., IBR Not DetectedHemadsorbingAgents—e.g., PI3 Not DetectedMycoplasmaLarge Volume, Direct Culture Not DetectedHoechst DNA Stain Not DetectedCertificate of Suitability Included

Additional assays performed (subject to change withoutnotice): Gamma Globulin, Alkaline Phosphatase, LactateDehydrogenase, Glutamic Pyruvic Transaminase (SGPT),Glutamic Oxaloacetic Transaminase (SGOT), pH, TotalProtein, Albumin, Blood Urea Nitrogen, Creatinine, TotalBilirubin, Sodium, Potassium, Calcium, Chloride, InorganicPhosphorus, Osmolality, Iron, Total Iron Binding Capacity(TIBC), and IgG.

Standard Fetal Bovine Serum

The vast expanse of the Pacific Ocean hasprotected New Zealand's islands from manyoutside influences, both geographically andbiologically. One of the benefits of thisisolation is that New Zealand has the fewestreported bovine diseases in the world and hasbeen determined to be free from BSE and FMD.This makes New Zealand FBS an excellentsource of bovine serum as it offers maximumsafety. New Zealand FBS is carefullycollected, processed, and filtered in our ownNew Zealand facility following our exactingstandards to ensure the highest quality andabsolute traceability. Our New Zealand FBSis filtered through three sequential 100 nm(0.1 Rm) pore-size rated filters.


,20 EU/mL


,25 mg/dl

SterilityTestingCurrent USP and EP)Bacteria & Fungi No GrowthVirusTesting (9 CFR 113.53)FluorescentAntibodyBluetongue Not DetectedBovine Adenovirus Not DetectedBovine Parvovirus Not DetectedBovine Respiratory Syncytial Virus Not DetectedBovine Viral Diarrhea Virus Not DetectedRabies Not DetectedReovirus Not DetectedCytopathogenic Agents—e.g., IBR Not DetectedHemadsorbing Agents—e.g., PI3 Not DetectedMycoplasmaLarge Volume, Direct Culture Not DetectedHoechst DNA Stain Not DetectedCertificate of Suitability Included

Additional assays performed (subject to change withoutnotice): Progesterone, Chloride, Insulin, InorganicPhosphorus, Parathyroid Hormone (PTH), Glucose,Cholesterol, Copper, High Density Lipo-Protein Cholesterol,Magnesium, Low Density Lipo-Protein Cholesterol,Selenium, Triglycerides, IgG, Gamma Globulin, pH,Phospholipids, Osmolality, Alkaline Phosphatase,Iron, Lactate Dehydrogenase, Total Iron Binding Capacity,Glutamic Pyruvic Transaminase (SGPT), Percent Saturation,Glutamic Oxaloacetic Transaminase (SGOT), Thiamine (B1),Riboflavin (B2), Pyridoxine (B6), Nicotinic Acid (Niacin),Vitamin H (Biotin), Vitamin A (Retinol), Carotene, TotalVitamin E (Tocopherol), Cyanocobalamin (B12),Pteroylglutamic Acid (Folate), Vitamin C, 1,25 DihydroxyVitamin D, Total Protein, Albumin, and Blood Urea Nitrogen.

New Zealand Foetal Bovine Serum

New Zealand FBS is now available in standardgrade, which offers price-conscious cellculturists the opportunity to use NZ-sourcedmaterial that fits their budget. New ZealandStandard FBS is filtered through threesequential 100 nm (0.1 Rm) pore-size rated filters.


For informationonly


For informationonly

SterilityTesting(Current USP and EP)Bacteria & Fungi No GrowthVirusTesting (9 CFR 113.53)FluorescentAntibodyBluetongue Not DetectedBovine Adenovirus Not DetectedBovine Parvovirus Not DetectedBovine Respiratory Syncytial Virus Not DetectedBovine Viral Diarrhea Virus TestedRabies Not DetectedReovirus Not DetectedCytopathogenic Agents—e.g., IBR Not DetectedHemadsorbing Agents—e.g., PI3 Not DetectedMycoplasmaLarge Volume, Direct Culture Not DetectedHoechst DNA Stain Not DetectedCertificate of Suitability Included


New Zealand Standard FoetalBovine Serum


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Australian FBS is also available in Standardgrade and is filtered through three sequential100 nm (0.1 Rm) pore-size rated filters.


For information only


For information only

SterilityTesting(Current USP and EP)Bacteria & Fungi No GrowthVirusTesting (9 CFR 113.53)FluorescentAntibodyBluetongue Not DetectedBovine Adenovirus Not DetectedBovine Parvovirus Not DetectedBovine Respiratory Syncytial Virus Tested

Bovine Viral Diarrhea Virus Not DetectedRabies Not DetectedReovirus Not DetectedCytopathogenic Agents—e.g., IBR Not DetectedHemadsorbing Agents—e.g., PI3 Not DetectedMycoplasmaLarge Volume, Direct Culture Not DetectedHoechst DNA Stain Not DetectedCertificate of Suitability Included


Standard Australian Foetal Bovine Serum

USDA Tested FBS is sourced from severalCentral American countries (Costa Rica,Honduras, Panama, Nicaragua, El Salvador, andGuatemala) which are all recognized as beingfree of BSE and FMD by the USDA. Serum fromCentral America is USDA safety tested and isfiltered through three sequential 100 nm (0.1Rm) pore-size rated filters.


,25 EU/mL


,25 mg/dl

SterilityTesting(Current USP and EP)Bacteria & Fungi No GrowthVirusTesting (9 CFR 113.53)FluorescentAntibodyBluetongue Not DetectedBovine Adenovirus Not DetectedBovine Parvovirus Not DetectedBovine Respiratory Syncytial Virus Not DetectedBovine Viral Diarrhea Virus TestedRabies Not DetectedReovirus Not DetectedCytopathogenic Agents—e.g., IBR Not DetectedHemadsorbingAgents—e.g., PI3 Not DetectedMycoplasmaLarge Volume, Direct Culture Not DetectedHoechst DNA Stain Not DetectedUSDA SafetyTesting PassedCertificate of Suitability Included

Additional assays performed (subject to change withoutnotice): Alkaline Phophatase, Lactate Dehydrogenase,Glutamic Pyruvic Transaminase (SGPT), Glutamic OxaloaceticTransaminase (SGOT), pH, Total Protein, Albumin, Blood UreaNitrogen, Creatinine, Total Bilirubin, Sodium, Potassium,Calcium, Chloride, Inorganic Phosphorus, Osmolality,Glucose, Gamma Globulin, and IgG.

Refer to Pages 164-168 for Australian and USDA Tested FBSvs. FBS growth promotion studies.

USDA Tested Fetal Bovine Serum

Our Characterized Australian FBS is sourced fromonly USDA or AQIS (AustralianQuarantine and Inspection Service) approvedabattoirs in Australia. We have found thatAustralian methods of animal husbandry areworld class. Animal nutrition and health careare unsurpassed. As with New Zealand,Australia is an island continent thus makinganimal disease control and management easierthan in most areas of the world. Australia is con-sidered to be free of BSE and FMD. Acomprehensive trail of paperwork ensures trace-ability. Our Characterized Australian FBSis USDA safety-tested. It is sterile filteredusing true pool technology through threesequential 100 nm (0.1 Rm) pore-size rated filters.


,25 EU/mL


,25 mg/dl

SterilityTesting(Current USP and EP)Bacteria & Fungi No GrowthVirusTesting (9 CFR 113.53)FluorescentAntibodyBluetongue Not DetectedBovine Adenovirus Not DetectedBovine Parvovirus Not DetectedBovine Respiratory Syncytial Virus Not DetectedBovine Viral Diarrhea Virus Not DetectedRabies Not DetectedReovirus Not DetectedCytopathogenic Agents—e.g., IBR Not DetectedHemadsorbing Agents—e.g., PI3 Not DetectedMycoplasmaLarge Volume, Direct Culture Not DetectedHoechst DNA Stain Not DetectedUSDA SafetyTestAntibodies Against:AkabaneVirus Not DetectedBluetongueVirus Not DetectedCertificate of Suitability Included

Additional assays performed (subject to change withoutnotice): Alkaline Phosphate, Lactate Dehydrogenase,Glutamic Pyruvic Transaminase (SGPT), Glutamic OxaloaceticTransaminase (SGOT), Total Protein, Albumin, Blood UreaNitrogen, Creatinine, Total Bilirubin, Sodium, Potassium,Calcium, Chloride, Inorganic Phosphorus, Glucose, Iron,Total Iron Binding Capacity (TIBC), Percent Saturation,Gamma Globulin, IgG, pH, and Osmolality.

Characterized Australian FoetalBovine Serum


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Thermo Scientific HyClone FBS from SouthAmerica complies with the EU regulationsand meets the requirements of most Asiancountries. South American FBS is filteredthrough three sequential 100 nm (0.1 Rm)pore-size rated filters. Not sold in the US;only for sale to European and Asian customers.


,10 EU/mL


,20 mg/dl

Sterility TestingBacteria & Fungi No Growth

Virus TestingFluorescent AntibodyBovine Viral Diarrhea Virus Not DetectedCytopathogenic Agents—e.g., IBR Not DetectedHemadsorbing Agents—e.g., PI3 Not DetectedMycoplasma Not Detected

Additional assays performed (subject to change withoutnotice): pH, Osmolality, Alpha Globulins, Gamma Globulins,Total Albumin, Sodium, Potassium, Chloride, Uric Acid,Calcium, Phosphorus, Alkaline Phosphatase, LDH, SCOT,Oestradiol, and Testosterone.

Research Grade Fetal Bovine Serum,South American Origin


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Fetal Bovine SerumDescription Part Number Packaging Storage TempDefined Fetal Bovine SerumDefined Fetal Bovine Serum, US Origin SH30070.01 50 mL -10°C

SH30070.02 100 mL -10°CSH30070.03 500 mL -10°C

Defined Fetal Bovine Serum, US Origin, Heat Inactivated SH30070.01HI 50 mL -10°CSH30070.02HI 100 mL -10°CSH30070.03HI 500 mL -10°C

Defined Fetal Bovine Serum, US Origin, Irradiated SH30070.03IR 500 mL -10°CCharacterized Fetal Bovine SerumCharacterized Fetal Bovine Serum, US Origin SH30071.01 50 mL -10°C

SH30071.02 100 mL -10°CSH30071.03 500 mL -10°C

Characterized Fetal Bovine Serum, US. Origin, Heat Inactivated SH30071.01HI 50 mL -10°CSH30071.02HI 100 mL -10°CSH30071.03HI 500 mL -10°C

Characterized Fetal Bovine Serum, US Origin, Irradiated SH30071.03IR 500 mL -10°CCharacterized Fetal Bovine Serum, Canadian Origin SH30396.02 100 mL -10°C

SH30396.03 500 mL -10°CCharacterized Fetal Bovine Serum, Canadian Origin, Heat Inactivated SH30396.02HI 100 mL -10°C

SH30396.03HI 500 mL -10°C

Standard Fetal Bovine SerumStandard Fetal Bovine Serum, US Origin SH30088.02 100 mL -10°C

SH30088.03 500 mL -10°CStandard Fetal Bovine Serum, US Origin, Heat Inactivated SH30088.02HI 100 mL -10°C

SH30088.03HI 500 mL -10°CStandard Fetal Bovine Serum, US Origin, Irradiated SH30088.03IR 500 mL -10°CStandard Fetal Bovine Serum, Canadian Origin SH30397.02 100 mL -10°C

SH30397.03 500 mL -10°CStandard Fetal Bovine Serum, Canadian Origin, Heat Inactivated SH30397.02HI 100 mL -10°C

SH30397.03HI 500 mL -10°C

New Zealand Foetal Bovine SerumNew Zealand Foetal Bovine Serum SH30406.01 100 mL -10°C

SH30406.02 500 mL -10°CNew Zealand Foetal Bovine Serum, Heat Inactivated SH30406.01HI 100 mL -10°C

SH30406.02HI 500 mL -10°CNew Zealand Foetal Bovine Serum, Irradiated SH30406.02IR 500 mL -10°CNew Zealand Standard Foetal Bovine SerumNew Zealand Standard Foetal Bovine Serum SH30559.02 100 mL -10°C

SH30559.03 500 mL -10°CNew Zealand Standard Foetal Bovine Serum, Heat Inactivated SH30559.02HI 100 mL -10°C

SH30559.03HI 500 mL -10°CNew Zealand Standard Foetal Bovine Serum, Irradiated SH30559.03IR 500 mL -10°C

SH30396.03IR 500 mL -10°CCharacterized Fetal Bovine Serum, Canadian Origin, Irradiated

SH30397.03IR 500 mL -10°CStandard Fetal Bovine Serum, Canadian Origin, Irradiated


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Fetal Bovine Serum (continued)Description Part Number Packaging Storage TempCharacterized Australian Fetal Bovine SerumCharacterized Australian Foetal Bovine Serum SH30084.02 100 mL -10°C

SH30084.03 500 mL -10°CSH30084.04 1000 mL -10°C

Characterized Australian Foetal Bovine Serum, Heat Inactivated SH30084.02HI 100 mL -10°CSH30084.03HI 500 mL -10°CSH30084.04HI 1000 mL -10°C

Characterized Australian Foetal Bovine Serum, Irradiated SH30084.02IR 100 mL -10°CSH30084.03IR 500 mL -10°CSH30084.04IR 1000 mL -10°C

Standard Australian Foetal Bovine SerumStandard Australian Foetal Bovine Serum SV30370.02 100 mL -10°C

SV30370.03 500 mL -10°CStandard Australian Foetal Bovine Serum, Heat Inactivated SH30370.02HI 100 mL -10°C

SH30370.03HI 500 mL -10°CStandard Australian Foetal Bovine Serum, Irradiated SH30370.02IR 500 mL -10°CUSDA-Tested Fetal Bovine SerumUSDA-Tested Fetal Bovine Serum SH30910.02 100 mL -10°C

SH30910.03 500 mL -10°CUSDA-Tested Fetal Bovine Serum, Heat Inactivated SH30910.02HI 100 mL -10°C

SH30910.03HI 500 mL -10°CUSDA-Tested Fetal Bovine Serum, Irradiated SV30014.03IR 500 mL -10°CResearch Grade Fetal Bovine SerumResearch Grade Fetal Bovine Serum, South American Origin, European Customers SV30160.02 100 mL -10°C

SV30160.03 500 mL -10°CResearch Grade Fetal Bovine Serum, Heat Inactivated South American Origin, European Customers SV30160.02HI 100 mL -10°C

SV30160.03HI 500 mL -10°CResearch Grade Fetal Bovine Serum, Irradiated, South American Origin, European Customers SV30160.03IR 500 mL -10°CResearch Grade Fetal Bovine Serum, South American Origin, China Customers SV30087.02 100 mL -10°C

SV30087.03 500 mL -10°CResearch Grade Fetal Bovine Serum, European Union Origin SV30143.02 100 mL -10°C

SV30143.03 500 mL -10°CSV30143.02HI 100 mL -10°CSV30143.03HI 500 mL -10°C


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Super Low IgG Fetal Bovine Serum

Processed to substantially reduce IgG (viaProtein G Chromatography) level, ThermoScientific HyClone Super Low IgG Fetal BovineSerum (FBS) is suitable for use in moreexacting applications. FBS contains naturallevels of immunoglobulin (IgG) that may betoo high for certain cell culture and proteinpurification applications. Serum from individualanimals may be screened for low content andpooled to produce large batches of FBS withlower levels of IgG. However, this may be quitea challenge due to the scarcity of such low IgGanimals and the logistics involved with thescreening and collection process. Evennaturally occurring low levels of IgG maystill be too high for certain applications.Comparative levels of IgG in bovine serumproducts are shown in the table below.

Certificate of Analysis

Specialty Fetal Bovine Serum

Charcoal/Dextran Treated Fetal BovineSerum, US Origin

Charcoal/Dextran Treated FBS is availablefor users requiring reduced levels of varioushormones. High quality FBS is processed usinga proprietary charcoal/dextran treatment thathas been shown to reduce the levels of manyhormones and growth factors. This serum isassayed for a variety of components before andafter treatment, thus assuring the efficacy ofthe treatment. Charcoal/Dextran FBS has beenshown to be effective in reducingsteroid levels for utilization in receptor studiesor estrogen related investigations. Otherstudies indicate that charcoal/dextrantreatments may be used to minimize lot-to-lotserum variability. Charcoal/Dextran FBS isfiltered through three sequential 100 nm(0.1 Rm) pore-size rated filters.


,10 EU/mL


,20 mg/dl

Sterility Testing(Current USP and EP)Bacteria & Fungi No Growth

Virus Testing (9 CFR 113.53)Fluorescent AntibodyBluetongue Not DetectedBovine Adenovirus Not DetectedBovine Parvovirus Not DetectedBovine Respiratory Syncytial Virus Not Detected

Bovine Viral Diarrhea Virus Not DetectedRabies Not DetectedReovirus Not DetectedCytopathogenic Agents—e.g. IBR Not DetectedHemadsorbing Agents—e.g. PI3 Not DetectedMycoplasmaLarge Volume, Direct Culture Not DetectedHoechst DNA Stain Not DetectedCertificate of Suitability Included

Additional assays performed (subject to change withoutnotice): Progesterone, Sodium, Testosterone, Potassium,Cortisol, Calcium, Corticosterone, Chloride, FollicleStimulating Hormone (FSH), Inorganic Phosphorus,Luteinizing Hormone (LH), Glucose, Insulin, Albumin,Triiodothyronine (T3), Blood Urea Nitrogen, Thyroxine (T4),Creatinine, Thyroid Stimulating Hormone (TSH), TotalBilirubin, Calcitonin, 17 Beta Estradiol, Estrone, AlkalinePhosphatase, Lactate Dehydrogenase, Glutamic PyruvicTransaminase (SGPT), Glutamic OxaloaceticTransaminase(SGOT), Thiamine (B1), Riboflavin (B2), Pyridoxine (B6),Nicotinic Acid (Niacin), Vitamin H (Biotin), Vitamin A(Retinol), Carotene, Total Vitamin E (Tocopherol),Cyanocobalamine (B12), Pteroylglutamic Acid (Folate),Viatmin C, 1,25 Dihydroxy Vitamin D, Total Protein, and IgG.

Testing (Release Criteria)IgG < 5µg/mLEndotoxin (LimulusAmebocyte Lysate - Gel Clot) < 10 EU/mLHemoglobin(Spectrophotometric) < 10 mg/dLSterility Testing(Current USP and EP)Bacteria & Fungi No GrowthVirusTesting (9 CFR 113.53)

FluorescentAntibody

Bluetongue Not DetectedBovine Adenovirus Not DetectedBovine Parvovirus Not DetectedBovine Respiratory Syncytial Virus Not DetectedBovine Viral Diarrhea Virus Not DetectedRabies Not DetectedReovirus Not DetectedCytopathogenic Agents - e.g. IBR Not DetectedHemadsorbing Agents - e.g. PI3 Not DetectedMycoplasma

(Large Volume,Direct Culture) Not Detected

(Hoechst DNA Stain) Not Detected

Additional Assays Performed: Albumin, Alkaline,Phosphatase, Blood Urea Nitrogen, Cholesterol, Creatinine,Gamma Globulin ,Glucose, Glutamic OxaloaceticTransaminase (SGOT), Glutamic Pyruvic Transaminase(SGPT), High Density Lipo-Protein Cholesterol, LactateDehydrogenase, Low Density Lipo-Protein Cholesterol,Osmolality pH Phospholipids, Total Bilirubin, Total Protein,Triglycerides, Calcium, Chloride, Copper, InorganicPhosphorus, Iron, Magnesium, Percent Saturation (Iron),Potassium, Selenium, Sodium, Total Iron Binding Capacity(TIBC), Carotene, Cyanocobalamine (B12), Insulin, NicotinicAcid (Niacin), Progesterone, Pteroylglutamic Acid (Folate),Pyridoxine (B6), Riboflavin (B2), Thiamine (B1), Total VitaminE (Tocopherol), Vitamin A (Retinol), Vitamin C, Vitamin D,1,25 Dihydroxy, Vitamin H (Biotin).

Product Average LgG LevelCalf serum 14.09 mg/mL

FBS 0.20 mg/mL

Naturally low IgG FBS 0.02 mg/mL

Super Low IgG FBS <0.005 mg/mL

HyClone Super Low IgG FBS performance is equivalent tonon-processed FBS and is suitable for applications requiringlower levels of specific antibody, such as propagation ofbovine viruses and monoclonal antibody production.

Test Specifications


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Lipid-Reduced FBS is the latest addition to ourspecialty fetal bovine serum (FBS) product line.It is a treated FBS product designed forresearchers who require lower levels of lipidsand other related components. Historically,researchers were required to purchase FBS andperform lipid-reducing treatments themselves.Some of these treatments involve the use ofhazardous or environmentally unfriendlysolvents followed by costly sterile filtrationprocedures. Lipid-reduced FBS is producedfrom our high quality raw FBS using aproprietary treatment and is filtered usingour proven 40 nm filtration system.

Additional assays performed (subject to change withoutnotice): Progesterone, Magnesium, Insulin, Selenium, IgG,Cholesterol, Potassium, High Density Lipo-ProteinCholesterol, Calcium, Low Density Lipo- Protein CholesterolChloride, Triglycerides, Copper, Gamma Globulin, Glucose,Phospholipids, Osmolality, Alkaline Phosphatase, Iron,Lactate Dehydrogenase, Total Iron Binding Capacity (TIBC),Glutamic Pyruvic Transaminase (SGPT), Percent Saturation,Glutamic Oxaloacetic Transaminase (SGOT), Thaimine (B1),Riboflavin (B2), Pyridoxine (B6), Nicotinic Acid (Niacin),Vitamin H (Biotin), Vitamin A (Retinol), Carotene, TotalVitamin E (Tocopherol), Cyanocobalamine (B12),Pteroylglutamic Acid (Folate), Vitamin C, 1,25 DihydroxyVitamin D, pH, Total Protein, Albumin, Blood Urea Nitrogen,Creatinine, Total Bilirubin, Sodium, and InorganicPhosphorus, Lipid Panel, and Chloride.

Lipid-Reduced Fetal Bovine Serum,US Origin

Cell culture seed-stock production, cellbanking, and growth of primary cell linesrequire the safest fetal bovine serum available.Super Safe FBS sourced from New Zealand,which has the fewest reported bovinediseases, along with gamma irradiation offersthe most protection against adventitiousagents without harming the growth factors inserum. Super Safe serum is filtered throughthree sequential 100 nm (0.1 Rm) pore-sizerated filters.

Additional assays performed (subject to change withoutnotice): Progesterone, Chloride, Insulin, Inorganic Phosphorus,Parathyroid Hormone (PTH), Glucose, Cholesterol, Copper,High Density Lipo-Protein Cholesterol, Magnesium, LowDensity Lipo-Protein Cholesterol, Selenium, Triglycerides,IgG, Gamma Globulin, pH, Phospholipids, Osmolality, AlkalinePhosphatase, Iron, Lactate Dehydrogenase, Total Iron BindingCapacity. Glutamic Pyruvic Transaminase (SGPT), PercentSaturation, Glutamic Oxaloacetic Transaminase (SGOT),Thiamine (B1), Riboflavin (B2), Pyridoxine (B6), Nicotinic Acid(Niacin), Vitamin H (Biotin), Vitamin A (Retinol), Carotene,Total Vitamin E (Tocopherol), Cyanocobalamin (B12),Pteroylglutamic Acid (Folate), Vitamin C, 1,25 DihydroxyVitamin D, Total Protein, Albumin, and Blood Urea Nitrogen.

Super Safe New Zealand Irradiated FetalBovine SerumDialyzed FBS is used in applications requiring

serum depleted of small molecules (less than10,000 mw) and is filtered through threesequential 100 nm (0.1 Rm) pore-size ratedfilters. A diafiltration process that reducesconcentrations of low molecular weightcomponents such as nucleotides and aminoacids that are necessary for alternativebiochemical survival pathways has beendeveloped for Thermo Scientific HyClone FBS.The process is reproducible and reduceshypoxanthine and thymidine concentrationsbelow detectable limits. While producing thesame results, a major difference betweendiafiltration and dialysis is that the drivingforce for diafiltration is hydrostatic pressure,rather than concentration gradients withdialysis. Monitoring glucose concentrationscarefully controls the diafiltration process.The glucose concentration, which isrepresentative of the extent of dialysis, isreported for each lot of serum.

Additional assays performed (subject to change withoutnotice): Progesterone, Magnesium, Insulin, Selenium, IgG,Cholesterol, Potassium, High Density Lipo-Protein Cholesterol,Calcium, Low Density Lipo- Protein Cholesterol Chloride,Triglycerides, Copper, Gamma Globulin, Glucose,Phospholipids, Osmolality, Alkaline Phosphatase, Iron, LactateDehydrogenase, Total Iron Binding Capacity (TIBC), GlutamicPyruvic Transaminase (SGPT), Percent Saturation, GlutamicOxaloacetic Transaminase (SGOT), Thaimine (B1), Riboflavin(B2), Pyridoxine (B6), Nicotinic Acid (Niacin), Vitamin H (Biotin),Vitamin A (Retinol), Carotene, Total Vitamin E (Tocopherol),Cyanocobalamine (B12), Pteroylglutamic Acid (Folate), VitaminC, 1,25 Dihydroxy Vitamin D, pH, Total Protein, Albumin, BloodUrea Nitrogen, Creatinine, Total Bilirubin, Sodium, andInorganic Phosphorus.

Dialyzed Fetal Bovine Serum, US Origin


,10 EU/mL


,10 mg/dl


Virus Testing (9 CFR 113.53)Fluorescent AntibodyBluetongue Not DetectedBovine Adenovirus Not DetectedBovine Parvovirus Not DetectedBovine Respiratory Syncytial Virus Not DetectedBovine Viral Diarrhea Virus Not DetectedRabies Not Detected

Not DetectedCytopathogenic Agents—e.g. IBR Not DetectedHemadsorbing Agents—e.g. PI3 Not DetectedMycoplasmaLarge Volume, Direct Culture Not DetectedHoechst DNA Stain Not DetectedCertificate of Suitability Included


,20 EU/mL


,25 mg/dl


Virus Testing (9 CFR 113.53)Fluorescent AntibodyBluetongue Not DetectedBovine Adenovirus Not DetectedBovine Parvovirus Not DetectedBovine Respiratory Syncytial Virus Not DetectedBovine Viral Diarrhea Virus Not DetectedRabies Not DetectedReovirus Not DetectedCytopathogenic Agents—e.g. IBR Not DetectedHemadsorbing Agents—e.g. PI3 Not DetectedMycoplasmaLarge Volume, Direct Culture Not DetectedHoechst DNA Stain Not DetectedCertificate of Suitability Included


,10 EU/mL

Hemoglobin (Spectrophotometric),10 mg/dl



Reovirus


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Research using embryonic stem (ES) cellscontinues to increase, and serum suitability iscritical for keeping these cells in their valuableundifferentiated state. Historically, researcherswere required to screen several lots of serumto find one suitable for ES research. ES(Murine) Screened FBS eliminates the need forprescreening serum, saving both time andmoney, while providing the assurance that theserum used has been screened by experiencedES cell culturists. The screening includesplating efficiency, colony morphology, andtoxicity tests. This protocol was adapted fromGene Targeting, A.L. Joyner, 1995. ES screenedFBS is selected from our highest qualityDefined FBS lots, and it is filtered throughsequential 40 nm (0.04 Rm) pore-size ratedfilters. Further heat inactiviation or irradiationprocessing can be done upon request.

Embryonic Stem (ES) Cell Screened FetalBovine Serum, US Origin

Additional assays performed (subject to change withoutnotice): Progesterone, Selenium, Insulin, IgG, Sodium,Cholesterol, Potassium, High Density Lipo- Protein Cholesterol,Calcium, Low Density Lipo-Protein Cholesterol, Chloride,Triglycerides, Magnesium, Gamma Globulin, Glucose,Phospholipids, Copper, Alkaline Phosphatase, Total IronBinding Capacity (TIBC), Lactate Dehydrogenase, PercentSaturation, Glutamic Pyruvic Transaminase (SGPT), GlutamicOxaloacetic Transaminase (SGOT), Thiamine (B1), Riboflavin(B2), Pyridoxine (B6), Nicotinic Acid (Niacin), Vitamin H (Biotin),Vitamin A (Retinol), Carotene, Total Vitamin E (Tocopherol),Cyanocobalamine (B12), Pteroylglutamic Acid (Folate), VitaminC, 1,25 Dihydroxy Vitamin D, pH, Total Protein, Albumin, BloodUrea Nitrogen, Creatinine, Total Bilirubin, InorganicPhosphorus, Osmolality, and Iron.


,10 EU/mL


,10 mg/dl


Virus Testing (9 CFR 113.53)Fluorescent AntibodyBluetongue Not DetectedBovine Adenovirus Not DetectedBovine Parvovirus Not DetectedBovine Respiratory Syncytial Virus Not DetectedBovine Viral Diarrhea Virus Not DetectedRabies Not DetectedReovirus Not DetectedCytopathogenic Agents—e.g., IBR Not DetectedHemadsorbing Agents—e.g., PI3 Not DetectedMycoplasmaLarge Volume, Direct Culture Not DetectedHoechst DNA Stain Not DetectedCertificate of Suitability Included

This screened FBS product is essential inensuring optimum performance when culturingundifferentiated human mesenchymal stemcells (hMSC). The screening involves growinghMSC over a minimum of three subculturegenerations with 10 percent serum. HumanMSC are then observed for evidence ofnutritional deficiency, cytotoxicity, ormorphological aberrations. The serum istested in parallel with a control lot which hasbeen proven to support the growth of hMSC.The selected lot must demonstrate equal orbetter doubling times when compared to thecontrol lot. Human MSC Screened FBS isselected from our highest Defined FBS and isfiltered through sequential 40 nm (0.4 Rm)pore-size rated filters. Further heatinactivation or irradiation processing can bedone upon request.

Human Mesenchymal Stem Cell ScreenedFetal Bovine Serum, US Origin

Tetracycline Screened Fetal BovineSerum, U.S. Origin


,10 EU/mL


,10 mg/dl



Tetracycline Screened FBS is designed forresearchers using tetracycline-regulated geneexpression systems in cultured cells. Thesesystems rely on the presence or absence oftetracycline in growth medium to either turnoff or turn on gene expression. The presenceof tetracycline in the serum may interfere withsuch systems. To eliminate the possibility ofinterference, we measure the tetracyclinecontent of several high quality Defined FBSlots using an ELISA system. Those lots withundetectable tetracycline levels are selectedas Tetracycline Screened FBS. TET ScreenedFBS is selected from our highest qualityDefined FBS and is filtered through sequential40 nm (0.4 Rm) pore-size rated filters. Furtherheat inactivation or irradiation processingcan be done upon request.

Additional assays performed (subject to change withoutnotice): Progesterone, Selenium, Insulin, IgG, Sodium,Cholesterol, Potassium, High Density Lipo-ProteinCholesterol, Calcium, Low Density Lipo-Protein Chloesterol,Chloride, Triglycerides, Magnesium, Gamma Globulin,Glucose, Phospholipids, Copper, Alkaline Phosphatase,Total Iron Binding Capacity (TIBC), Lactate Dehydrogenase,Percent Saturation, Glutamic Pyruvic Transaminase (SGPT),Glutamic Oxaloacetic Transaminase (SGOT), Thiamine (B1),Riboflavin (B2), Pyridoxine (B6), Nicotinic Acid (Niacin),Vitamin H (Biotin), Vitamin A (Retinol), Carotene, TotalVitamin E (Tocopherol), Cyanocobalamine (B12),Pteroylglutamic Acid (Folate), Vitamin C 1,25 DihydroxyVitamin D, pH, Total Protein, Albumin, Blood Urea Nitrogen,Creatinine, Total Bilirubin, Inorganic Phosphorus, Osmolality,and Iron.


,10 EU/mL


,10 mg/dl



Additional assays performed (subject to change withoutnotice): Progesterone, Selenium, Insulin, IgG, Sodium,Cholesterol, Potassium, High Density Lipo-ProteinCholesterol, Calcium, Low Density Lipo-Protein Chloesterol,Chloride, Triglycerides, Magnesium, Gamma Globulin,Glucose, Phospholipids, Copper, Alkaline Phosphatase,Total Iron Binding Capacity (TIBC), Lactate Dehydrogenase,Percent Saturation, Glutamic Pyruvic Transaminase (SGPT),Glutamic Oxaloacetic Transaminase (SGOT), Thiamine (B1),Riboflavin (B2), Pyridoxine (B6), Nicotinic Acid (Niacin),Vitamin H (Biotin), Vitamin A (Retinol), Carotene, TotalVitamin E (Tocopherol), Cyanocobalamine (B12),Pteroylglutamic Acid (Folate), Vitamin C 1,25 DihydroxyVitamin D, pH, Total Protein, Albumin, Blood Urea Nitrogen,Creatinine, Total Bilirubin, Inorganic Phosphorus, Osmolality,and Iron.


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Insect Cell Screened Fetal Bovine Serum,US OriginWith the increasing popularity of baculovirusexpression vector systems (BEVS), moreresearchers are culturing insect cells. Ourhighest quality Defined FBS is screened toidentify lots that offer optimal performancewith BEVS. This screening involves comparingcell yield and viability using insect cells grownin various test lots for four passages. Thesetest lots are compared to a control lot that hasdemonstrated the ability to support robustgrowth of insect cells. Insect Cell ScreenedFBS is selected from our highest qualityDefined FBS, and it is filtered throughsequential 40 nm (0.04 Rm) pore-size ratedfilters. Further heat inactivation or irradiationprocessing can be done upon request.


,10 EU/mL


,10 mg/dl



Additional assays performed (subject to change withoutnotice): Progesterone, Selenium, Insulin, IgG, Sodium,Cholesterol, Potassium, High Density Lipo- ProteinCholesterol, Calcium, Low Density Lipo-Protein Cholesterol,Chloride, Triglycerides, Magnesium, Gamma Globulin,Glucose, Phospholipids, Copper, Alkaline Phosphatase,Total Iron Binding Capacity (TIBC), Lactate Dehydrogenase,Percent Saturation, Glutamic Pyruvic Transaminase (SGPT),Glutamic Oxaloacetic Transaminase (SGOT), Thiamine (B1),Riboflavin (B2), Pyridoxine (B6), Nicotinic Acid (Niacin),Vitamin H (Biotin), Vitamin A (Retinol), Carotene, TotalVitamin E (Tocopherol), Cyanocobalamine (B12),Pteroylglutamic Acid (Folate), Vitamin C, 1,25 DihydroxyVitamin D, pH, Total Protein, Albumin, Blood Urea Nitrogen,Creatinine, Total Bilirubin, Inorganic Phosphorus, Osmolality,and Iron.


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Specialty Fetal Bovine SerumDescription Part Number Packaging Storage Temp

Super Low IgG Fetal Bovine Serum, US Origin 0 SH30898.02 100 mL -10°CSH30898.03 500 mL -10°C

Super Low IgG Fetal Bovine Serum, US Origin, Heat Inactivated 0 SH30898.02HI 100 mL -10°CSH30898.03HI 500 mL -10°C

Charcoal/Dextran Treated Fetal Bovine SerumCharcoal/Dextran Treated Fetal Bovine Serum, US Origin SH30068.01 50 mL -10°C

SH30068.02 100 mL -10°CSH30068.03 500 mL -10°C

Charcoal/Dextran Treated Fetal Bovine Serum, US Origin, Heat Inactivated SH30068.01HI 50 mL -10°CSH30068.02HI 100 mL -10°CSH30068.03HI 500 mL -10°C

Charcoal/Dextran Treated Fetal Bovine Serum, US Origin, Irradiated SH30068.03IR 500 mL -10°CDialyzed Fetal Bovine SerumDialyzed Fetal Bovine Serum, US Origin SH30079.01 50 mL -10°C

SH30079.02 100 mL -10°CSH30079.03 500 mL -10°C

Dialyzed Fetal Bovine Serum, US Origin, Heat Inactivated SH30079.01HI 50 mL -10°CSH30079.02HI 100 mL -10°CSH30079.03HI 500 mL -10°C

Dialyzed Fetal Bovine Serum, US Origin, Irradiated SH30079.03IR 500 mL -10°CLipid-Reduced Fetal Bovine SerumLipid-Reduced Fetal Bovine Serum, US Origin SH30855.01 50 mL -10°C

SH30855.02 100 mL -10°CSH30855.03 500 mL -10°C

Lipid-Reduced Fetal Bovine Serum, US Origin, Heat Inactivated SH30855.01HI 50 mL -10°CSH30855.02HI 100 mL -10°CSH30855.03HI 500 mL -10°C

Lipid-Reduced Fetal Bovine Serum, US Origin, Irradiated SH30855.03IR 1000 mL -10°CSuper Safe New Zealand Fetal Bovine SerumSuper Safe New Zealand Fetal Bovine Serum, Irradiated SH30406.02IR 500 mL -10°CEmbryonic Sterm Cell Screened Fetal Bovine SerumES Screened FBS, US Origin SH30070.02E 100 mL -10°C

SH30070.03E 500 mL -10°CHuman Mesenchymal Stem Cell Screened Fetal Bovine SerumHuman Mesenchymal Stem Cell Screened Fetal Bovine Serum, US Origin SH30070.03M 500 mL -10°CInsect Cell Screened Fetal Bovine SerumInsect Cell Screened Fetal Bovine Serum, US Origin SH30070.03I 500 mL -10°CTetracycline Screened Fetal Bovine SerumTetracycline Screened Fetal Bovine Serum, US Origin SH30070.03T 500 mL -10°C

Super Low IgG Fetal Bovine Serum, US Origin, Irradiated 0 SH30898.03IR 500 mL -10°C

Super Low IgG Fetal Bovine Serum


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Iron-Supplemented Bovine Calf Serum iscollected by venipuncture from formula-fedveal calves (16 - 22 weeks at time ofcollection), which produce exceptionally highlevels of transferrin. We carefully supplementthis serum with iron to load serum transferrinlevels to physiological levels. Aftersupplementation, our Iron-SupplementedBovine Calf Serum contains three to four timesas much available iron and transferrin as FBSor equine serum. Many researchers havefound this product to be an excellent andcost-effective alternative to FBS. Growthpromotion studies have demonstrated manyapplications for Iron-Supplemented BovineCalf Serum and show how it often outperformsFBS in many applications. As with all our calfserum products, this product is sterile filteredusing three sequential 100 nm (0.1 Rm)pore-size rated filters.

Test SpecificationEndotoxin(LimulusAmebocyteLysate—GelClot)

,10 EU/mL


,10 mg/dl



Additional assays performed (subject to change withoutnotice): Insulin, Gamma Globulin, Alkaline Phosphatase,Lactate Dehydrogenase, Glutamic Pyruvic Transaminase(SGPT), Glutamic Oxaloacetic Transaminase (SGOT), pH, TotalProtein, Albumin, Blood Urea Nitrogen, Creatinine, TotalBilirubin, Sodium, Potassium, Calcium, Chloride,Phosphorus, Osmolality, Iron, Total Iron Binding Capacity(TIBC), Percent Saturation, Glucose, and IgG.

Iron-Supplemented Bovine Calf Serum,US Origin Our quality calf serum is also available without

iron supplementation and is filtered throughthree sequential 100 nm (0.1 Rm) pore-sizerated filters.

Additional assays performed (subject to change withoutnotice): Insulin, Gamma Globulin, Alkaline Phosphatase,Lactate Dehydrogenase, Glutamic Pyruvic Transaminase(SGPT), Glutamic Oxaloacetic Transaminase (SGOT), pH, TotalProtein, Albumin, Blood Urea Nitrogen, Creatinine, TotalBilirubin, Sodium, Potassium, Calcium, Chloride,Phosphorus, Osmolality, Iron, Total Iron Binding Capacity(TIBC), Percent Saturation, Glucose, and IgG.

Bovine Calf Serum, US Origin

To produce our Cosmic Calf Serum, we startwith our high quality Iron-SupplementedBovine Calf Serum and add growth promotingfactors. The result is a superior bovine calfserum fortified with iron and naturally derivedcomponents for increased performance with abroad spectrum of cell types. All componentsof Cosmic Calf are sourced in the U.S. and aretraceable. Studies have shown Cosmic Calfoutperforms Iron-Supplemented Bovine CalfSerum and FBS in several applications, andperforms exceptionally well with CHO-KI cellsand CHO derivatives. Cosmic Calf serum isfiltered through three sequential 100 nm(0.1 Rm) pore-size rated filters.


,10 EU/mL




Additional assays performed (subject to change withoutnotice): Gamma Globuline, Alkaline Phosphatase, LactateDehydrogenase, Glutamic Pyruvic Transaminase (SGPT),Glutamic Oxaloacetic Transaminase (SGOT), pH, TotalProtein, Albumin, Blood Urea Nitrogen, Creatinine, TotalBilirubin, Sodium, Potassium, Calcium, Chloride, IncorganicPhosphorus, Osmolality, Iron, Total Iron Binding Capacity(TIBC), Percent Iron Saturation, Glucose, and IgG.

Cosmic Calf Serum, US Origin

Test SpecificationEndotoxin(Limulus AmebocyteLysate— Gel Clot)

,10 EU/mL


Sterility Testing(Current USP and EP)Bacteria & Fungi No GrowthVirus Testing (9 CFR 113.53)Fluorescent AntibodyBluetongue Not DetectedBovine Adenovirus Not DetectedBovine Parvovirus Not DetectedBovine Respiratory Syncytial Virus Not DetectedBovine Viral Diarrhea Virus Not DetectedRabies Not DetectedReovirus Not DetectedCytopathogenic Agents—e.g., IBR Not DetectedHemadsorbing Agents—e.g., PI3 Not DetectedMycoplasmaLarge Volume, Direct Culture Not DetectedHoechst DNA Stain Not DetectedCertificate of Suitability Included

Bovine Calf Serum


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Historically, the quality of newborn bovine calfserum available from some serum suppliershas been compromised as a result of outdatedand careless collection procedures. The samecareful collection and processing procedures,including venipuncture, are used for ournewborn calf serum, as well as all our bovineserum products. Newborn Calf is filteredthrough three sequential 100 nm (0.1 Rm)pore-size rated filters and is sourced fromanimals that are typically less than 10 days old.


,50 EU/mL


,25 mg/dl


Virus Testing (9 CFR 113.53)Fluorescent AntibodyBluetongue Not DetectedBovine Adenovirus Not DetectedBovine Parvovirus Not DetectedBovine Respiratory Syncytial Virus Not DetectedBovine Viral Diarrhea Virus Not DetectedRabies Not DetectedReovirus Not DetectedCytopathogenic Agents—e.g., IBR Not DetectedHemadsorbing Agents—e.g., PI3 Not DetectedMycoplasmaLarge Volume, Direct Culture Not DetectedHoechst DNA Stain Not Detected

Additional assays performed (subject to change withoutnotice): Gamma Globulin, Alkaline Phosphatase, LactateDehydrogenase, Glutamic Pyruvic Transaminase (SGPT),Glutamic Oxaloacetic Transaminase (SGOT), pH, TotalProtein, Albumin, Blood Urea Nitrogen, Creatinine, TotalBilirubin, Sodium, Potassium, Calcium, Chloride, InorgaincPhosphorous, Osmolality, Iron, Total Iron Binding Capacity(TIBC), Percent Saturation, Glucose, and IgG.

Refer to Pages 164-168 for calf serum vs. U.S. FBS growthpromotion studies.

Newborn Bovine Calf Serum, US Origin

Additional assays performed (subject to change withoutnotice): Gamma Globulin, Alkaline Phosphatase, LactateDehydrogenase, Glutamic Pyruvic Transaminase (SGPT),Glutamic Oxaloacetic Transaminase (SGOT), Total Protein,Albumin, Blood Urea Nitrogen, Creatinine, Total Bilirubin,Sodium, Potassium, Calcium, Chloride, InorganicPhosphorous, Glucose, pH, Osmolality, Iron, Total IronBinding Capacity (TIBC), Percent Saturation, and IgG.

New Zealand Newborn Bovine Calf Serum

As with our New Zealand FBS products, allNew Zealand bovine serum products arecarefully collected, processed and filteredin New Zealand and offer maximum safetyagainst bovine diseases. We follow the samestandards in processing our New Zealandserum as with our FBS to ensure the highestquality and absolute traceability. Calf (newborn)products are sourced from animals that are lessthan 10 days old.

Bovine Growth Serum (BGS) was developed forcell culturists who are concerned about theavailability of fetal bovine serum and areseeking an affordable and viable alternative.Growth promotion studies demonstrate thatBGS is a superior performing FBS replacement.BGS consists of high quality Thermo ScientificHyClone Defined Bovine Calf Serum, supple-mented with chemically defined componentsincluding vitamins, amino acids, trace metals,and other small molecules to stimulate cellgrowth and proliferation. It is filtered throughthree sequential 100 nm (0.1 Rm) pore-sizerated filters.


,10 EU/mL


,10 mg/dl



Assays (Subject to change without notice): Insulin, GammaGlobulin, Alkaline Phosphatase, Lactate Dehydrogenase,Glutamic Pyrivic Transaminase (SGPT), Glutamic OxaloaceticTransaminase (SGOT), pH, Total Protein, Albumin, Blood UreaNitrogen, Creatinine, Total Bilirubin, Sodium, Potassium,Calcium, Chloride, Phosphorus, Osmolality, Iron, Total IronBinding Capaicty (TIBC), Percent Saturation, Glucose,and IgG.

Bovine Growth Serum, US Origin


,100 EU/mL


,50 mg/dl




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Additional Assays performed (subject to change withoutnotice): Gamma Globulin, Alkaline Phosphatase,Progesterone, Lactate Dehydrogenase, Glutamic PyruvicTransaminase (SGPT), Glutamic Oxaloacetic Transaminase(SGOT), Total Protein, Albumin, Blood Urea Nitrogen,Creatinine, Total Bilirubin, Testosterone, Cortisol,Corticosterone, Insulin, Triiodothyronine (T3), Thyroxine )T4),Parathyroid Hormone (PTH), Calcitonin, Sodium, Potassium,Calcium Chloride, Inorganic Phosphorous, Glucose, pH,Osmolality, Iron, Total Iron Binding Capacity (TIBC), PercentIron Saturation, and IgG.

New Zealand Cosmic Calf Serum


,10 EU/mL


,10 mg/dl



Assays (Subject to change without notice): Insulin, GammaGlobulin, Alkaline Phosphatase, Lactate Dehydrogenase,Glutamic Pyrivic Transaminase (SGPT), Glutamic OxaloaceticTransaminase (SGOT), pH, Total Protein, Albumin, Blood UreaNitrogen, Creatinine, Total Bilirubin, Sodium, Potassium,Calcium, Chloride, Phosphorus, Osmolality, Iron, Total IronBinding Capaicty (TIBC), Percent Saturation, Glucose,and IgG.

New Zealand Bovine Growth Serum


,100 EU/mL


,50 mg/dl


Virus Testing (9 CFR 113.53)Fluorescent AntibodyBluetongue Not DetectedBovine Adenovirus Not DetectedBovine Parvovirus Not DetectedBovine Respiratory Syncytial Virus Not DetectedBovine Viral Diarrhea Virus Not DetectedRabies Not DetectedReovirus Not DetectedCytopathogenic Agents—e.g., IBR Not DetectedHemadsorbingAgents—e.g., PI3 Not DetectedMycoplasmaLarge Volume, Direct Culture Not DetectedHoechst DNA Stain Not DetectedCertificate of Suitability Included


New Zealand Iron-SupplementedNewborn Serum


,50 EU/mL


,30 mg/dl




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Bovine Calf SerumDescription Part Number Packaging Storage TempIron-Supplemented Bovine Calf SerumIron-Supplemented Bovine Calf Serum, US Origin SH30072.02 100 mL -10°C

SH30072.03 500 mL -10°CSH30072.04 1000 mL -10°C

Iron-Supplemented Bovine Calf Serum, US Origin, Heat Inactivated SH30072.02HI 100 mL -10°CSH30072.03HI 500 mL -10°CSH30072.04HI 1000 mL -10°C

Iron-Supplemented Bovine Calf Serum, US Origin, Irradiated SH30072.03IR 500 mL -10°CSH30072.04IR 1000 mL -10°C

Bovine Calf SerumBovine Calf Serum, US Origin SH30073.02 100 mL -10°C

SH30073.03 500 mL -10°CSH30073.04 1000 mL -10°C

Bovine Calf Serum, US Origin, Heat Inactivated SH30073.02HI 100 mL -10°CSH30073.03HI 500 mL -10°CSH30073.04HI 1000 mL -10°C

Bovine Calf Serum, US Origin, Irradiated SH30073.03IR 500 mL -10°CSH30073.04IR 1000 mL -10°C

Cosmic Calf SerumCosmic Calf Serum, US Origin SH30087.02 100 mL -10°C

SH30087.03 500 mL -10°CSH30087.04 1000 mL -10°C

Cosmic Calf Serum, US Origin, Heat Inactivated SH30087.02HI 100 mL -10°CSH30087.03HI 500 mL -10°CSH30087.04HI 1000 mL -10°C

Cosmic Calf Serum, US Origin, Irradiated SH30087.03IR 500 mL -10°CSH30087.04IR 1000 mL -10°C

Bovine Growth SerumBovine Growth Serum, US Origin SH30541.02 100 mL -10°C

SH30541.03 500 mL -10°CSH30541.04 1000 mL -10°C

Bovine Growth Serum, US Origin, Heat Inactivated SH30541.02HI 100 mL -10°CSH30541.03HI 500 mL -10°CSH30541.04HI 1000 mL -10°C

Bovine Growth Serum, US Origin, Irradiated SH30541.03IR 500 mL -10°CSH30541.04IR 1000 mL -10°C

Newborn Bovine Calf SerumNewborn Bovine Calf Serum, US Origin SH30118.02 100 mL -10°C

SH30118.03 500 mL -10°CSH30118.04 1000 mL -10°C

Newborn Bovine Calf Serum, US Origin, Heat Inactivated SH30118.02HI 100 mL -10°CSH30118.03HI 500 mL -10°CSH30118.04HI 1000 mL -10°C

Newborn Bovine Calf Serum, US Origin, Irradiated SH30118.03IR 100 mL -10°CSH30118.04IR 500 mL -10°C


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New Zealand Bovine SerumDescription Part Number Packaging Storage TempNew Zealand Newborn Bovine SerumNew Zealand Newborn Bovine Serum SH30401.01 500 mL -10°C

SH30401.02 1000 mL -10°CSH30401.03 3000 mL -10°C

New Zealand Newborn Bovine Serum, Heat Inactivated SH30401.01HI 500 mL -10°CSH30401.02HI 1000 mL -10°C

New Zealand Newborn Bovine Serum, Irradiated SH30401.01IR 500 mL -10°CSH30401.02IR 1000 mL -10°C

New Zealand Iron-Supplemented Calf SerumNew Zealand Iron-Supplemented Calf Serum SH30626.01 100 mL -10°C

SH30626.02 500 mL -10°CSH30626.03 1000 mL -10°C

New Zealand Iron-Supplemented Calf Serum, Heat Inactivated SH30626.01HI 100 mL -10°CSH30626.02HI 500 mL -10°CSH30626.03HI 1000 mL -10°C

New Zealand Iron-Supplemented Calf Serum, Irradiated SH30626.02IR 500 mL -10°CSH30626.03IR 1000 mL -10°C

New Zealand Bovine Growth SerumNew Zealand Bovine Growth Serum SH30591.01 100 mL -10°C

SH30591.02 500 mL -10°CSH30591.03 1000 mL -10°C

New Zealand Bovine Growth Serum, Heat Inactivated SH30591.01HI 100 mL -10°CSH30591.02HI 500 mL -10°CSH30591.03HI 1000 mL -10°C

New Zealand Bovine Growth Serum, Irradiated SH30591.02IR 500 mL -10°CSH30591.03IR 1000 mL -10°C

New Zealand Cosmic Calf SerumNew Zealand Cosmic Calf Serum SH30413.01 100 mL -10°C

SH30413.02 500 mL -10°CSH30413.03 1000 mL -10°CSH30413.04 3000 mL -10°C

New Zealand Cosmic Calf Serum, Heat Inactivated SH30413.02HI 500 mL -10°CSH30413.03HI 1000 mL -10°CSH30413.04HI 3000 mL -10°C

New Zealand Cosmic Calf Serum, Irradiated SH30413.02IR 500 mL -10°CSH30413.03IR 1000 mL -10°C


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Our FBS alternatives are designed to deliverboth cost-effectiveness and high performance.FBS replacements can take the place of FBSat a reduced cost and are readily available. AllThermo Scientific HyClone FetalClone productsare sterile filtered using three sequential100 nm (0.1 Rm) pore-size rated filters.

FetalClone I is optimized for the growth ofhybridomas; and IgG levels are comparableto those found in FBS. Although FetalClone Iwas designed for use with hybridomas, manycustomers have successfully cultured severalcell lines using FetalClone I.


,10 EU/mL


,20 mg/dl



FetalClone I, US Origin

Additional assays performed (subject to change withoutnotice): Gamma Globulin, Alkaline Phosphatase, LactateDehydrogenase, Glutamic Pyruvic Transaminase (SGPT),Glutamic Oxaloacetic Transaminase (SGOT), pH, TotalProtein, Albumin, Blood Urea Nitrogen, Creatinine, TotalBilirubin, Sodium, Potassium, Calcium, Chloride, InorganicPhosphorus, Osmolality, Iron, Total Iron Binding Capacity(TIBC), Percent Iron Saturation, Glucose, and IgG.

FetalClone II is optimized for the growth ofCHO cells and derivatives, offering the samebasic formulation as our FetalClone I, plusadditional growth factors and supplements.No adaptation is required and suggestedconcentrations are the same as those usedwith FBS.


,10 EU/mL


,20 mg/dl



Additional assays performed (subject to change withoutnotice): Gamma Globulin, Alkaline Phosphatase, LactateDehydrogenase, Glutamic Pyruvic Transaminase (SGPT),Glutamic Oxaloacetic Transaminase (SGOT), pH, TotalProtein, Albumin, Blood Urea Nitrogen, Creatinine, TotalBilirubin, Sodium, Potassium, Calcium, Chloride, InorganicPhosphorus, Osmolality, Iron, Total Iron Binding Capacity(TIBC), Percent Iron Saturation, Glucose, and IgG.

FetalClone II, US Origin

FetalClone III is the most widely applicableof our FetalClone family and is designed toinclude fibroblasts in its applications.


,10 EU/mL


,20 mg/dl



Additional assays performed (subject to change withoutnotice): Gamma Globulin, Alkaline Phoaphatase, LactateDehydrogenase, Glutamic Pyrivic Transaminase (SGPT),Glutamic Oxaloacetic Transaminase (SGOT), pH, TotalProtein, Albumin, Blood Urea Nitrogen, Creatinine, TotalBilirubin, Sodium, Potassium, Calscium, Chloride, InorganicPhosphorus, Osmolality, Iron, Total Iron Binding Capacity(TIBC), Percent Iron Saturation, Glucose, and IgG.

FetalClone III, US Origin

Fetal Bovine Serum Alternatives

This product has been successfully used with the cell linesshown on Page 153.


86

Alpha Calf Fraction provides hybridoma userswith a cost-effective medium supplement thatpromotes growth and antibody production. Thisproduct is produced from our bovine calf serumusing a non-ethanol proprietary process toreduce immunoglobulins and protein tolevels very similar to those of FBS. Alpha CalfFraction typically contains less than 200 Rg/mLIgG. It is available with and without iron.


,10 EU/mL


,20 mg/dl


Virus Testing (9 CFR 113.53)Fluorescent AntibodyBluetongue Not DetectedBovine Adenovirus Not DetectedBovine Parvovirus Not DetectedBovine Respiratory Syncytial Virus Not DetectedBovine Viral Diarrhea Virus Not DetectedRabies Not DetectedReovirus Not DetectedCytopathogenic Agents—e.g., IBR Not DetectedHemadsorbing Agents—e.g., PI3 Not DetectedMycoplasmaLarge Volume, Direct Culture Not DetectedHoechst DNA Stain Not Detected

Additional assays performed (subject to change withoutnotice): Gamma Globulin, Alkaline Phosphatase, LactateDehydrogenase, Glutamic Pyruvic Transaminase (SGPT),Glutamic Oxaloacetic Transaminase (SGOT), pH, Albumin,Blood Urea Nitrogen, Creatinine, Total Bilirubin, Sodium,Potassium, Calcium, Chloride, Inorganic Phosphorus, Iron,Total Iron Binding Capacity (TIBC), Percent Saturation,and Glucose.

Alpha Calf Fraction


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Fetal Bovine Serum AlternativesDescription Part Number Packaging Storage TempFetalClone IFetalClone I SH30080.02 100 mL -10°C

SH30080.03 500 mL -10°CFetalClone I, Heat Inactivated SH30080.02HI 100 mL -10°C

SH30080.03HI 500 mL -10°CFetalClone I, Irradiated SH30080.03IR 500 mL -10°CFetalClone IIFetalClone II SH30066.02 100 mL -10°C

SH30066.03 500 mL -10°CFetalClone II, Heat Inactivated SH30066.02HI 100 mL -10°C

SH30066.03HI 500 mL -10°CFetalClone II, Irradiated SH30066.03IR 500 mL -10°CFetalClone IIIFetalClone III SH30109.02 100 mL -10°C

SH30109.03 500 mL -10°CFetalClone III, Heat Inactivated SH30109.02HI 100 mL -10°C

SH30109.03HI 500 mL -10°CFetalClone III, Irradiated SH30109.03IR 500 mL -10°CAlpha Calf FractionAlpha Calf Fraction, Iron-Supplemented SH30076.02 100 mL -10°C

SH30076.03 500 mL -10°CAlpha Calf Fraction, Iron-Supplemented, Heat Inactivated SH30076.02HI 100 mL -10°C

SH30076.03HI 500 mL -10°CAlpha Calf Fraction, Iron-Supplemented, Irradiated SH30076.03IR 500 mL -10°CAlpha Calf Fraction, without Iron SH30212.02 100 mL -10°C

SH30212.03 500 mL -10°CAlpha Calf Fraction, without Iron, Heat Inactivated SH30212.02HI 100 mL -10°C

SH30212.03HI 500 mL -10°CAlpha Calf Fraction, without Iron, Irradiated SH30212.03IR 500 mL -10°C


88

Donor Equine Serum is carefully collected fromselected U.S. donor herds that receive regularveterinary inspection and care. The animals arefed an enriched diet to ensure proper nutritionand are fasted prior to bleeding to reduce lipidconcentration. The horses are bled in anenclosed facility that is specially equipped foraseptic collection. Coggins tests for equineinfectious anemia are routinely performed oneach horse. Donor Equine Serum is sterilefiltered using three sequential 100 nm (0.1 Rm)pore-size rated filters.

Additional assays performed (subject to change withoutnotice): Gamma Globuline, Alkaline Phosphatase, LactateDehydrogenase, Glutamic Pyruvic Transaminase (SGPT),Glutamic Oxaloacetic Transaminase (SGOT), pH, TotalProtein, Albumin, Blood Urea Nitrogen, Creatinine, TotalBilirubin, Sodium, Potassium, Calcium, Chloride, InorganicPhosphorous, Osmolality, iron, Total Iron Binding Capacity(TIBC), Percent Saturation, and Glucose.

Donor Equine Serum, US Origin


,10 EU/mL


,10 mg/dl


Virus Testing (9 CFR 113.53)Fluorescent AntibodyEquine Herpes Virus Not DetectedEquine Arteritis Not DetectedBovine Viral Diarrhea Virus Not DetectedRabies Not DetectedReovirus Not DetectedCytopathogenic Agents—e.g. IBR Not Detected

Coggins (Equine Infectious Anemia)Not Detected

MycoplasmaLarge Volume, Direct Culture Not DetectedHoechst DNA Stain Not Detected

New Zealand Adult Bovine serum is collected,processed, and filtered in New Zealand.This serum is sterile filtered through 200 nm(0.2 Rm) filters.

Additional assays performed (subject to change withoutnotice): Gamma Globulin, Alkaline Phosphatase, LactateDehydrogenase, Glutamic Pyruvic Transaminase (SGPT),Glutamic Oxaloacetic Transaminase (SGOT), Total Protein,Albumin, Blood Urea Nitrogen, Creatinine, Total Bilirubin,Sodium, Potassium, Calcium, Chloride, Inorganic Phosphorous,Glucose, pH, Osmolality, Iron, Total Iron Binding Capacity(TIBC), Percent Saturation, and IgG.

Adult Bovine Serum New Zealand Origin

Sera Products

Donor Adult Bovine serum (ABS) is sourced inthe US, from a closed herd maintained onUSDA-licensed premises. Cattle from theseclosed herds are used exclusively for theproduction of donor bovine blood products.They receive regular veterinary inspections andcare. Prior to entering the herd, the cattle arequarantined for 30 days and are tested fortuberculosis, brucellosis, and bluetongue virus.Donor Adult Bovine serum is sterile filteredusing three sequential 100 nm (0.1Rm) pore-size rated filters.


,50 EU/mL


,25 mg/dl




Donor Adult Bovine Serum, US Origin


,100 EU/mL





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Porcine Serum is sourced from New Zealandand filtered in the United States. This productis sterile filtered through multiple 200 nm(0.2 Rm) pore-size rated filters and is gammairradiated at 25-40 kGy after filtration.


,100 EU/mL


,50 mg/dl


Virus Testing (9 CFR 113.53)Fluorescent AntibodyTransmissableGastroenteritis Virus

Not Detected

Porcine Adenovirus Not DetectedPorcine Parvovirus Not DetectedBovine Viral Diarrhea Virus Not DetectedRabies Not DetectedHemagglutinating Encephalitis Virus Not DetectedCircovirus Not DetectedCytopathogenic Agents—e.g. IBR Not DetectedHemadsorbing Agents—e.g. PI3 Not DetectedMycoplasmaLarge Volume, Direct Culture Not DetectedHoechst DNA Stain Not DetectedGamma Irradiation 25-40 kGy

Additional assays performed (subject to change withoutnotice): Gamma Globulin, Alkaline Phosphatase, LactateDehydrogenase, Glutamic Pyruvic Transaminase (SGPT),Glutamic Oxaloacetic Transaminase (SGOT), pH, TotalProtein, Albumin, Blood Urea Nitrogen, Creatinine, TotalBilirubin, Sodium, Potassium, Calcium, Chloride, InorganicPhosphorous, Osmolality, and Glucose.

Porcine Serum, New Zealand Origin

This product is sourced from New Zealand’sSouth Pacific Sera and is processed in NewZealand. Source animals are aged six to 36months at time of collection and the serum issterile filtered using three sequential 100 nm(0.1 Rm) pore-size rated filters.


,5 EU/mL


For Information Only




Donor Bovine Serum,New Zealand Origin


90

Description Part Number Packaging Storage TempDonor Adult Bovine SerumDonor Adult Bovine Serum, US Origin SH30075.02 100 mL -10°C

SH30075.03 500 mL -10°CDonor Adult Bovine Serum, US Origin, Heat Inactivated SH30075.02HI 100 mL -10°C

SH30075.03HI 500 mL -10°CDonor Adult Bovine Serum, US Origin, Irradiated SH30075.03IR 500 mL -10°CDonor Bovine Serum, New Zealand Origin SH30417.01 100 mL -10°C

SH30417.02 500 mL -10°CSH30417.03 1000 mL -10°C

Donor Bovine Serum, New Zealand Origin, Heat Inactivated SH30417.01HI 100 mL -10°CSH30417.02HI 500 mL -10°CSH30417.03HI 1000 mL -10°C

Donor Bovine Serum, New Zealand Origin, Irradiated SH30417.02IR 500 mL -10°CSH30417.03IR 1000 mL -10°C

Adult Bovine SerumAdult Bovine Serum, New Zealand Origin SH30409.02 500 mL -10°C

SH30409.03 1000 mL -10°CSH30409.04 3000 mL -10°C

Adult Bovine Serum, New Zealand Origin, Heat Inactivated SH30409.02HI 500 mL -10°CSH30409.03HI 1000 mL -10°C

Adult Bovine Serum, New Zealand Origin, Irradiated SH30409.03IR 1000 mL -10°CAdult Bovine Serum, New Zealand Origin, Non-Sterile SH30402.01 3000 mL -10°C

SH30402.02 10 L -10°CSH30402.03 12.5 L -10°C

Donor Equine SerumDonor Equine Serum, US Origin SH30074.02 100 mL -10°C

SH30074.03 500 mL -10°CDonor Equine Serum, US Origin, Heat Inactivated SH30074.02HI 100 mL -10°C

SH30074.03HI 500 mL -10°CDonor Equine Serum, US Origin, Irradiated SH30074.03IR 500 mL -10°CWear Testing Fluid for Artifical Joints 30Wear Testing Fluid for Artificial Joints 30, US Origin SH30856.03 500 mL -10°C

SH30856.04 1000 mL -10°CWear Testing Fluid for Artifical Joints 20Wear Testing Fluid for Artificial Joints 20, US Origin, Heat Inactivated SH30891.03 500 mL -10°C

SH30891.04 1000 mL -10°CPorcine SerumPorcine Serum, New Zealand Origin SH30908.03 500 mL -10°C

SH30908.04 1000 mL -10°CPorcine Serum, New Zealand Origin, Heat Inactivated SH30908.03HI 500 mL -10°C

SH30908.04HI 1000 mL -10°C

Serum Products

Porcine Serum, New Zealand Origin, Irradiated SH30908.03IR 500 mL -10°CSH30908.04IR 1000 mL -10°C


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The European Agency for the Evaluation ofMedicinal Products (EMEA) has issued twodirectives that apply to the use of bovine serumin the production of medicinal products.

• EMEA/CVMP/743/00-FINAL from theCommittee for Veterinary MedicinalProducts (CVMP) Guideline on Requirementsand Controls Applied to Bovine Serum Usedin the Production of ImmunologicalVeterinary Medicinal Products

• EMEA/CPMP/BWP/1793/02 from theCommittee for Proprietary MedicinalProducts (CPMP) Note for Guidance on theUse of Bovine Serum in the Manufacture ofHuman Biological Medicinal Products

To support medicinal manufacturers who utilizebovine serum in their processes, a line ofEMEA conforming products that meet thescope of both directives has been created.

Guideline on Requirements and ControlsApplied to Bovine Serum Used in theProduction of Immunological VeterinaryMedicinal Products

The following is an excerpt fromEMEA/CVMP/743/00-FINAL, whichsummarizes the scope of the directive:

"Starting materials of animal origin arenecessary for the production of immunologicalveterinary medicinal products. Attempts shouldbe made to reduce the use of bovine serum inthe production of immunological veterinarymedicinal products and whenever possible,the use of nonruminant materials is preferred.

It is, however, recognized that materials ofanimal origin, including bovine serum, are stillessential ingredients of the cell culture mediaused in the production of many veterinaryvaccines. Different risks are associated withthe use of such starting materials. Indeed, the

nature and quality of the bovine serum canprofoundly influence the quality of themanufacturing process and of the finishedproduct."

Note for Guidance on the Use of BovineSerum in theManufacture of HumanBiological Medicinal Products

The following is an excerpt fromEMEA/CPMP/BWP/1793/02, whichsummarizes the scope of the directive:

"This Note for Guidance outlines the generalprinciples, which should be applied to thecontrol of quality and safety of bovine serumused during the manufacture of humanbiological medicinal products includingvaccines and biotech products. This includesserum used directly during production cellgrowth and serum used during cell growth priorto a production phase, for example in thegrowth of cells prior to vaccine production.Serum that is used minimally, such as in theestablishment of a master cell bank, duringcryopreservation or during a developmentalphase, such as during transfection in thegeneration of genetically engineered cells,also falls within the scope of this Note forGuidance."

We recognize the need for high quality,traceable bovine serum, which is tested(and inactivated as required) to meet bothdirectives. Where applicable, our EMEAConforming products are tested per EuropeanPharmacopoeia protocols and irradiated at therequired minimum dose. These EMEAConforming products provide medicinalmanufacturers with the assurance of receivinghigh quality, consistent product that conformsto the requirements. Complete versions of bothdirectives can be found atwww.emea.eu.int.

In recognizing the challenges in changing theregulatory filings, we also offer an option tothose serum users who need to meet thehuman guideline requirements, but prefer tocontinue using the same products (no changesto the product part number) with the additionof three tests.

These products are ordered using the non-EMEApart numbers and are tested per the informationshown. Standard serum products that requireEMEA/CPMP/BWP/1793/02 Conformance are

provided with the standard Certificate ofAnalysis, in addition to an addendum, whichincludes the additional testing results. It is thecombination of both documents that provideassurance that the product purchased isEMEA/CPMP/BWP/1793/02 Conforming.

EMEA/CVMP/743 /00 andEMEA/CPMP/BWP/1793/02 Conforming

EMEA Conforming Serum Products

Test SpecificationEndotoxin(Limulus Amebocyte Lysate—Gel Clotand EP Ph. Eur. 2.6.14)

See non-EMEAequivalent

Hemoglobin (spectrophotometric)See non-EMEA

equivalent

Sterility Testing(Current USP and EP Ph.Eur. 2.6.1) Bacteria & Fungi

No Growth

Virus Testing (Full EMEA and 9CFR113.53)Fluorescent AntibodyBluetongue Not DetectedBovine Adenovirus Not DetectedBovine Parvovirus Not DetectedBovine Respiratory Syncytial Virus Not DetectedBovine Viral Diarrhea Virus Not DetectedBovine Leukemia Virus Not DetectedRabies Not DetectedReovirus Not Detected

Cytopathogenic Agents—e.g. IBRNot Detected

Hemadsorbing Agents—e.g. PI3 Not DetectedMycoplasma(Points to Consider and EP Ph. Eur. 2.6.7)Large Volume, Direct Culture Not DetectedHoechst DNA Stain Not Detected

Ouchterlony/Double ImmunodiffusionSatisfactory

Bovine Viral Diarrhea VirusAntibodies Pass

Interference/Comparative TitrationNo Interference

Quantitation TestedGamma Irradiation )30 kGy

()30 Mrad)Post-Irradiation TestingBovine Viral Diarrhea Virus Not DetectedAntibodies Pass

Interference/Comparative TitrationNo Interference

Quantitation Not DetectedCertificate of Suitability Included


92

EMEA/CPMP/BWP/1793/02 Conforming EMEA Conforming Sera ProductsDescription Part Number Packaging Storage TempDefined Fetal Bovine Serum (FBS), U.S.Origin (non-EMEA equivalent SH30070)

SH30531.02 100 mL -10°CSH30531.03 500 mL -10°C

Characterized FBS, U.S. Origin(non-EMEA equivalent SH30071)

SH30532.02 100 mL -10°CSH30532.03 500 mL -10°C

Characterized Australian FBS (non-EMEAequivalent SH30084)


New Zealand FBS (non-EMEA equivalentSH30406)

SH30611.01 100 mL -10°CSH30611.02 500 mL -10°C

Iron-Supplemented Bovine Calf Serum,U.S. Origin (non-EMEA equivalentSH30072)


Bovine Calf Serum, U.S. Origin(non-EMEA equivalent SH30073)


Cosmic Calf Serum, U.S. Origin(non-EMEA equivalent SH30087)


New Zealand Newborn Bovine Serum(non-EMEA equivalent SH30401)


Donor Adult Bovine Serum, U.S. Origin(non-EMEA equivalent SH30075)

SH30527.02 100 mL -10°CSH30527.03 500 mL -10°C

New Zealand Adult Bovine Serum(non-EMEA equivalent SH30409)

SH30528.03 1000 mL -10°CSH30528.04 2000 mL -10°C

New Zealand Donor Bovine Serum(non-EMEA equivalent SH30417)


FetalClone III (non-EMEA equivalentSH30109)

SH30530.02 100 mL -10°CSH30530.03 500 mL -10°C

New Zealand Cosmic Calf (non-EMEAequivalent SH30413)

SH30625.02 500 mL -10°CSH30625.03 1000 mL -10°C

New Zealand Bovine Calf Serum IronSupplemented (non-EMEA equivalentSH30626)


Test Specification

Endotoxin(Limulus Amebocyte Lysate—Gel Clot)

See productdescriptions

Hemoglobin (Spectrophotometric)See productdescriptions

Sterility Testing(Current USP and EP Ph. Eur. 2.6.1)Bacteria & Fungi No GrowthVirus Testing (9 CFR 113.53)Fluorescent AntibodyBluetongue Not DetectedBovine Adenovirus Not DetectedBovine Parvovirus Not DetectedBovine Respiratory Syncytial Virus Not DetectedBovine Viral Diarrhea Virus Not DetectedRabies Not DetectedReovirus Not Detected

Cytopathogenic Agents—e.g. IBR Not Detected

Hemadsorbing Agents—e.g. PI3 Not DetectedMycoplasma (per EP Ph. Eur. 2.6.7)Large Volume, Direct Culture Not DetectedHoechst DNA Stain Not Detected

Bovine Diarrhea Virus Testing Post-Irradiation Serum Inhibitory Test(Interference/Comparative Titration)

No Interference

Certificate of Suitability Included


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Labtainers

Thermo Scientific HyClone LabtainerSystems have a Convenient 2D ‘pillow’ designwith ports at one end and a handle at the other,and are a space saving and an ergonomicalternative to solid-walled carboys.

BPC Chambers are constructed of ThermoScientific HyClone CX5-14 film, providing thesame contact surface as other ThermoScientific HyClone BPCs. These BPC's arefreezable to -80°C.

Standard product configurations cover a widerange of industry standard connection systems,and are available in unit volumes ranging from50 mL to 200 L.

We also offer handling systems for transportand storage.

BPCs 2D Pillow Design, End Dispense

Applications:• Collection, storage and transport of sterileliquids

• Delivery of media to small-scalebiotechnology production systems

• Formulation and filling of sterile media,buffers and other process liquids

• Chromatography feed and fraction collection• Bioreactor and fermentation feed andharvest

• Transportation and storage of bulkintermediate product

• Freezing/thawing, heat inactivation andirradiation of product

2 PortsPack of 10

Line 1 Female Luer Lock, PolypropyleneTubing:Length: 12” (30 cm)IDxOD:1⁄8”x1⁄4”(3.2mmx6.4mm)

Line 2 Male Luer Lock, PolypropyleneTubing:Length: 12” (30 cm)IDxOD:1⁄8”x1⁄4”(3.2mmx6.4mm)

Part # Size Dimensions (L xW)

SH30658.11 50 mL 4.6” x 5.4”(11.7 cm x 13.7 cm)

SH30658.12 100 mL 5.8” x 5.6”(14.7 cm x 14.2 cm)

SH30658.13 250 mL 7.5” x 5.9”(19.1 cm x 15 cm)

SH30658.14 500 mL 10.4” x 6.8”(26.4 cm x 17.3 cm)

SH30658.15 1 L 11.7” x 7.9”(29.7 cm x 20.1 cm)

SH30658.16 2 L 13.7” x 9.6”(34.8 cm x 24.4 cm)

Line 1 Female Luer Lock, PolypropyleneTubing:Length: 12” (30 cm)IDxOD:1⁄8”x1⁄4”(3.2mmx6.4mm)

Line 2 MPC Insert, PolycarbonateNo Tubing


SH30662.11 50 mL 4.6” x 5.4”(11.7 cm x 13.7 cm)

SH30662.12 100 mL 5.8” x 5.6”(14.7 cm x 14.2 cm)

SH30662.13 250 mL 7.5” x 5.9”(19.1 cm x 15 cm)

SH30662.14 500 mL 10.4” x 6.8”(26.4 cm x 17.3 cm)

SH30662.15 1 L 11.7” x 7.9”(29.7 cm x 20.1 cm)

SH30662.16 2 L 13.7” x 9.6”(34.8 cm x 24.4 cm)

Line 1 Female Luer Lock, PolypropyleneNo Tubing

Line 2 Male Luer Lock, PolypropyleneNo Tubing


SH30657.11 50 mL 4.6” x 5.4”(11.7 cm x 13.7 cm)

SH30657.12 100 mL 5.8” x 5.6”(14.7 cm x 14.2 cm)

SH30657.13 250 mL 7.5” x 5.9”(19.1 cm x 15 cm)

SH30657.14 500 mL 10.4” x 6.8”(26.4 cm x 17.3 cm)

SH30657.15 1 L 11.7” x 7.9”(29.7 cm x 20.1 cm)

SH30657.16 2 L 13.7” x 9.6”(34.8 cm x 24.4 cm)

2 Ports 2 Ports


94

BPCs, End Dispense



Line 3 Injection PortNo Tubing


SH30709.05 2 L 11.6” x 12.2”(29.5 cm x 31 cm)

SH30709.01 5 L 14.8” x 13.1”(37.6 cm x 33.3 cm)

SH30709.02 10 L 24.5” x 11.8”(62.2 cm x 30 cm)

SH30709.03 20 L 25.8” x 17”(65.5 cm x 43.2 cm)

SH30709.04 50 L 32.5” x 23”(82.6 cm x 58.4 cm)

Line 1 Male Luer Lock, PolypropyleneTubing:Length: 12” (30 cm)ID x OD: 1⁄4” x 3⁄8”(6.4 mm x 9.5 mm), C-Flex ADCF

Line 2 Female Luer Lock, PolypropyleneTubing:Length: 12” (30 cm)ID x OD: 1⁄4” x 3⁄8”(6.4 mm x 9.5 mm), C-Flex ADCF

Line 3 Female Luer Lock, PolypropyleneTubing:Length: 12” (30 cm)ID x OD: 1⁄8” x 1⁄4”(3.2 mm x 6.4 mm)

Line 1 MPC Insert, PolycarbonateTubing:Length: 24” (61 cm)ID x OD: 3⁄8” x 5⁄8”,(9.5 mm x 15.9 mm), C-Flex ADCF

Line 2 MPC Body, PolycarbonateTubing:Length: 24” (61 cm)ID x OD: 3⁄8” x 5⁄8”,(9.5 mm x 15.9 mm), C-Flex ADCF

Line 3 Female Luer Lock, Polypropylene Tubing:Length: 24” (61 cm)ID x OD: 1⁄8” x 1⁄4”,(3.2 mm x 6.4 mm), C-Flex ADCF

Line 1 MPC Insert, PolycarbonateTubing:Length: 12” (30 cm)ID x OD: 3⁄8” x 1⁄2”(9.5 mm x 12.7 mm), C-Flex ADCF

Line 2 MPC Insert, PolycarbonateTubing:Length: 12” (30 cm)ID x OD: 3⁄8” x 1⁄2”(9.5 mm x 12.7 mm), C-Flex ADCF

Line 3 PluggedNo Tubing


SH30713.05 2 L 11.6” x 12.2”(29.5 cm x 31 cm)

SH30713.01 5 L 14.8” x 13.1”(37.6 cm x 33.3 cm)

SH30713.02 10 L 24.5” x 11.8”(62.2 cm x 30 cm)

SH30713.03 20 L 25.8” x 17”(65.5 cm x 43.2 cm)

SH30713.04 50 L 32.5” x 23”(82.6 cm x 58.4 cm)


SH30712.05 2 L 11.6” x 12.2”(29.5 cm x 31 cm)

SH30712.01 5 L 14.8” x 13.1”(37.6 cm x 33.3 cm)

SH30712.02 10 L 24.5” x 11.8”(62.2 cm x 30 cm)

SH30712.03 20 L 25.8” x 17”(65.5 cm x 43.2 cm)

SH30712.04 50 L 32.5” x 23”(82.6 cm x 58.4 cm)


SH30714.05 2 L 11.6” x 12.2”(29.5 cm x 31 cm)

SH30714.01 5 L 14.8” x 13.1”(37.6 cm x 33.3 cm)

SH30714.02 10 L 24.5” x 11.8”(62.2 cm x 30 cm)

SH30714.03 20 L 25.8” x 17”(65.5 cm x 43.2 cm)

SH30714.04 50 L 32.5” x 23”(82.6 cm x 58.4 cm)

3 PortsSingle Pack

3 Ports

3 Ports 3 Ports

Line 1 MPC Insert, PolycarbonateTubing:Length: 24” (61 cm)ID x OD: 3⁄8” x 5⁄8”(9.5mm x 15.9 mm)

Line 2 MPC Body, PolycarbonateTubing:Length: 24” (61 cm)ID x OD: 3⁄8” x 5⁄8”(9.5mm x 15.9 mm)

Line 3 Female Luer Lock, PolycarbonateTubing:Length: 26” (66 cm)ID x OD: 1⁄8” x 1⁄4”(3.2mm x 6.4 mm)

Product # Size Outer Container

SH30667.01 50 L SV50076.02

SH30667.02 100 L SV50076.03

SH30667.03 200 L SV50076.04

3 Ports


95

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Protocol Table of Contentsfor Thermo Scientific HyClone Products

Page

Passage of adherent cell lines (subculture) 96

Viability Assay 97HyQTase and Trypsinization Protocol 98Hemocytometer Counting 99

Generation of Growth Curve 100

Plating Efficiency 101

Cryopreservation of Cells 102

Thawing Cryopreserved Cells 103

CDM4CHO 104

SFM4CHO 105

SFM4CHO-A 106

SFM4CHO-Utility 107

CDM4NS0 108

CDM4MAb 109

ADCF-MAb 110

SFM4MAb 111

SFM4MAb-Utility 112

LS1000 113

LS250 113

CDM4PERMAb 114

CDM4Retino 115

CDM4HEK293 116

SFM4Transfx-293 117

SFM4HEK293 118

SFM4MegaVir 119

SFX-Insect 120

SFX-Insect rAutographa 121

SFX-Insect Production 121

SFM4Insect 122

Cell Boost Process Supplement Preparation 123

HyQSphere Microcarriers-Siliconizing Glassware 124

HyQSphere Microcarriers-Cell Culture Initiation 125

HyQSphere Microcarriers-Harvesting Cells from 200 mL Microcarrier Cultures 126

HyQSphere Microcarriers-Microcarrier Sampling Technique 127

HyQSphere (HLX II-170) Microcarriers-Cell Culture Initiation 128

HyQSphere (HLX II-170) Microcarriers-Harvesting Cells Microcarrier Culture 129

HyQSphere (HLX II-170) Microcarriers-Microcarrier Sampling Technique 130

AdvanceSTEM Protocols PDF Library 131


96

General Cell Culture—Protocol

Passage of Adherent Cell Lines (Subculture)Adapted from Freshney, R.I., 1994. Culture of Animal Cells: a Manual of Basic Technique.3rd Ed. Wiley-Liss. New York. USA

OverviewRemove medium from culture container, rinsewith PBS, expose cells to Trypsin, incubate,stop Trypsin activity by adding serumcontaining medium, dissociate cells, diluteand return to incubator.

1. Pipette off spent medium and discard.

2. Add PBS (10 mL/75 cm2 flask) to the flask,being careful not to disturb the monolayer.Rinse the monolayer by gently rocking theflask back and forth. Remove the PBS anddiscard.

3. Add Trypsin (3 mL/75 cm2 flask) and rock theflask to ensure that the entire monolayer iscovered with the Trypsin solution.

4. Incubate until the cells begin to detach,usually 3–5 min. Care should be taken toavoid leaving cells exposed to the Trypsinlonger than necessary. Care should also betaken that the cells not be forced to detachprematurely, as this may result in clumping.

5. Add serum-containing medium (3–10 mL/75 cm2 flask) and pipette the cells up anddown until the cells are dispersed intoa single cell suspension.

6. Count the cells either by hemocytometeror Coulter counter and dilute to theappropriate concentration for seeding.

7. Add the appropriate volume of cellsuspension to a new flask containingmedium.

8. Place flask in incubator, loosen the cap1⁄4 turn to allow for gas exchange.

DiscussionSerum contains Trypsin inhibitors. Thus it isimportant to remove traces of serum instep two.

The serum in the medium in step five willinactivate the remaining Trypsin and preventcell damage. If dealing with a serum-freemedium, it may be necessary to use analternative Trypsin inhibitor such as soybeanTrypsin inhibitor. The incubation time for theTrypsin and the degree of force required toget the cells into single cell suspension variesbetween cell lines. It may be necessary toadjust these parameters to suit yourparticular cells.


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Viability AssayAdapted from Freshney, R.I., 1994. Culture of Animal Cells: A Manual of Basic Technique.3rd Ed. Wiley-Liss. New York. USAViability assays measure the percentage of acell suspension that is viable. This is generallyaccomplished by a dye exclusion stain, wherecells with an intact membrane are able toexclude the dye while cells without an intactmembrane take up the coloring agent.

The dye used for exclusion stain is usuallytrypan blue but erythrosin and naphthaleneblack have also been used. A dye uptake staincan be used to measure viability as well. In thiscase, the dye is normally taken up by viablecells but not by the non-viable cells. Diacetylfluorescein is an example of a dye used for dyeuptake assays. The trypan blue dye exclusionis commonly used and a protocol for thisprocedure is included here:

1. Prepare a cell suspension of the cells to beassayed (about 106 cells/mL).

2. Prepare a 1:1 dilution of the suspensionusing a 0.4% trypan blue solution.(SV30084.01)

3. Load the counting chambers of ahemocytometer with the dilution (seeHemocytometer Counting).

4. Let sit for 1–2 minutes (do no leave longeras viable cells may die and begin to take upthe dye).

5. Count the number of stained cells and totalnumber of cells following the procedure forHemocytometer Counting.

6. The calculated percentage of unstainedcells will represent the percentage ofviable cells.


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HyQTase and Trypsinization ProtocolProcedure for detaching cells from cultureware to allow subculturing and expansion.Procedure

1. Microscopically examine cells to ensure 60–90% confluency, normal cell morphology,and lack of microbial contamination.

2. Thaw cell detachment solution (eitherTrypsin SH30236.01 or HyQTase SV30030.01to be used.

3. Aseptically decant media from culturewareand wash the cell monolayer or substratetwice with sterile PBS, removing as muchfluid as possible after the final rinse.

4. Add detachment solution to culturewareusing sterile pipette and aseptic techniquesat 5–10 mL per 75 cm2 surface area. Gentlyrock the closed cultureware to completelyimmerse the cell monolayer.

5. Examine the cells for detachment every2–3 min. The time required for completedetachment may vary from 5–15 min.depending on cell line. Some cell lines mayrequire centrifugation and/or incubation at37°C for 5–10 min.

6. Neutralization:

a. If using HyQTase, no neutralization isrequired. Once all cells are detached,aseptically decant off HyQTase solution,and then simply count cells and passageas usual.

b. If using Trypsin, aseptically decant Trypsinfrom cultureware. Then add complete,serum-containing medium or soybeanTrypsin inhibitor at approximately thesame volume as the volume of Trypsinused. Pipette the culture medium orsoybean inhibitor up and down severaltimes to break up any cell clumps andwash the sides of cultureware. Ifsoybean Trypsin inhibitor is used, cellsshould be placed in a centrifuge tube andcentrifuged at 100 x g for approximatelyfive minutes. Once centrifugation isfinished, re-suspend cells in theirrespective complete culture medium.It is important to obtain a single cellsuspension at this stage.

7. Count cells and passage as usual.


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Hemocytometer CountingAdapted from Mather, J.P., and P.E. Roberts, 1998. Introduction to Cell and Tissue Culture:Theory and Technique. Plenum Press. New York and London

The hemocytometer is commonly used todetermine the concentration of cells in a cellsuspension.

Materials1. Cell suspension2. Hemocytometer with cover-slip (improved

Neubauer)3. Tally counter4. Pasteur pipettes5. Microscope

Procedure1. Place the coverslip over the hemocytometer

counting chamber and using a Pasteurpipette, place a drop of the cell suspensionat the edge of the "V" shape of the chamber.Allow the suspension to be drawn into thechamber by capillary action. Care should betaken not to overfill or underfill the chamber.Fill the opposite chamber in the samemanner.

2. Place the chamber on the microscope stage.

3. The hemocytometer consists of nine 1 mmsquares divided into smaller squares. Oneof the 1 mm squares represents a volume of0.1 mm3 or 10-4mL. Using the 10X objective,count the number of cells in a 1 mm squarearea. If there are fewer than 100 cells in asquare mm, two or more 1 mm square areasshould be counted and the results averaged.

4. Use the same procedure to count the cellson the other side of the hemocytometer.

5. To calculate the concentration of the cells,first calculate the average of all 1 mm2

areas counted and apply this formula:c=n/v where:c = cell concentration in cells/mLn = avg. number of cells/mm2 areav = volume counted = 104

Thus: c = n x 104


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Generation of Growth CurveAdapted from Mather, J.P., and P.E. Roberts, 1998. Introduction to Cell and TissueCulture: Theory and Technique. Plenum Press. New York and London.

Generation of a growth curve can be useful inevaluating the growth characteristics of a cellline. From a growth curve, the lag time,population doubling time, and saturationdensity can be determined.

Materials1. Cells2. Tissue culture dishes or flasks3. Growth medium4. Trypsin

Procedure1. Trypsinize and centrifuge the cells.

2. Resuspend the pellet in 5 mL of mediumand count the cells (see HemocytometerCounting).

3. Dilute the cell suspension in order to havean appropriate amount of medium and cellsto achieve a seeding density of 2 x 103

cells per cm2 of surface area. Cell seedingdensities vary by cell line.

4. Mix well and seed the dishes/flasks withthe appropriate amount of diluted cellsuspension.

5. Count some of the leftover cell suspensionin order to determine the actual seedingdensity.

6. Put the plates in an incubator.

7. Count the duplicate plates every 24 hours.

8. Plot the results on a log-linear scale. Thepopulation-doubling time can be determinedby identifying a cell number along theexponential phase of the curve, tracing thecurve until that number has doubled, andcalculating the time between the two.


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Plating EfficiencyAdapted from Mather, J.P., and P.E. Roberts, 1998. Introduction to Cell and Tissue Culture:Theory and Technique. Plenum Press. New York and London.A plating efficiency is a measure of the numberof colonies originating from single cells. It isa very sensitive test and is often used fordetermining the nutritional requirements ofcells, testing serum lots, measuring the effectsof growth factors, and toxicity testing.

Materials1. Cells2. Cell culture plates or flasks3. Centrifuge tubes4. Growth medium5. Trypsin6. 10% formalin7. 0.1% crystal violet

Procedure

1. Trypsinize the cells (see Passage ofAdherent Cell Lines, ) andcentrifuge the cells.

2. Resuspend in 5–10 mL or growth mediumand count the cell suspension (seeHemocytometer Counting).

3. Calculate the volume of the suspension thatwould be required to achieve concentrationsof 2, 10, and 20 cells/cm2 of surface area.

4. Plate the appropriate volume of suspensionin duplicate culture plates/flasks. It isimportant to mix the suspension prior toplating to ensure an even suspensionof cells.

5. Incubate the cells for approximately 10days. This time will vary depending on thecell line and the conditions. The coloniesshould be visible with the naked eye butshould not be joining together.

6. Wash the plates/flasks with PBS, cover thecells with 10% formalin and fix for 10 min.

7. Remove the formalin, add crystal violet tocover the cells and let sit 10 min.

8. Rinse with water until no additional colorcomes off the flask/plate.

9. Count the colonies and calculate a platingefficiency. Plating efficiency = number ofcolonies formed/number of cells platedx 100.


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Cryopreservation of Cells1. Cultures to be cryopreserved should be healthy,

free from contamination, and should bemaintained in log phase growth for severaldays before freezing.

2. Grow attaching cell culture to late log phase,trypsinize and centrifuge. If freezing suspensioncells, only centrifugation is necessary.

3. Resuspend cells in sterile serum-containingculture medium containing 10% v/vdimethylsulfoxide (DMSO). Work shouldproceed quickly to minimize the length of timethe cells are exposed to DMSO in the liquidstate. The highest purity DMSO should be usedand, preferably, it should come from a bottlethat has not been previously opened or exposedto light for long periods of time.

4. Place the appropriate volume and cell numberinto cryopreservation ampules—usually 2 x 105

to 5 x 106 cells/1 mL ampule. Plastic or glassampules may be used. However, plasticampules with external silicone seals functionbest when kept above liquid nitrogentemperature (i.e. in the vapor phase). Immersioninto the liquid nitrogen phase can result in liquidnitrogen entering the ampule and the contentsof the ampule spraying out during the defrostingprocedure. If storage in liquid nitrogen ispreferred, plastic ampules with internal O ringsperform satisfactorily. Glass ampules offer thebest results due to the secure seal and therapidity with which the ampule can bedefrosted, thereby allowing for higher cultureviability. However, they can be inconvenient touse due to the requirements of flame sealing.

5. Place the ampules in a controlled-rate freezerand cool at a rate of 1°C/minute. If acontrolled-rate freezing apparatus is notavailable, adequate results can be obtained by:

a. Placing the ampule inside a one-inch foaminsulated box and keeping the box at -70°Cfor 12 hrs.

b. Cooling the ampules in the liquid nitrogenphase using a liquid nitrogen canister insert.

c. Placing the ampules in an isopropanol baththat is subsequently cooled in a -70°Cfreezer.

d. Placing the ampules directly into a -20°Cfreezer for several hours and thentransferring to a -70°C freezer for furthercooling.

e. Placing the ampules directly into a-70°C freezer.

The last two methods (d and e) are not ideal sincethe culture viability can be affected and result inthe loss of sensitive populations. These methodsshould only be used when no other options areavailable.

6. After freezing, the ampules should betransferred to a liquid nitrogen-fill storagevessel. Prolonged storage at temperaturesabove -135°C will result in decreased viabilities.


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Thawing Cryopreserved Cells1. Defrost the vial in a 37°C water bath with

constant, moderate agitation, until ice in theampule is no longer visible.

2. Continue to warm the ampule in the waterbath for 30 seconds with gentle agitation.

3. Immediately disinfect the vial with70% ethanol.

4. Working in a hood, open the vial andtransfer the contents to a sterile 15 mL tube.Note: if thawing a glass vial, open the vialby wrapping the disinfected vial in a sterilegauze pad and breaking the neck of thepre-scored ampule.

5. Add 1.5 mL of culture medium (serumcontaining medium) that has beenprewarmed to 37°C.

6. Allow to stand for five minutes.

7. Add 3 mL of prewarmed culture medium andallow to stand for five minutes.

8. Add 6 mL of prewarmed culture medium andallow to stand for five minutes.

9. Centrifuge the suspended cells at200 x g for 10 minutes.

10. Decant the medium and gently resuspendthe cell pellet in 25 mL of culture mediumand transfer into one 75 cm2 culture flask.

11. Observe the cells microscopically toestimate cell viability and then placethem in an incubator.

12. After overnight incubation, the cells shouldbe observed so that the viability may againbe evaluated. A trypan blue dye exclusionstain may be appropriate when preciseviability assay is desired.


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Application-Specific Technical Information—Protocol

CDM4CHOProduct HandlingReconstitution of CDM4CHO DPM (SH30556)

1. While stirring, add (SH30556) to cell culturegrade water at 90% of final preparationvolume (17.2 g/L). Mix until dissolved.

2. Add 1.0 g/L Pluronic F68 and 2.2 g/Lsodium bicarbonate. Mix until dissolved.

3. Bring vessel to final volume with cellculture grade water. Allow solution to mixfor 10-20 min.

4. Check pH and osmolality. Expected values:pH.............................7.1–7.5Osmolality................280–320 mOsm/kg

5. Sterile filter into desired container usinga 0.2 micron sterile filter.

Note: SH30556 does not contain L-glutamine.Recommended concentration: 4 mM. Liquidand DPM CDM4CHO should be stored at 2-8°Caway from light.

General Culture Recommendations1. Cultures should be incubated at 37°C andin a 5% CO2 environment.

2. Maintain adapted cells by establishingmid-logarithmic growth phase sub-culturingschedule.

3. Suggested seeding density of cultures:2.0 x 105 cells/mL; Viability should be )90%.

Direct Adaptation1. Transfer cells grown in current mediumdirectly into CDM4CHO at 2.0 x 105

cells/mL.

2. When viable cell density reaches1.0–1.5 x 106 cells/mL, subculture the cells.

3. Cells should be sub-cultured every48–96 hrs.

4. If cell viability drops below 80%, proceedto sequential adaptation.

Sequential AdaptationMedium Preparation: Dilute serum-containingmedium with an equal volume of CDM4CHO.This preparation will be referred to as theSequential Adaptation Medium (SAM).Prepare twice the volume of medium neededfor the culture vessel in use (i.e., for a T-75flask using 25 mL of medium, prepare 50 mL ofSAM). Prior to each subculture, warm mediumto 37°C.

1. Subculture the cells into SAM at a seedingconcentration of 2.0 x 105 cells/mL. For bestresults, the culture should be ~70%confluent.

2. When the cells reach a density of1.0–1.5 x 106 cells/mL, sub-culture into anequal mixture of SAM and fresh CDM4CHOat a seeding density of 2.0 x 105 cells/mL.

Cryopreservation1. CDM4CHO adapted cells can becryopreserved in a medium consisting of a1:1 ratio of fresh and conditionedCDM4CHO. To this add DMSO at a finalconcentration of 7.5%.

QC Test Specification1Appearance Clear SolutionOsmolality 280–320 mOsm/kgpH 7.1–7.5Sterility No Growth

(bacteria or fungi)Endotoxin P10.0 EU/mL1

Application Growth Promotion11Refer to certificate of analysis for results.


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SFM4CHOProduct HandlingReconstitution of SFM4CHO DPM (SH30518)1. While stirring, add (SH30518) to cell culturegrade water at 90% of final preparationvolume (19.83 g/L). Mix until dissolved.

2. Add 1.0 g/L Pluronic F68 and 2.2 g/L sodiumbicarbonate. Mix until dissolved.

3. Bring vessel to final volume with cellculture grade water. Allow solution to mixfor 20 min.

4. Check pH and osmolality. Expected values:pH...........................7.0–7.4Osmolality..............280–320 mOsm/kg

5. Sterile filter into desired container using a0.2 micron sterile filter.

Note: (SH30518) does not contain L-glutamine.Recommended concentration: 4 mM. Liquidand DPM SFM4CHO should be stored at2-8°C away from light.




Direct Adaptation1. Transfer cells grown in current mediumdirectly into SFM4CHO at 2.0 x 105 cells/mL.


3. Cells should be sub-cultured every48–96 hours.


Sequential AdaptationMedium Preparation: Dilute serum-containingmedium with an equal volume of SFM4CHO.This preparation will be referred to as theSequential Adaptation Medium (SAM). Preparetwice the volume of medium needed for theculture vessel in use (i.e., for a T-75 flask using25 mL of medium, prepare 50 mL of SAM). Priorto each subculture, warmmedium to 37°C.

1. Subculture the cells into SAM at a seedingconcentration of 2.0 x 105 cells/mL. Forbest results, the culture should be ~70%confluent.

2. When the cells reach a density of1.0–1.5 x 106 cells/mL, sub-culture into anequal mixture of SAM and fresh SFM4CHOat a seeding density of 2.0 x 105 cells/mL.

Cryopreservation1. SFM4CHO adapted cells can becryopreserved in a medium consisting ofa 1:1 ratio of fresh and conditionedSFM4CHO. To this add DMSO at a finalconcentration of 7.5%.



Application Growth Promotion11 Refer to certificate of analysis for results.


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SFM4CHO-AProduct• SFM4CHO-A, Liquid with L-Glutamine—SH30821

• SFM4CHO-A, Liquid without L-Glutamine—SH30820

• SFM4CHO-A, Powder without L-Glutamine—SH30819

Features• Animal-derived component free (ADCF)formulation

•Metabolically designed for high cell yieldand recombinant protein production

• Designed for adherent culture applications• Component traceability• Manufactured under cGMP

Specifications• Protein-Free• Does not contain phenol red• Contains 4 mM L-glutamine (SH30821 only)•Without L-glutamine (SH30820 andSH30819)

QC Testing




Suggested PreparationReconstitution of SFM4CHO-A Powder(SH30819)1..While stirring, add SH30819 to cellculture grade water at 90% of finalpreparation volume (14.5 g/L). Mix untildissolved.

2. Add 1.2 g/L sodium bicarbonate. Mixuntil dissolved.

3. Add any additional supplements. Mixuntil dissolved. Note: SFM4CHO-APowder (SH30819) does not containL-glutamine.

4. Bring vessel to final volume with cellculture grade water. Allow solution to mixfor 10-20 minutes.

5. Check pH and osmolality. Expectedvalues:pH 7.5-7.9Osmolality 280-320 mOsm/kg


SFM4CHO-AMedium NotesSH30819 and SH30820 do not contain L-gluta-mine. Recommended L-glutamine supplemen-tation is 4 mM. Liquid and powderSFM4CHO-A should be stored at 2-8°C awayfrom light. Cultures maintained in SFM4CHO-Aat 37°C in a 5% CO2 environment will equili-brate to a pH of 7.1-7.3.

Culture Recommendations1. Stock cultures can be maintained serum-free in SFM4CHO-A or in Thermo Scien-tific HyClone Ham’s F-12 with 10% FBS.

2. Remove spent medium from midlogarith-mic growth phase stock culture and washstock culture with DPBS.

3. To dissociate stock culture add a mini-mum amount of Thermo Scientific HyQ-Tase enzymatic dissociation solution. (e.g.2.0-2.5 mL for a T75 flask; 1.0-1.5 mL for aT25 flask). Note: Dissociated culture canbe diluted in SFM4CHO-A for counting.

4. Count culture and reseed in freshSFM4CHO-A at a density of 10,000-20,000 cells/cm2.

5. Incubate cultures in at 37°C in a 5% CO2environment.

6. Perform a complete medium exchangefollowing cell attachment. Typically cellswill attach within 2-4 hours. It is recom-mended that medium exchange takeplace from 2-24 hours post subculturing

Cell MaintenanceMaintain stock cultures in mid-logarithmicgrowth phase. Typically stock cultures shouldbe sub-cultured every 72-96 hours or whenthey are 70-85% confluent.

Adaptation to SFM4CHO-AFollowing above Culture Recommendations,adaptation can be performed directly. Stockcultures can be maintained for extendedpassages in SFM4CHO-A.

CryopreservationSFM4CHO-A adapted cells can be cryopre-served in a medium consisting of a 1:1 ratio offresh and conditioned SFM4CHO-A followingcommon laboratory cryopreservation protocols.A recommended final DMSOconcentration is 7.5%.


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SFM4CHO-UtilityProduct HandlingReconstitution of SFM4CHO-UtilityDPM (SH30517)

Note: SFM4CHO-Utility is a two-part powder.

1. While stirring, add (SH30539) (Base Powder)to cell culture grade water at 90% of finalpreparation volume (9.8 g/L). Mix untildissolved.

2. After SH30539 has dissolved add SH30540(Main Powder) to vessel (6.0 g/L). Mix untildissolved.

3. Add 1.0 g/L Pluronice F68 and 2.2 g/Lsodium bicarbonate. Mix until dissolved.

4. Bring vessel to final volume with cell culturegrade water. Allow solution to mix for10-20 min.



Note: SH30517 does not contain L-glutamine.Recommended concentration: 6 mM. Liquidand DPM SFM4CHO-Utility should be stored at2-8°C away from light.




Direct Adaptation1. Transfer cells grown in current mediumdirectly into SFM4CHO-Utility at 2.0 x 105

cells/mL.2. When viable cell density reaches1.0–1.5 x 106 cells/mL, subculture the cells.

3. Cells should be sub-cultured every48–96 hrs.


Sequential AdaptationMedium Preparation: Dilute serum-containingmedium with an equal volume ofSFM4CHO-Utility. This preparation will bereferred to as the Sequential AdaptationMedium (SAM). Prepare twice the volume ofmedium needed for the culture vessel in use(i.e., for a T-75 flask using 25 mL of medium,prepare 50 mL of SAM). Prior to eachsubculture, warm medium to 37°C.

1. Subculture the cells into SAM at a seedingconcentration of 2.0 x 105 cells/mL. For bestresults, the culture should be ~70%confluent.

2. When the cells reach a density of1.0–1.5 x 106 cells/mL, sub-culture into anequal mixture of SAM and freshSFM4CHO-Utility at a seeding densityof 2.0 x 105 cells/mL.

Cryopreservation1. SFM4CHO-Utility adapted cells can becryopreserved in a medium consisting of a1:1 ratio of fresh and conditionedSFM4CHO-Utility. To this add DMSO at afinal concentration of 7.5%.





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CDM4NSOProduct HandlingReconstitution of CDM4NSO DPM (SH30578)1. While stirring, add 21.5 g/L of (SH30578) tocell culture grade water (room temperature)at 90% of final preparation volume.

2. Add 0.5 g/L Pluronic F68 and 3.2 g/L sodiumbicarbonate. Minimummixing time: 20 min.

3. Bring vessel to final volume with cell culturegrade water. Allow solution to mix for 10-20mins.


5. Sterile filter into desired container using a0.2 micron sterile filter.Note: CDM4NS0 does not containL-glutamine. Recommended concentration:6 mM for non-GS NS0 cells.


2. The caps on culture flasks should beloosened and adequate vessel headspaceshould be given to provide gas exchange.

3. Seeding densities should be ~3.0 x 105

cells/mL after cells are adapted. Cellsshould typically be subcultured every 3–5days, as necessary.

Direct Adaptation1. Transfer cells grown in current mediumdirectly into CDM4NS0 at5.0 x 105 cells/mL.


3. Cells should be subcultured every48–96 hrs.


Sequential Adaptation1. Transfer cells grown in current medium intoCDM4NS0 at a ratio of 1:1 using a seedingdensity of 5.0 x 105 cells/mL.

2. Incubate culture until two populationdoublings are observed. Subculture cells bymixing equal volumes of cell suspension inconditioned medium and fresh CDM4NS0(1:1 ratio).

3. Continue to subculture the cells using thismethod until the previously used mediumis reduced below 0.05% concentration andcell viability is )85%.

Note: A seeding density of 5.0 x 105 cells/mLduring adaptation increases success.

Cryopreservation1. CDM4NS0 adapted cells can becryopreserved in fresh CDM4NS0supplemented with 10.0% DMSO.





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CDM4MAbProduct HandlingGeneral Culture Recommendations

1. Cultures should be incubated at 37°C andin a 5% CO2 environment.



cells/mL. Cells should typically besubcultured every 2–4 days, as necessary.

Direct Adaptation1. Transfer cells grown in current medium

directly into CDM4MAb at2.0 x 105 cells/mL.

Note: Increasing the seeding density at theonset of adaptation can improve successratios.


3. Cells should be subcultured every 48–96 hrs.4. If cell viability drops below 80%, proceed

to sequential adaptation.

Sequential Adaptation1. Transfer cells grown in current medium into

CDM4MAb at a ratio of 1:1 using a seedingdensity of 2.0 x 105 cells/mL.

2. Incubate culture until two populationdoublings are observed. Subculture cells bymixing equal volumes of cell suspension inconditioned medium and fresh CDM4MAb(1:1 ratio).


Cryopreservation1. CDM4MAb adapted cells can be

cryopreserved in a medium consisting ofa 1:1 ratio of fresh and conditionedCDM4MAb. To this mixture, add DMSO ata final concentration of 7.5%.





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ADCF-MAbProduct Handling

General Culture Recommendations1. Cultures should be incubated at 37°C and in

a 5% CO2 environment.2. The caps on culture flasks should be

loosened and adequate vessel headspaceshould be given to provide gas exchange.

3. Seeding densities should be~1.0 x 105 cells/mL. Cells should typically besubcultured every 2–4 days, as necessary.

Direct Adaptation1. Transfer cells grown in current medium

directly into ADCF-MAb at 2.0 x 105

cells/mL.

Note: Increasing the seeding density at theonset of adaptation can improve successratios.


3. Cells should be subcultured every 48–96 hrs.4. If cell viability drops below 80%, proceed to

sequential adaptation.

Sequential Adaptation1. Transfer cells grown in current medium into

ADCF-MAb at a ratio of 1:1 using a seedingdensity of 2.0 x 105 cells/mL.

2. Incubate culture until two populationdoublings are observed. Subculture cells bymixing equal volumes of cell suspension inconditioned medium and freshADCF-MAb (1:1 ratio).

3. Continue to subculture the cells using thismethod until the previously used medium isreduced below 0.05% concentration andcell viability is )85%.

Cryopreservation

1. ADCF-MAb adapted cells can becryopreserved in a medium consisting of a1:1 ratio of fresh and conditionedADCF-MAb. To this mixture, add DMSO ata final concentration of 7.5%.





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SFM4MAbProduct HandlingReconstitution of SFM4MAb DPM (SH30535)1. While stirring, add SH30535 to cell culturegrade water at 90% of final preparationvolume (18.62 g/L). Mix until dissolved.

2. Add 2.3 g/L sodium bicarbonate. Mix untildissolved.

3. Bring vessel to final volume with cell culturegrade water. Allow solution to mix for10–20 mins.



Note: SH30535 does not contain L-glutamine.Recommended concentration: 6 mM. Liquidand DPM SFM4MAb should be stored at 2–8°Caway from light.




cells/mL. Higher densities (e.g., 5.0 x 105

cells/mL) may facilitate quicker adaptation.

Direct Adaptation1. Transfer cells grown in current mediumdirectly into SFM4MAb at 5.0 x 105

cells/mL.2. When viable cell density reaches 2–4 x 106

cells/mL, subculture the cells.3. Cells should be subcultured every 72-96 hrs.4. If cell viability drops below 80%, proceed tosequential adaptation.

Sequential Adaptation1. Transfer cells grown in current medium intoSFM4MAb at a ratio of 1:1 using a seedingdensity of 5.0 x 105 cells/mL.

2. Incubate culture until two populationdoublings are observed. Subculture cells bymixing equal volumes of cell suspension inconditioned medium and fresh SFM4MAb(1:1 ratio).


Cryopreservation1. SFM4MAb adapted cells can becryopreserved in a medium consisting of a1:1 ratio of fresh and conditionedSFM4MAb. To this add DMSO at a finalconcentration of 7.5%.


(bacteria or fungi)Endotoxin P10.0 EU/mL1Application Growth Promotion11Refer to certificate of analysis for results.


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SFM4MAb-UtilityProduct HandlingReconstitution of SFM4MAb-UtilityDPM (SH30550)1. While stirring, add (SH30550) to cell culturegrade water at 90% of final preparationvolume (19.4 g/L). Mix until dissolved.

2. Add 2.3 g/L sodium bicarbonate. Mix untildissolved.

3. Bring vessel to final volume with cell culturegrade water. Allow solution to mix for10–20 min.



Note: (SH30550) does not contain L-glutamineor Pluronic F68. Recommended L-glutamineconcentration: 6 mM. Addition of 0.1% PluronicF68 may be needed for some cultureapplications. Liquid and DPM SFM4MAb-Utilityshould be stored at 2-8°C away from light.



3. Seeding densities should be~2.5 x 105 cells/mL. Higher densities(e.g., 5.0 x 105 cells/mL) may facilitatequicker adaptation.

Direct Adaptation1. Transfer cells grown in current mediumdirectly into SFM4MAb-Utility at5.0 x 105 cells/mL.

2. When viable cell density reaches2–4 x 106 cells/mL, subculture the cells.

3. Cells should be subcultured every 72-96 hrs.4. If cell viability drops below 80%, proceed tosequential adaptation.

Sequential Adaptation1. Transfer cells grown in current medium intoSFM4MAb-Utility at a ratio of 1:1 using aseeding density of 5.0 x 105 cells/mL.

2. Incubate culture until two populationdoublings are observed. Subculture cells bymixing equal volumes of cell suspension inconditioned medium and freshSFM4MAb-Utility (1:1 ratio).


Cryopreservation1. SFM4MAb-Utility adapted cells can becryopreserved in a medium consisting of a1:1 ratio of fresh and conditionedSFM4MAb-Utility. To this add DMSO at afinal concentration of 7.5%.





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LS1000IntroductionLS1000 is a chemically defined, ADCFcholesterol supplement specifically designed tobe added as a fed-batch supplement or to sterileliquid media at the time of use. This 1000Xconcentrated supplement has been formulatedusing a proprietary complexing process forenhanced, concentrated cholesterol delivery.

LS1000 has been developed for fed-batchapplications. Due to its unique properties, itcan be added multiple times to cultures as afed-batch supplement. LS1000 can also beused to prepare complete cell culture media.After dilution into cell culture media, however,the preparation cannot be re-filtered andshould be used immediately.

While each application of LS1000 will beunique, suggested supplementation ratio is1:1000. Fed-batch additions beginning at day3–4 of cell culture, followed by subsequentadditional feeds, have been shown to boostmonoclonal antibody expression.

Features• Animal-derived component free• Contains 2.5 mg/mL synthetic cholesterol• Supports high cell density fed-batch

bioreactor cultures

QC Test Specification1

Appearance Clear SolutionOsmolality FIO1

Sterility No Growth(bacteria or fungi)

Endotoxin FIO1

1Refer to certificate of analysis for results.

IntroductionLS250 is a chemically defined, ADCF lipidsupplement specifically developed throughMetabolic Pathway Design to stimulate cellgrowth and monoclonal antibody (MAb)production in cholesterol auxotrophic NS0 celllines. This 250X concentrated lipid supplementhas been formulated using a proprietary lipidcomplexing process for enhanced stability andfilterability.

LS250 has been designed to supplement avariety of serum-free media, and may be usedin the preparation of complete liquid mediafrom dry powdered media (i.e., it may befiltered after dilution in cell culture media).

LS250 is not a fed-batch supplement. It hasbeen designed to enable hybridoma/myelomaserum-free media deficient in cholesterol tomeet the cholesterol requirements ofauxotrophic NS0 cells.

While each application of LS250 will beunique, its suggested supplementation ratio is1:250.

Features• Animal-derived component free• Developed specifically for NS0 cell cultures• Provides cholesterol and fatty acids

necessary for optimal NS0 cell culture


Appearance Clear SolutionOsmolality FIO1

Sterility No Growth(bacteria or fungi)

Endotoxin FIO1

1Refer to certificate of analysis for results.


LS250


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CDM4PERMAbQC Test Specification1Appearance Clear Yellowish SolutionOsmolality 290-340 mOsm/kgpH 7.0-7.4Sterility No GrowthEndotoxin P1.0 EU/mLApplication Growth Promotion1 Refer to certificate of analysis for results.

Product HandlingReconstitution of CDM4PERMAb DPM(SH30872)1. While stirring, add SH30872 to cell culturegrade water (20-25°C) at 90 percent of finalpreparation volume (17.3 g/L). Mix untildissolved. Medium should be a clear,golden solution at this point.

2. Add 0.5 g/L Pluronic F68 and 3.2 g/L sodiumbicarbonate. Ensure eachcomponent has completely dissolvedbefore adding the next component.

3. Bring vessel to final volume with cellculture grade water. Allow solution to mixfor 10-20 min.

4. Adjust pH to between 7.0 and 7.2 byadding 1N NaOH, or 1N HCl dropwiseto solution.

5. Check osmolality. Expected value:290-340 mOsm/kg


Note: SH30872 does not contain L-glutamine.Recommended concentration: 4 mM. Liquidand DPM CDM4PERMAb should be stored at 2-8°C away from light.

General Cell Culture MaintenanceCultures should be incubated at 37°C and ina 5 percent CO2 environment. Maintainadapted cells by establishing a passageschedule that allows the cells to be passedwhile in log growth phase. PER.C6 (PCR.C6 is atrademark of Crucell NV) cells cultivated inCDM4PERMAb for routine maintenance shouldbe subcultured every 72 to 96 hr. The passageschedule and seeding density may be adjustedto optimize performance. The recommendedpopulation seeding density of new cultures forgeneral maintenance is 0.3 x 106 cells/mL. Theculture viability of an adapted culture shouldremain greater than 90%. However, duringadaptation from serum-containing mediumviabilities may be slightly lower than 90%.

Cells should exhibit a population doublingtime of approximately 24 to 30 hr. If therecommended population seed density of0.3 x 106 cells/mL is used, cultures typicallyreach peak cell population densities ofbetween 8.0 to 12.0 x 106 cells/mL, dependingupon the specific clone used. Doubling timesduring an adaptation period may be higher.

CryopreservationCDM4PERMAb adapted cells can becryopreserved in a medium consisting of a 1:1ratio of fresh and conditioned CDM4PERMAb.To this add DMSO at a final concentration of7.5 percent.


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CDM4RetinoQC Test Specification1

Appearance Clear SolutionOsmolality 290–340 mOsm/kgpH 7.0–7.4Sterility No Growth



Product HandlingReconstitution of CDM4Retino DPM (SH30519)1. While stirring, add 13.7 g/L of SH30519 tocell culture grade water (room temperature)at 90% of final preparation volume.

2. Add 1.0 g/L Pluronic F68 and 2.0 g/L sodiumbicarbonate. Allow 20 min. to mix.


4. Check pH and osmolality. Expected values:pH 7.0–7.4 Osmolality 290–340 mOsm/kg


Note: CDM4Retino DPM does not containL-glutamine. Recommended concentration:4 mM.




cells/mL after cells are adapted. Cellsshould typically be subcultured every 3-5days, as necessary.

Direct Adaptation1. Transfer cells grown in current mediumdirectly into CDM4Retino at 5.0 x 105

cells/mL.2. When viable cell density reaches2.0-4.0 x 106 cells/mL, subculture the cells.



Sequential Adaptation1. Transfer cells grown in current mediuminto CDM4Retino at a ratio of 1:1 using aseeding density of 5.0 x 105 cells/mL.

2. Incubate culture until two populationdoublings are observed. Subculture cells bymixing equal volumes of cell suspension inconditioned medium and freshCDM4Retino (1:1 ratio).

3. Continue to subculture the cells using thismethod until the previously used medium isreduced below 0.05% concentration and cellviability is )85%.

Cryopreservation1. CDM4Retino adapted cells can becryopreserved in a 1:1 mixture of fresh andconditioned CDM4Retino supplementedwith 10.0% DMSO.


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CDM4HEK293Product HandlingReconstitution of CDM4HEK293 DPM(SH30859)1. While stirring, add SH30859 to cell culturegrade water (20-25°C) at 90 percent of finalpreparation volume (19.5 g/L). Mix untildissolved. Medium should be a clear, goldensolution at this point.

2. Add 1.0 g/L Pluronic F68 and 2.0 g/L sodiumbicarbonate. Ensure eachcomponent has completely dissolved beforeadding the next component.


4. Adjust pH to between 7.0 and 7.2 by adding1N NaOH, or 1N HCl drop-wise to solution.



Note: SH30859 does not contain L-glutamine.Recommended concentration: 4 mM. Liquidand DPM CDM4HEK293 should be stored at2-8°C away from light.

General Cell Culture MaintenanceCultures should be incubated at 37°C and in a5 percent CO2 environment. Maintain adaptedcells by establishing a passage schedule thatallows the cells to be passed while in loggrowth phase. HEK 293 cells cultivated inCDM4HEK293 should be subcultured every72 to 96 hr. The passage schedule and seedingdensity may be adjusted to optimizeperformance. The recommended cell seedingdensity of new cultures for generalmaintenance is 3.0 x 105 cells/mL. The cultureviability of an adapted culture should remaingreater than 90 percent. However, duringadaptation from serum-containing mediumviabilities may be slightly lower than90 percent.

Cells should exhibit a population doubling timeof approximately 20 to 24 hr. If therecommended seed density of3.0 x 105 cells/mL is used, cultures typicallyreach approximately 2.5 to 3.5 x 106 cells/mLafter 72 hr. and 5.0 to 6.0 x 106 cells/mL after96 hr. Doubling times during an adaptationperiod may be higher. Seed stock does notrequire centrifugation for spent mediumremoval unless the seeding volume is greaterthan 50 percent of the culture working volume.This may occur during adaptation, but shouldnot be the case during general culturemaintenance.

CryopreservationCDM4HEK293 adapted cells can becryopreserved in a medium consisting of a 1:1ratio of fresh and conditioned CDM4HEK293.To this add DMSO at a final concentration of7.5 percent.

QC Test Specification1Appearance Clear SolutionOsmolality 290-340 mOsm/kgpH 7.0-7.4Sterility No GrowthEndotoxin P1.0 EU/mLApplication Growth Promotion1 Refer to certificate of analysis for results.


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SFM4Transfx-293 ProtocolProduct HandlingReconstitution of SFM4Transfx-293 DPM(SH30861)1. While stirring, add SH30861 to cell culturegrade water (20-25°C) at 90 percent of finalpreparation volume (19.5 g/L). Mix untildissolved. Medium should be a clear, goldensolution at this point.

2. Add 1.0 g/L Pluronic F68 and 2.0 g/L sodiumbicarbonate. Ensure eachcomponent has completely dissolved beforeadding the next component.


4. Adjust pH to between 7.0 and 7.2 by adding1N NaOH, or 1N HCl drop-wise to solution.



Note: SH30861 does not contain L-glutamine.Recommended concentration: 4 mM. Liquidand DPM CDM4HEK293 should be stored at2-8°C away from light.

General Cell Culture MaintenanceCultures should be incubated at 37°C.Maintain adapted cells by establishing apassage schedule that allows the cells to bepassed while in log growth phase. HEK 293cells cultivated in SFM4Transfx-293 should besubcultured every 72 to 96 hr. The passageschedule and seeding density may be adjustedto optimize performance. The recommendedcell seeding density of new cultures for generalmaintenance is 3 x 105 cells/mL. The cultureviability of an adapted culture should remaingreater than 90 percent. However, duringadaptation from serum-containing mediumviabilities may be slightly lower than90 percent.

Cells should exhibit a population doubling timeof approximately 24 hr. If the recommendedseed density of 3.0 x 105 cells/mL is used,cultures typically reach approximately2.5 to 3.5 x 106 cells/mL after 72 hr. and5.0 to 6.0 x 106 cells/mL after 96 hr. Doublingtimes during an adaptation period may behigher. Seed stock does not requirecentrifugation for spent medium removalunless the seeding volume is greater than 50percent of the culture working volume. Thismay occur during adaptation, but should notbe the case during general culture maintenanceand in a 5 percent CO2 environment.

CryopreservationSFM4Transfx-293 adapted cells can becryopreserved in a medium consisting of a1:1 ratio of fresh and conditioned SFM4Transfx-293. To this add DMSO at a final concentrationof 7.5 percent.

QC Test Specification1Appearance Clear SolutionOsmolality 290-340 mOsm/kgpH 7.0-7.4Sterility No GrowthEndotoxin P1.0 EU/mLApplication Growth Promotion1 Refer to certificate of analysis for results.


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SFM4HEK293Product HandlingReconstitution of SFM4HEK293 DPM(SH30522)1. While stirring, add 18.4 g/L of SH30522 tocell culture grade water (room temperature)at 90% of final preparation volume.

2. Add 1.0 g/L Pluronic F68 and 2.0 g/L sodiumbicarbonate. Allow 20 min. to mix.


4. Check pH and osmolality. Expected values:pH..............................7.0–7.4Osmolality.................290–340 mOsm/kg


Note: SFM4HEK293 DPM does not containL-Glutamine. Recommended concentration:4 mM.

General Culture Recommendations1. Cultures should be incubated at 37°C and ina 5% CO2 environment.



cells/mL after cells are adapted. Cellsshould typically be subcultured every 3-5days, as necessary.

Direct Adaptation1. Transfer cells grown in current mediumdirectly into SFM4HEK293 at5.0 x 105 cells/mL.

2. When viable cell density reaches2.0-4.0 x 106 cells/mL, subculture the cells.


4. If cell viability drops below 80%, proceed tosequential adaptation.

Sequential Adaptation1. Transfer cells grown in current medium intoSFM4HEK293 at a ratio of 1:1 using aseeding density of 5.0 x 105 cells/mL.

2. Incubate culture until two populationdoublings are observed. Subculture cells bymixing equal volumes of cell suspension inconditioned medium and freshSFM4HEK293 (1:1 ratio).


Cryopreservation1. SFM4HEK293 adapted cells can becryopreserved in a 1:1 mixture of fresh andconditioned SFM4HEK293 supplementedwith 10.0% DMSO.


Appearance Clear SolutionOsmolality 290–340 mOsm/kgpH 7.0–7.4Sterility No Growth




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SFM4MegaVirProduct HandlingReconstitution of SFM4MegaVir DPM(SH30587)1. While stirring, add 16.1 g/L of SH30587 tocell culture grade water (room temperature)at 90% of final preparation volume.

2. Add 2.95 g/L sodium bicarbonate. Allow20 min. to mix.

3. Bring vessel to final volume with cellculture grade water. Allow solution to mixfor 10–20 min.



Note: SFM4MegaVir DPM does not containL-Glutamine. Recommended concentration:4 mM.

General Culture MaintenanceThe following procedure may be used formaintaining cells in SFM4MegaVir:1. Remove liquid medium from eachculture vessel.

2. Carefully rinse cell monolayer once usingMagnesium- and Calcium-free PhosphateBuffered Saline.

3. Add minimum amount of Thermo ScientificHyClone HyQTase (Cat V SV30030.01)(enough to just "wet" monolayer) to eachvessel and incubate for 3 minutes.

4. Following the incubation observe monolayerfor cell detachment. When cells arecompletely detached, you may remove theHyQTase by centrifugation for 10 min. at 100x g. Removal of HyQTase is notnecessary for subculturing.

5. Cell counting may be accomplished usingvarious methods (i.e., hemacytometer orCoulter).

6. Seed new vessels with approximately2.5 x 104 cell/cm2. Incubate at 37°C, 5%CO2 until culture reaches 80–90%confluency.

7. When initiating roller bottle cultures theroller bottle apparatus should be set to1 rpm until the cells have attached firmlyto the roller bottle surface.

Cryopreservation1. SFM4MegaVir adapted cells can becryopreserved in a 1:1 mixture of fresh andconditioned SFM4MegaVir supplementedwith 7.5% DMSO.


(bacteria or fungi)

Endotoxin P10.0 EU/mL1



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SFX-InsectProduct Handling

Reconstitution of SFX-Insect DPM (SH30350)1. While stirring, add SH30350 to cell culturegrade water at 90% of final preparationvolume (41.32 g/L). Mix until dissolved.

2. Add 0.35 g/L sodium bicarbonate and1.5 mL glycerol. Mix until dissolved.

3. Adjust pH to 6.1–6.4, if necessary.4. Bring vessel to final volume with cell culturegrade water. Allow solution to mix for10–20 min.



Note: SH30350 contains 10 mM L-glutamine.Liquid and DPM SFX-Insect should be stored at2–8°C away from light.

General Culture Recommendations1. Cultures should be incubated at 27°C andin an ambient gas environment.


3. Seeding densities should be~5.0 x 105 cells/mL. Higher densities(e.g., 10.0 x 105 cells/mL) may facilitatequicker adaptation.

Direct Adaptation1. Transfer cells grown in current mediumdirectly into SFX-Insect at 5.0 x 105 cells/mL.

2. When viable cell density reaches2–4 x 106 cells/mL, subculture the cells.

3. Cells should be subcultured every72-96 hours.


Sequential Adaptation1. Transfer cells grown in current medium intoSFX-Insect at a ratio of 1:1 using a seedingdensity of 5.0 x 105 cells/mL.

2. Incubate culture until two populationdoublings are observed. Subculture cells bymixing equal volumes of cell suspension inconditioned medium and fresh SFX-Insect(1:1 ratio).


Cryopreservation1. SFX-Insect adopted cells can becryopreserved in a medium consisting ofa 1:1 ratio of fresh and conditioned SFXInsect. To this add DMSO at a finalconcentration of 7.5%.


Appearance Clear SolutionOsmolality 350–380 mOsm/kgpH 6.1-6.4Sterility No Growth




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SFX-InsectProduction of rAutographa californica Multi-Nuclear Polyhedrosis Virus (rAcMNPV) Stock

1. Plant Sf9 cells in shakers, Fernbachs orbioreactor at 5 x 105 cells/mL and grow for3–5 days in SFX-Insect until the cell densityreaches 3–6 x 106 cells/mL with 95%viability.

2. Remove the spent medium by aspiration fromsedimented/concentrated cells and resuspendto original volume in the resident vessel.

3. Subculture the cells and plant1–2 x 106 cells/mL into appropriate vesselsusing fresh SFX-Insect. Incubate the cells inincubator for one hour for physiological andphysiochemical equilibrium. Perform cellcount which should be about 95–98%viable. Infect the cells with withMOI = 0.5 of rAcMNPV.

4. Incubate for 48 hr. and harvest sample fortitration by PFU. (Budded Virus)

5. Concentrate the virus, plaque titrate andcheck for expression vs. standard control.Stabilize the virus with glutamates or othersuitable stabilizers and quick freeze usingdry ice—ETOH method. Store the stockvirus at 80°C or liquid N2.


SFX-InsectProduction of rProtein Using BEVS1. Plant Sf9 or High Fiveq cells in shakers,Fernbachs or bioreactor at 5 x 105 cells/mLand grow for 3–5 days in SFX-Insect untilthe cell density reaches 4–8 x 106 cells/mLwith 95% viability.

2. Remove spent medium by aspiration fromsedimented/concentrated cells or hollowfiber and resuspend the cells to originalvolume in a resident vessel.

3. Subculture the cells from the residentvessel by planting 2 x 106 to 5 x 106 cells/mLinto appropriate production vessels usingfresh Thermo Scientific HyClone SFX-Insect.Incubate the cells for one hour forphysiological and physio-chemicalequilibrium. Perform viable cell count whichshould be about 95%–98% viable. Infectthe cells with desired MOI (0.5–2.5).

4. Harvest at optimum expression time.Upstream processing: Cell separation usingcross-flow filtration, HF or centrifugation.

5. Concentration of secreted rprotein(s) fromspent medium. Cell lysis/disruption toliberate intracellular rprotein(s).

6. Preparation for Chromatography (pH andconductivity adjustment, buffer exchange,filtration and process testing such asSDSPAGE, BIACOR, LAL, and Sterility).

7. Initiate downstream processing:

a) Primary & Polishing Chromatography:Buffer exchange, elution/gradient, IEC,HIC, GFC, affinity.

b) Product into final formulation bufferfollowed by dialysis, diafiltration &concentration. Testing for productrecovery, sterility.

8. Fill & Storage: Store as liquid, frozen orlyophilized. QC, QA


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Introduction

SFM4Insect™ provides superior growth with

minimal adaptation. This medium has been

successfully tested in a variety of culture

systems including traditional and disposable

bioreactors for production of recombinant

proteins using the Baculovirus Expression

Vector System (BEVS).

SFM4Insect can be found in many biotechnol-

ogy fields, including the production of human

and animal biopharmaceuticals, diagnostic

reagents and bioagricultural products. Due to

increased attention on producing biological

representative of their native form, our serum-

free media (SFM) focuses on supporting mam-

malian and invertebrate cell culture platforms.

Some of the critical cell culture applications

of these platforms include the expression of

recombinant proteins, monoclonal antibodies,

viral vectors for gene therapy and viral

vaccines. These applications constitute the

majority of biopharmaceutical research,

development and manufacturing employing

eukaryotic cell culture.

Key Features• ADCF and protein-free formulation• Metabolically designed for high cell yieldand recombinant protein production

• Complexed lipids for enhanced stability• Supports multiple insect cell lines• Supports direct adaptation• High-performance growth in multiple culturesystems including high-density bioreactors

• Component traceability• Manufactured in cGMP and ISO compliantfacility

• Available as liquid or powder• Available in bottles, BioProcess Containers(BPC), or custom packaging

Application Information1. Cultures should be incubated at 27°C and inanambient gas environment.

2. The caps on culture flasks should be loos-ened and adequate vessel headspaceshould be given ton provide gas exchange.

3. Seeding densities should be ~5.0 x 105cells/mL.

4. Higher densities (e.g., 10.0 x 105 cells/mL)may facilitate quicker adaptation.

Direct Adaptation1. Transfer cells grown in current medium di-rectly into SFM4Insect at 5.0 x 105 cells/mL.

2. When viable cell density reaches 2-4 x 106cells/mL, subculture the cells.

3. Cells should be subcultured every 72-96hours.

4. If cell viability drops below 80%, proceed tosequential adaptation.

Sequential Adaptation1. Transfer cells grown in current medium intoSFM4Insect at a ratio of 1:1 using a seedingdensity of 5.0 x 105 cells/mL.

2. Incubate culture until two population dou-blings are observed. Subculture cells by mix-ing equal volumes of cell suspension inconditioned medium and fresh SFM4Insect(1:1 ratio).

3. Continue to subculture the cells using thismethod until the previously used medium isreduced below 0.05% concentration and cellviability is >85%.

Cryopreservation1. SFM4Insect adapted cells can be cryopre-served in a medium consisting of a 1:1 ratioof fresh and conditioned SFM4Insect. To thisadd DMSO at a final concentration of 7.5%.

Specifications• Protein-free• Contains Pluronic F-68• Does not contain phenol red• Contains 10 mM L-glutamine


SFM4Insect


Appearance Clear SolutionOsmolality 355–385 mOsm/kgpH 6.1-6.4Sterility No Growth




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Cell Boost Process Supplement PreparationFed-Batch SupplementationThese formulations have been designed for usein many feed formats. Preparations and feedrates shown below are general suggestions.Optimization of feeding strategies isrecommended.

Method 1 Preparation — 3.5% Solution1. To 900 mL of cell culture grade water add 35

g of Cell Boost.2. Allow to stir for 20-30 min.3. Adjust pH from 7.0 to 7.4, if desired.4. QS to 1 L and 0.2 Rm sterile filter.

3.5% Solution Suggested Application:Start culture at a reduced volume according toanticipated feeds. Begin feeding at mid to lategrowth-phase. Continue supplementation dailyuntil final volume is reached. It is recommendedto monitor L-Glutamine separately andsupplement as needed.

Suggested Supplementation• Cell Boost 1—100-200 mL/L• Cell Boost 2—100-200 mL/L• Cell Boost 3—20-200 mL/L• Cell Boost 4—100-200 mL/L• Cell Boost 5—20-200 mL/L• Cell Boost 6—20-200 mL/L


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HyQSphere Microcarriers—Siliconizing GlasswareFor siliconizing glassware, use Sigmacotee,catalog no. SL-2, or an equivalent. According tothe manufacturer's instructions, the product isready to use without dilution. The followingprocedure is provided by Sigma:

1. Begin the procedure by cleaning allglassware to be used.

2. Apply the Sigmacote product, thoroughlycoating the surface of all the cleanglassware to be exposed to microcarriers.(For example, the inside of the glass vessel.)

3. Drain excess Sigmacote from the glasswareand allow to air dry (18–24 hrs. typically).

4. Wash and rinse the glassware again beforea final thorough rinsing with cell culturegrade water (SH30529).


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HyQSphere Microcarriers—Cell Culture InitiationThis brief pertains to HyQSphere FACT 102L, CGEN 102L, Pro-F 102L, P Plus 102L,and P 102L MicrocarriersCell Culture InitiationThis procedure is based on a 200 mL HyQSpheremicrocarrier spinner culture. Twenty (20) gramsper liter is recommended as a starting point;therefore, for a 200 mL culture, 4 g ofmicrocarriers will be used.

1. To avoid cell attachment to the glassware,treat with a silicone solution. For siliconizingglassware, use Sigmacote, catalog no.SL-2, or equivalent. Follow themanufacturer's instruction, summarizedbelow.

a. Start with clean glassware.b. Apply the Sigmacote product, thoroughlycoating the surface of all the glasswareto be exposed to microcarriers.

c. Drain excess Sigmacote from theglassware and allow to air dry(18–24 hrs. typically).

d. Wash and rinse the glassware againbefore a final thorough rinsing withDI water.

2. Suspend 4 g of microcarriers in cellculture grade water (SH30529) or PhosphateBuffered Saline (PBS), calcium andmagnesium free (SH30526) beforeautoclaving at 121°C for at least 30 min.

3. Discard the autoclave liquid, rinse ifrequired, and then equilibrate themicrocarriers by suspending in culturemedia for at least 60 min. in an incubator.

4. The cell inoculum is typically within a rangeof 1.5 to 4.0 x 105 per mL. For the 200 mLculture using a seeding density of2.0 x 105 cells per mL, 4.0 x 107 cellsare required.

5. Add the cells to the warmmicrocarrier-mediasuspension. Add warmed media to bringvolume to 200 mL.

a. For cells with high plating efficiency,determine which one of several stir ratesmay be optimal to get even distribution,depending on the cell type.

• Stir the spinner flask at 45–60 rpmthroughout the incubation period (whenusing MDCK, Vero, primary CEFs, or otherrobust continuous cell lines).

b. For cells with low plating efficiency orcultures using animal protein-free media,initiate the culture incubation periodusing an intermittent stir cycle, stirring asslowly as possible while keeping themicrocarriers in suspension.

• For example, one minute stirring at45–60 rpm, then off for 7–10 min. Avoidoxygen deprivation of the cells. After thecells attach, bring to full volume withwarm media.

6. Maintain the cells as required by theirgrowth characteristics. Media exchangemay be required.


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HyQSphere Microcarriers—Harvesting Cells from 200 mL Microcarrier CulturesThis brief pertains to HyQSphere FACT 102L, CGEN 102L, Pro-F 102L, P Plus 102L,and P 102L Microcarriers1. Transfer the 200 mL culture from the

incubator to a biological safety hood,allowing the microcarriers to settle. Avoidprolonged oxygen deprivation to the cells.

2. Pipette the media from the settledmicrocarrier pack.

3. Thoroughly rinse the cell-laden microcarriersby adding 50–100 mL of Thermo ScientificHyClone Dulbecco's Phosphate BufferedSaline (DPBS), calcium and magnesium free(SH30028) to the vessel, taking care not todouse the microcarriers, thereby dislodgingthe cells.

a. Stir the culture at 40 rpm, roomtemperature, for about 15 min.

4. After removing the second rinse of DPBS,add 25 mL (perhaps less) of ThermoScientific HyClone Trypsin (SH30236) orThermo Scientific HyQTasenonmammalian cell dissociation reagent(SV30030.01) allowing the culture to stir at40 rpm for 15 min. (Time may vary dependingon the actual temperature.)

Note: To harvest a clean, single, viable cellsuspension, use the lowest concentration ofTrypsin or HyQTase possible: titrating theTrypsin or HyQTase in well plates within arange of 0.05 to 0.25%.

Note: Titrate the Trypsin or HyQTase at37°C, room temperature, and at a lower tem-perature (e.g., 10°C) to determine the optimaltemperature for harvesting a viable, single cellsuspension. The lowest temperature may beoptimal for the cell viability.

5. Make certain the cell type will tolerate thePBS used for rinsing. We recommend tissueculture DPBS.

6. After the cells are off the microcarriers,pipette the microcarrier-cell suspensiongently up and down to dislodge the cellsinto a single cell suspension. (A 24–50 mLpipette is convenient for this. An alternatemethod for larger culture volumes: circulatethe microcarrier slurry through a peristalticpumped loop of sterile tubing.)


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HyQSphere Microcarriers—Microcarrier Sampling Technique(Instructions for taking representative samples from a stir vessel)This brief pertains to HyQSphere FACT 102L, CGEN 102L, Pro-F 102L, P Plus 102L,and P 102L Microcarriers1. Transfer the culture vessel from the

incubator to the biological safety hood,placing the vessel on a stir plate set at60 rpm.

2. With the culture in the stir mode, removethe cap from one side-arm of the vessel.Lower the extended pipette tip of theThermo Scientific Finnpipette assembly(or equivalent) mid-point into the cultureand free of the impeller assembly whiledepressing the sample button, then releasethe sample button, allowing the culturesample to quickly fill the pipette. Transferthe sample to a 15 mL conical tube.

Note: The bead pack is part of thesample volume removed from the culture.

3. Centrifuge the sample for 5–10 min. at 900rpm. For precision and volume reference,transfer the media taken from the sampleto a graduated cylinder documenting thevolume. (For example, 9.5 mL of media wastaken from the 10 mL sample, leaving0.5 mL of microcarriers.) Remove liquid.

4. Rinse the microcarrier pack twice byadding 5 mL of Thermo Scientific HyCloneDulbecco's Phosphate Buffered Saline(DPBS) (SH30028) to the tube, swirlingseveral times before removing thesupernatant. Centrifuge for 5-10 min.between rinses to form a microcarrierpack before removing the liquid.

Note: For this step, conducted at roomtemperature, volume accuracy isunimportant.

5. To remove the cells from the microcarriers,add the correct known volume (seereference volume step 3) of Trypsin orThermo Scientific HyQTasenonmammalian cell dissociation reagent(0.05% to 0.25%) to the tube containing therinsed cell-microcarriers and allow to set for10 min. or so at the desired temperature.Using the Finnpipette, gently pull thesample into the pipette and dispense,repeating the in-out motion several times,thereby creating a single cell suspension.

6. The sample is now ready to count orprocess further. As an alternative, cellnuclei counts can be done as well.

Note: As noted in the literature, microcarrieraggregates tend to skew cell counts, makingsamples difficult to measure.


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HyQSphere (HLX II-170) Microcarriers Cell Culture InitiationThis procedure is based on a 200 mL ThermoScientific HyQSphere HLX II-170 microcarrierspinner culture. Twenty (20) grams per liter isrecommended as a starting point; therefore, fora 200 mL culture, 4 g of microcarriers will beused.

1. To avoid cell attachment to the glassware,treat with a silicone solution. For siliconizingglassware, we recommend the use ofSigmacote, catalog no. SL-2, availablefrom Sigma Chemical Co. Follow themanufacturer's instructions, summarizedbelow.

a. Start with clean glassware.b. Apply the Sigmacote product, thoroughlycoating the surface of all the glasswareto be exposed to microcarriers.

c. Drain excess Sigmacote from theglassware and allow to air-dry (18–24hours typically).

d. Wash and rinse the glassware againbefore a final thorough rinsing withDI water.

2. Suspend 4 g of HLX II-170 microcarriers inThermo Scientific HyClone Cell Culture GradeWater (SH30259) or Thermo ScientificHyClone Phosphate Buffered Saline (PBS),magnesium and calcium free (SH30526),before autoclaving at 121°C for at least30 min.

3. Discard the autoclave liquid, rinse ifrequired, and then equilibrate themicrocarriers by suspending in culturemedia for at least 60 min. in an incubator.

Note: HLX II-170 microcarriers absorb PhenolRed contained in media, therefore mediawithout Phenol Red is recommended for mediaprofusion or long-term culture periods.

4. The cell inoculum is typically within a rangeof 1.5 to 4.0 x 105 per mL. For the 200 mLculture using a seeding density of2.0 x 105 cells per mL, 4.0 x 107 cellsare required.

5. Add the cells to the warmmicrocarrier-media suspension. Addwarmed media to bring volume to 200 mL.

a. For cells with high plating efficiency,determine which one of several stir ratesmay be optimal to get even distributiondepending on the cell type. Stir the spinnerflask at 45–60 rpm throughout theincubation period (when using MDCK,Vero, primary CEFs and other robustcontinuous cell lines).

b. For cells with low plating efficiency orcultures using animal protein-free media,initiate the culture incubation periodusing an intermittent stir cycle, stirring asslowly as possible while keeping themicrocarriers in suspension. For example,one minute stirring at 45–60 rpm, then offfor 7–10 min. Avoid oxygendeprivation of the cells. After the cellsattach, bring to full volume withwarmed media.

6. Maintain the cells as required by theirgrowth characteristics. Media exchangemay be required.


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HyQSphere (HLX II-170) Microcarriers Harvesting Cellsfrom 200 mL Microcarrier Cultures1. Transfer the 200 mL culture from theincubator to a biological safety hood,allowing the microcarriers to settle.Because HLX II-170 microcarriers have arelatively high specific gravity (1.10) theytend to settle quickly. Avoid prolongedoxygen deprivation to the cells.

2. Remove the media from the settledmicrocarrier pack by carefully pouring themedia through a side-arm of the vessel.With care, few microcarriers are discardedwith the media, because of the higherspecific gravity of the HLX II-170microcarriers.

3. Thoroughly rinse the cell-ladenmicrocarriers by adding 50–100mL of DPBS(Thermo Scientific HyClone Dulbecco'sPhosphate Buffered Saline Solution, Ca-Mgfree (SH30028.03) to the vessel, taking carenot to douse the microcarriers and therebydislodging cells. Stir the culture at 40 rpm,room temperature, for about 15 min.

4. Repeat the rinse cycle.

5. After removing the second rinse of DPBS,add 25 mL (perhaps less) of Thermo ScientificHyClone Trypsin (SH30236.01) or HyQTasenonmammalian cell dissociation reagent(SV30030.01) allowing the culture to stir at 40rpm for 15 min. Time may varydepending on the actual temperature.a. To harvest a clean, single, viable cellsuspension, use the lowest concentrationof Trypsin possible: by first titrating theTrypsin or HyQTase in well plates withina range of 0.05 to 0.25%.b. Titrate the Trypsin at 37°C, roomtemperature and at a lower temperature(e.g., 10°C) to determine the optimaltemperature for harvesting a viable,single cell suspension. The lowesttemperature may be optimal for thecell viability.c. Make certain the cell type will toleratethe PBS used for rinsing. We recommendtissue culture DPBS.

6. After the cells are off the microcarriers,pipette the microcarrier-cell suspensiongently up and down to dislodge the cellsinto a single cell suspension. A 24–50 mLpipette is convenient for this. (As analternate method for larger volumes,circulate the microcarrier slurry through aperistaltic pumped loop of sterile tubing.)


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HyQSphere (HLX II-170) Microcarriers MicrocarrierSampling TechniqueFor taking representative samples from anHLX II- 170 stir vessel:1. Transfer the culture vessel from the

incubator to the biological safety hood,placing the vessel on a stir plate set at60 rpm.

2. With the culture in the stir mode, removethe cap from one side-arm of the vessel.Lower the extended pipette tip of theFinnpipettee assembly (or equivalent)midpoint into the culture and free of theimpeller assembly while depressing thesample button, then release the samplebutton, allowing the culture sample toquickly fill the pipette. Transfer the sampleto a 15 mL conical tube.

Note: the bead pack is part of the samplevolume removed from the culture.

3. Allow the microcarriers to settle in thesample tube before removing the mediafrom the microcarrier pack using theFinnpipette. For precision and volumereference, transfer the media taken from thesample to a graduated cylinder documentingthe volume. (For example, 9.5 mL of mediawas taken from the 10 mL sample leaving0.5 mL of microcarriers.)

4. Rinse the microcarrier pack twice by adding5 mL of DPBS (SH30028) to the tube, swirlingseveral times before removing the super-natant. For this step, conducted at room tem-perature, volume accuracy isunimportant.

5. To remove the cells from the microcarriers,add the correct known volume (seereference volume step 3) of Trypsin (0.05%to 0.25%) to the tube containing the rinsedcell microcarriers and allow to set for 10min. or so at the desired temperature. Usingthe Finnpipette, gently pull the sample intothe pipette and dispense, repeating thein-out motion several times thereby creatinga single cell suspension.

6. The sample is now ready to count orprocess further. As an alternative, cellnuclei counts can be done as well.

Note: As noted in the literature, microcarrieraggregates tend to skew cell counts, makingsamples difficult to measure.


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AdvanceSTEM Protocol PDF LibraryFor complete protocol library, go to www.thermoscientific.com/certificatesearch


132

Chinese Hamster Ovary Cells for the Production of Recombinant Glycoproteins 133

Lipids in Cell Culture Media 135

Adherent Growth of CHO cells in SFM4CHO-A medium using Corning CellBIND Surface Culture Flasks 138

Metabolically Designed Serum-Free Media For HEK293 and PER. C6 Cell Lines 139

Production of Poliovirus Using Vero Cells Cultured in Thermo Scientific HyClone SFM4MegaVir 142

A Novel Process for rProtein Production Using BEVS 144

Growth comparison studies between FBS and other serum products 148

Cell Lines Grown Successfully in Thermo Scientific HyClone FetalClone I, II, and III 153

Heat Inactivation— Are You Wasting Your Time? 154

Viral Safety in Serum for Cell Culture Use 158

Thermo Scientific HyClone Single-Use Bioreactor (S.U.B.) for Vero Cell Culture on Microcarriers 165

Technical Information Table of Contents

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Chinese Hamster Ovary (CHO) cells are avaluable tool for producing recombinant proteinswith "native" mammalian glycosylation patterns.Following their introduction in the 1950s, CHOcell lines have become a workhorse formanufacturing recombinant proteins. Geneticengineers can modify CHO cells to produceproteins with "mammalian" post-translationalmodifications, and through gene amplificationcan selectively produce high levels ofrecombinant proteins.

Process development, using CHO cell lines,focuses on achieving the maximum amount ofactive product. Optimization of the amount ofactive product can be achieved in at least twobasic ways. The first way is to increase thespecific productivity (i.e., the product per cell)through cell line development. Cell linedevelopment may include both sub-cloning thecell line to select higher producing clones anduse of gene amplification. Drug resistance hasbeen the tool of choice in the biopharmaceuticalindustry to induce gene amplification.

By combining the gene of interest with aselectable gene, increased production levelscan be accomplished. Using productivity-per-cellapproaches, scientists have increasedexpression levels more than 1,000-fold(Schimke, 1982). Two gene amplificationsystems used in CHO cells are the dihydrofolatereductase (DHFR) system using methotrexate(MTX) resistance, and the glutamine synthetase(GS) system using methionine sulfoxamine(MSX) resistance. The DHFR enzyme catalyzesthe conversion of folate to tetrahydrofolate(Figure 1).

This precursor is necessary for the de novosynthesis of purines, pyrimidines, and glycine(Goeddel, 1990). Methotrexate is a drug which

is similar (i.e., an analog) to folate. MTX bindsto DHFR, thereby inhibiting the production oftetrahydrofolate. With insufficient levels ofDHFR, cells are deprived of nucleosideprecursors (hypoxanthine and thymidine) anddie. Gene amplification is a technique used bymolecular biologists. They transform cells withrecombinant DNA consisting of the gene ofinterest closely linked to the gene for DHFR.Then during gene amplification the cells arecultured in increasingly higher levels of MTX.Those CHO cells that have increased copies ofthe DHFR gene, and therefore higher levels ofthe enzyme, are selected.

The GS/MSX system is similar to theDHFR/MTX system. The GS enzyme catalyzesthe production of glutamine from glutamate andammonia (Figure 2). Methionine sulfoxaminebinds to the GS enzyme and prevents theproduction of glutamine. Gene amplificationoccurs when cells are subjected to increasingconcentrations of MSX.

A second way to achieve higher recombinantprotein yields is to increase the cell yield (i.e.,cells per volume) of the process. This may beaccomplished through process development(e.g., batch, fed-batch, perfusion, etc.) andmedium development. By increasing the cellsper volume per day, higher levels of productmay be produced.

To demonstrate the utility of this proteinproduction system, we used CHO cell line(ATCC® CRL-10154 CHO 5/9 M! 3-18), whichproduces human macrophage colony stimulatingfactor (hm-CSF). This cell line was producedusing a DHFR-CHO DG44 cell clone as theparental line. Gene amplification wasperformed using the DHFR/MTX system.The cell growth and protein production kineticsof this cell line were studied using a powdered

medium developed for CHO cells bya Thermo Fisher Scientific Researchand Development team. Standardculturing procedures for CHO cellswere used for these evaluations(Table 1).

Initial evaluations compared

commercially available serum-free and protein-free media to Themo Scientific HyClonePF-CHOMPS (SH30333) using the CHO 5/9 M!3-18 cell line. Representative results obtainedin these comparisons are shown in Table 2and Figure 3.

Table 1: Evaluation Parameters for usewith Shaker and Spinner CulturesTemperature 37°CCO2 Atmosphere 5%Agitation Shaker 100–130 rpm

Spinner 60–80 rpmOxygen AtmosphericBuffering 2 g/L Sodium

Bicarbonate

Table 2: Comparison of SH30333 vsCompetitor MediumParameter SH30333Maximum CellDensity (Mean)

212.1% Higher

MaximumPopulation Doubling Time(Mean)

19.6% Higher

Maximum ProteinProduction (Mean)

74.6% Higher

Application-Specific Technical Information—Application Notes

Chinese Hamster Ovary Cells for the Productionof Recombinant GlycoproteinsCamire, Joseph. Art to Science, Vol. 19, No. 1; Logan, UT, 2000

Analysis of growth promotion and hm-CSFproductivity kinetics showed a maximumspecific productivity (Figure 4) of1.26 pg/cell/day. These data indicate thatincreased protein expression with this cellclone is cell-growth associated; that is, themaximum specific productivity is seen duringlog-phase growth. Additional studies analyzedextended cell performance (Figure 5).In the case of this clone, cells were culturedfor more than 70 days with approximately 68cumulative population doublings (CPD). TheCPD was linear over the course of the studyindicating good stability of the cell line in thismedium. Following these evaluations, cultureswere scaled up to increase the total proteinyield by increasing the cell population density.

PF-CHOMPS medium is a two-part powder,protein-free medium designed for suspensiongrowth of CHO cell lines. The medium wasdesigned to be "regulatory friendly" by limitinganimal-derived components to just two, i.e.,cod liver oil and cholesterol. This productcomes in a range of standard packagingconfigurations (Table 3).


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Some typical components that maybe added at the time of liquidpreparation are: sodiumbicarbonate, L-glutamine (forcell clones which require thiscomponent for growth), andPluronic F68. L-glutaminesupplementation is recommendedfor DHFR systems.

We anticipate that users will evaluate themedium prior to scale-up procedures. Werecommend using cells that have beenpreviously adapted to the medium to eliminatecarryover variables from prior sera or media.

Through customer collaborations, we havesuccessfully optimized PF-CHOMPS tospecific CHO clones. Contact your salesrepresentative to inquire about availabilityof these services.

We anticipate that users will evaluate themedium prior to scale-up procedures. Werecommend using cells that have beenpreviously adapted to the medium.

References1. Schimke, R.T. (ed.). 1982. Gene AmplificationColdspring Harbor Laboratory, Cold SpringHarbor, N.Y.

2. Goeddel, D.V. (ed.). 1990. Methods inEnzymology, Vol. 185 pp. 543-551.

The medium is designed to be used with awide range of CHO cell lines and productionprocesses, from straight batch to fed-batch tocontinuous perfusion cultures. The formulation,which is deficient in nucleic acids andprecursors (i.e., hypoxanthine and thymidine), isdesigned for use with the dihydrofolatereductase (DHFR) selection system andmethotrexate (MTX) induced gene amplification.The medium is also deficient in L-glutaminefor use with the glutamine synthetase (GS)selection system and the methioninesulfoxamine (MSX) gene amplification system.

PF-CHOMPS can be purchased in lot sizes aslarge as 400,000 L. This reduces QA/QC cost;by reducing the number of lots needing tests.

Table 3. Standard PackagingConfigurationCatalog Number Package SizeSH30333.01 5 LSH30333.02 10 LSH30333.03 50 LSH30333.04 100 LSH30333.05 1,000 L

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Lipids in Cell Culture MediaWilliam Whitford and John ManwaringIntroductionRequirements for better performance, a desireto avoid serum, and improved understandingof culture systems are inspiring fresh interestin supplementing cell-culture media withidentified lipids.

Surprisingly, there is no universally accepteddefinition of "lipid." Originally, scientistsconsidered all naturally occurring compoundsthat are soluble in non-polar solvents such asbenzene, to be lipids. For cell culturists, a morepractical definition is "water-insolublebiomolecules, biosynthetically or functionallyrelated to fatty acids and their derivatives."This definition includes fatty acids, sterols,triacylglycerols, glycerophospholipids, andsphingolipids, but excludes steroid hormones,fat-soluble vitamins, and petroleum products.There is logic to both definitions, and eitherwill suffice in this article. By any definition, cellculturists have a special concern about lipids:Their limited solubility in media makes addingsufficient quantities a challenge.

Lipids function in three roles. They serve asenergy stores, as structural components ofcellular membranes, and in transport andsignal systems. Cell and organelle membranescontain much of a cell's lipids. Major categoriesof biological lipids include glycerophospholipids,e.g., phosphatidyl choline; sterols, e.g.,cholesterol in animals and phytosterols inplants; sphingolipids, e.g., ceramides; andvarious lipoprotein complexes. Structuralfunctions of many lipids, such as therequirement of cholesterol for animal cellmembrane fluidity, have been extensivelydescribed. Many signaling systems rely uponlipid-containing complexes, such as the familiarABO blood type determinant from the lacto-and neolacto-series of glycosphingolipids.

Cellular RequirementsIn any living system an "essential nutrient" isa compound that the organism requires forgrowth and reproduction, and which theorganism cannot produce. Cells can synthesizemost of the dozens of lipids they require forprimary cellular functions. In mammals, though,two fatty acids (linoleic and -linolenic acid) havebeen proven to be essential.

For decades scientists have reported on the lipidrequirements of particular cell lines as culturedin an excess of lipids, usually from FBS. Withthe more widespread use of serum-free andchemically defined (CD) media, researchers arenow looking at the requirements of cellsmaintained in minimal complements ofidentified lipids. Also, a distinction is emergingbetween required media components andoptimal components and levels. Apparently,much of the work performed with cells adaptedto media with high lipid levels provided aninaccurate picture of the actual requirement ofcells in culture. Many known serum-free media(SFM) formulations lack one or both of linoleicand -linolenic acid, yet sustain indefinite cellgrowth and full function. It appears that these"essential" fatty acids are not essential in mostanimal cell culture (Grammatikos et al. 1994).

Supplementation of cell culture systems withparticular non-essential fatty acids,phospholipids, and sterols significantlyimproves performance. Providing cells withappropriate preformed lipids, even when notessential, reduces the need for their biosynthesisby the cell. The result is more efficientmetabolism. This is especially evident wherethe rate of cell division is important, or wherethe cell produces high levels of transgenicproduct (Manwaring, et al., in press). Somecultured cells are truly auxotrophs for particularlipids, meaning that these lipids are "essential"to them. For example NS0, a common myelomacell line, requires large amounts of cholesterolbut is incapable of producing it. In this case,the phenotype is caused by NS0's loss of anenzyme in the cholesterol synthesis pathway.

Media SupplementationPeople have been devising heuristic formulasfor working with lipid dispersions for millennia.Churning butter and adding an egg to a failingrecipe are examples of applying simpletechniques without necessarily understandingthe chemistry behind them. At the other extreme,modern industries such as pharmaceuticals, foodprocessing, agrochemicals, and cosmetics oftenapproachoil andwater dispersion issueswithsophisticated technologies. Interfacial andcolloidal chemistry, hydrocarbonchainpacking,and lyotropic and thermotropic mesophase

behavior have all been brought to bear againstlipid dispersion issues. (Larsson, 1994).

Whatever approach or combination is used, thegoal of cell-culture media supplementation isto disperse select lipids such that they arenontoxic, are taken up by the cells in a controlledfashion, can be micro-filtered, and remain stableupon storage at 5 to 50°C for up to a year.

These requirements can be met in three ways:1. Adsorb the lipid to a soluble carrier molecule,2. Devise a formula that drives lipid self-

assembly to the required particle size, and3. Disperse and stabilize a lipid mixture to a

particle of sufficient transient sizeand stability.

Each of these approaches is a science in itsown right, and all have been used indeveloping cell culture media (Table 1).

Adsorption to a CarrierAnimal serum, the original vehicle for providinglipids to cells in culture, uses proteins ascarriers of every lipid required by mammaliansystems. FBS, the most common serum in cellculture, contains very high levels of lipids.For example, FBS contains approximately300 Rg/mL cholesterol and 30 Rg/mL oleic acid.The major lipid carrier proteins in sera include:(1) Albumin, a globular protein with manydistinct hydrophobic moieties and whichrepresents over 50% of the protein in serum, and(2) Four classes of apoprotein-containinglipoproteins: chylomicrons, carrying tryacylglycerides; very low-density lipoproteins(VLDL), carrying fatty acids; and low- andhigh-density lipoproteins (LDL and HDL) thatare principally involved in cholesterol transport.The high level and diversity of lipids in sera issometimes detrimental. For example, cellscultured in serum are constantly exposed to anumber of steroids, making it difficult todetermine the specific effects of each.

Serum extracts and lipid-rich fractions arepopular products for adding high concentrationsof serum lipids to media. Commercially availablefractions of animal serum contain a number ofserum lipids, including cholesterol, fatty acids,and phopholipids bound to select serumproteins. While the ratio of lipids remains fixed,


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LiposomesSpheres of lipids in the lamellar phase with anaqueous core are variously referred to as lipidbilayer vesicles or liposomes. All nutrient lipidscarried by liposomes must be components ofthe lipid lamellae. It is sometimes possible tocombine lamellae-forming polar lipids withnutrient lipids such that the phase behavior andintrinsic curvature of the mixture generates li-posomes of suitable size and stability. Forexample, cholesterol or fatty acids canintercalate within the acyl chains of polarlipids, such as phosphatidyl choline. Thetemperature, pH, and tonicity of cell culturemedia usually remains within a narrow range.This allows the use of such principles as sterichindrance, hydrogen bonding, and electrostaticcharge to be exploited in developing stablepreparations. Although mixtures of polar andnutrient lipids so formulated are conceivable,published attempts mostly report failure.

Emulsions andMicroemulsionsThese two related forms of filterable dispersionscan have a term of physical stability comparableto that of the media itself.

Emulsions are kinetically (non-equilibrium)stable dispersions. They are produced by firstreducing particle size through introduction ofhydrodynamic force, and then stabilizing thesurface of the particles. Stabilization isaccomplished by the addition of suchamphophiles as polar lipids and certainpeptides or polymers, or by modulating theparticle surface charge.

Microemulsions may be formed by usingdetergents to generate mixed micellescontaining the lipid of interest at their core.The key to developing such microscale andthermodynamically stable dispersions is to finda surfactant that is neither too toxic nor toodisruptive to cell membranes. The surfactantand lipids must form filterable structures ofacceptable size and stability at normaltemperatures. Concepts such as the critical

micelle concentration (CMC) and hydrophilic/lipophilic balance (HLB) are useful here.

A number of SFM in current use have their lipidcomponents dispersed by these means. Ineither case, the amphiphile surrounds andstabilizes a particle of lipid, presenting itshydrophilic region to the aqueous media.

MaterialsNutrient lipids are available from a variety ofsources and vendors. Enriched lipid fractionsextracted from such diverse starting materialsas animal serum, sheep's wool, fish oil, andsoybeans are commercially available. Individuallipids purified from these naturally occurring richsources are also available. Chemical synthesisand derivitization is used to produce those lipidsthat are either rarely found in nature, or that areabundant only in unacceptable sources. Forexample, in formulating animal component freemedia, cholesterol, commonly obtained fromsheep's wool, must instead be synthesized fromnon-animal origins.

Tween® 80, a non-ionic detergent; pluronicacid, a block co-polymer; phosphatidyl choline(or lecithin); and protein hydrolysates, such asthose from beef or soy, have proven to be themost popular materials for dispersing non-polarlipids in serum and protein-free (PF) cell culturemedia. Lipid particle surface and interfacialenergies must be overcome in both thegeneration of metastable emulsions and inaccelerating the equilibrium of microemulsions.This is accomplished in instruments capable ofgenerating extreme hydrodynamic force whileminimizing heat production (e.g., the EmulsiFlex-C50, Avestin, Ottawa, ON). The addition ofchemical antioxidants, such as -lipoic acid and-tocopherol, can reduce the peroxidation ofpolyunsaturates in the formulation. Proceduresthat limit the introduction of free oxygen alsohelp in this regard. Interestingly, many lipidsseem to be exquisitely protected from oxidationwhen complexed with cyclodextrin(Kim, et al., 2000).

these IgG-free and albumin-reducedsupplements allow variation of the total lipidconcentration, and combination with othersupplements. They are of significant value insome serum-dependent systems by, forexample, reducing material costs and improvingperformance. Notably, they work well with someSFM, but not at all with others.

Bovine serum albumin (BSA) is a commonvehicle for lipids. It is commercially available ina variety of purities and formats. All basicpreparations contain high levels of serum lipids,especially fatty acids and lecithin. It is possibleto create or purchase a reduced lipid BSA. Sucha preparation has the capacity to adsorb asignificant amount of added lipids of choice.

Cyclodextrins, naturally occurring circularpolymers of glucopyranose, increase thesolubility of lipids. Their function is similar toprotein adsorptive systems, in that they encaseor chelate lipids in a more water-solublemolecule and thereby increase the lipids'aqueous solubility. The addition of various sidegroups to the cyclodextrin molecule increasesthe cyclodextrin's solubility in water to nearly50%w/v, while leaving the hydrophobic,lipid-active central cavity intact. Two newerproducts, Thermo Scientific HyClone LS1000 andThermo Scientific HyClone LS250,successfully utilize cyclodextrin to solubilizecholesterol and fatty acids for supplementationof cell culture media. LS1000 is designed to beadded at culture initiation, or as a component infed batch cultures (Figure 1). LS250 is optimizedfor media supplementation at culture initiation.Dilutions in media may be filtered and are stablein appropriate storage for months.

Lipid DispersionsLiposomes, emulsions, and microemulsionsare forms of lipid dispersions used in mediasupplementation.

Table 1. Various lipid dispersion technologies; their potential and limitations.

PhysicalStability

ADCFPotential

PFPotential

CDPotential

FormulationAdjustable

Serum High No No No MinimallySerum Extracts High No No No NoAlbumins High rAlbumin No Nearly SomewhatEmulsions Low Yes Yes Yes SignificantMicelles High Yes Yes Yes SomewhatLiposomes Med Yes Yes Yes SomewhatCyclodextrin High Yes Yes Yes YesFFiigguurree 11.. CCyyccllooddeexxttrriinn--bbaasseedd ccuullttuurree ffeeeeddiinngg..

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ApplicationsLipids currently referred to in the literature forsupplementation include cholesterol; cod liveroil; soybean oil; and oleic, linoleic, and palmiticacid. Applications for lipid-based mediasupplementation include essential andperformance-enhancing lipids in most CDformulations, special requirements such as theneed for high levels of cholesterol in NS0based transgenic producers, and fed-batchprocedures in bioreactors (Mahadevan, 2003).

ConclusionSystems for delivering lipids have evolved asthe culture requirements of research andindustry changed. Earlier, supplementationwith animal sera provided an effective answer.As concerns about animal-derived productsincreases, media producers move furthertowards serum-free, animal-derived componentfree, and CD technologies. The elimination ofserum, with its complement of natural lipidcarriers, initially posed significant problems tomedia supplementation. Fortunately, these arebeing solved through the approaches andtechniques outlined in this paper.

References1. Grammatikos SI, Subbaiah PV, Victor TA &Miller WM. 1994. Diverse effects ofessential (n-6 and n-3) fatty acids oncultured cells. Cytotechnology 15:31–50.

2. Kim SJ, Park GB, Kang CB, Park SD, JungMY, Kim JO, Ha YL. 2000. Improvement ofoxidative stability of conjugated linoleic acid(CLA) by microencapsulation in cyclodex-trins. J. Agric. Food Chem. 48:3922-3929.

3. Larsson K. 1994. Lipids—Molecularorganization, physical functions andtechnical applications. The Oily Press Ltd;Dundee, Scotland.

4. Mahadevan, MD. 2003. Bioreactor Processselection for large-scale manufacturing ofmonoclonal antibodies—tradeoffs andrecommendations.Bioprocessing Journal2:3, 25-31.

5. Manwaring J, Barnett B, Pence B, and Whit-ford W. NS0 and derivatives: MAb produc-tion in large-scale SFM formats.Animal cell technology: Proceedings ofthe 18 ESACT Meeting, Granada. Spain,May 11–14, 2003. Kluwer AcademicPublishers (in Press)


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Adherent Growth of CHO Cells in SFM4CHO-A Medium UsingCorning CellBIND Surface Culture Flasks

Cell Line:CHO-K1Chinese Hamster Ovary Cell Line

Medium:SFM4CHO-AHam’s F12 with 10% FBSCompetitor CHO AttachmentMedium

CultureFlask:

Standard Flask: TCT25 CorningTissue Culture Treated 25 cm2

Loose lid during cultureCorning CellBIND surface flask:25 cm2vent capTight lid during culture

Incubator: 37°C; 5% CO2

Culture: 6 mL per flask batch culture

IntroductionOne of the most important cell lines used inthe production of recombinant proteins is theChinese Hamster Ovary (CHO) cell line. CHOcell lines can be cultured adherently to asubstrate or in suspension. Traditionally, theadherent culture of CHO cells has requiredserum-containing medium. Regulatory con-cerns surrounding the use of animal-derivedcomponents in the production of therapeuticproteins is driving improvements in culturingparameters of adherent CHO cell lines.Standard CHO serum-free media formula-tions have failed to equal serum-containingmedium in adherence, spreading and growthusing standard tissue-culture surfaces.

Thermo Scientific HyClone SFM4CHO-A, wasdeveloped for culturing adherent CHO cellsusing the Corning CellBINDsurface, whichshows superior adherence, spreading andgrowth compared to standard Tissue CultureTreated (TCT) culture surfaces.

Materials and MethodsDiscussionTypically CHO cell lines can achievebetween 200,000-400,000 cells/cm2 inserum-containing medium using standardTCT cell culture surfaces. SFM4CHO-A,serum-free medium developed for adherentculture, when combined with the CorningCellBIND surface improved cell densities intothe 400,000-600,000 cells/cm2 range (Figure1). Improvements in growth can be observedwhen comparing standard TCT and CorningCellBIND surface flasks in serum-freeculture in SFM4CHO-A medium (Figure 2).Improvements can also be observed inattachment and spreading as seen in theexample photomicrographs (Figure 3).Exceptional growth is also seen whencomparing serum-free growth of CHO cells inSFM4CHO-A and a competitor serum-freemedium (Figure 4).

ConclusionCHO cells were successfully grownadherently in serum-free SFM4CHO-A onboth standard TCT and Corning CellBINDsurfaces. Using HyClone SFM4CHO-A andthe Corning CellBIND surface, CHO cellssignificantly improved adhesion, spreadingand growth compared to serum-free cultureon standard TCT surfaces. This combinationalso exceeded the performance of serumcontaining medium when culturing CHO cells.

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Metabolically Designed Serum-Free MediaFor HEK293 and PER. C6 Cell LinesCard, Cory. Art to Science, Vol. 22, No. 1; Logan, UT 2003

AbstractProduction of human and animal therapeuticshas become increasingly stringent in order toensure products free of adventitious agentsand immunogenic components. This trendleaves two options for culture medium: the useof high quality serum that has been producedusing the most rigorous processing availablewith extensive quality testing; or the use ofserum-free media with limited or no animal-derived components. Herein we describe thedevelopment and performance characteristicsof serum-free, animal component-free mediafor HEK293 and retina-derived cell lines.

IntroductionThe human embryonic kidney (HEK) 293 cellline is popular for the production of recombinanthuman proteins and viral vectors used in genetherapy. Other uses include high-throughputscreening for drug discovery, and research incell cycle, gene expression, metabolism, andreceptor binding. Graham et al. transformedprimary human embryonic kidney cells withsheared adenovirus type 5 DNA, producing theHEK293 cell line1. HEK293 cells are readilyinfected by adenovirus and produce high titersof human adenovirus. The transformationallows adenovirus vectors with an E1-deletion(the E1 gene is necessary for viral replication)to replicate using the E1 gene present in thehost HEK293 cells.

New cell lines are being developed that mayoffer advantages over HEK293 cells in theproduction of adenoviral vectors free of

replication-comPETEnt adenovirus (RCA).The PER.C6 cell line, a retina-derived cell lineowned by Crucell N.V. (Leiden, the Netherlands),was developed to minimize the creation ofE1-containing RCA.

In addition to applications in gene therapy,these human-derived cell lines are becomingincreasingly popular for the production ofrecombinant human proteins. The "human"posttranslational modifications performed byHEK293 and PER.C6 cells make them desirablefor recombinant protein production.

Traditionally, serum has been the principalsource of attachment factors, transport andbinding proteins and growth factors in cellculture media. Serum also provides protectionagainst shear, toxins, proteolysis, and freeradicals2. The components of a serum-freemedium must replace these functions tosupport growth and productivity. Developmentof the medium takes into account the intendeduse of the cells and their metabolicrequirements. Not only do nutritionalrequirements vary for different cell types, butalso significant variation occurs within thesame cell line when used for different purposes.

Materials and Methods

Stock cultures of HEK293 cells (ATCCCRL-1573) were maintained at 37°C with 5%CO2 in Thermo Scientific HyClone Dulbecco'sModified Eagle's Medium (DMEM) (SH30022)supplemented with 10% Thermo ScientificHyClone Defined Fetal Bovine Serum SH30070)in T-162 tissue culture flasks (Costar™ 3150).Under these conditions HEK293 cells grow asadherent cells in monolayers

(Figure 1). Prior to culture in serum-free media,the cells were adapted to suspension culture.Attached cells were detached using ThermoScientific HyClone Trypsin/EDTA (SH30042),and pelleted by centrifugation at 100 x G forfive minutes. The pellet was then resuspendedby addition of DMEM (with reduced calcium)supplemented with 5% Thermo ScientificHyClone Cosmic Calf Serum (SH30087). Thisbasal medium/serum combination was foundto support suspension HEK293 cells better thanDMEM supplemented with FBS. A viable cellcount was performed using trypan blueexclusion stain and an improved Neubauerchamber hemacytometer.

Cells were seeded at a density of 3.0 x 105

viable cells/ mL into 125 mL Erlenmeyer shakeflasks (Corning 430421). The working volumewas initially maintained at 20 mL to helpreduce cell aggregation. Shake flasks wereplaced into a shaker apparatus and incubator,and maintained at an agitation rate of 150 rpm,37°C with 5% CO2. Every two or three days celldensity and viability were recorded. Celldensities were maintained between 3.0 x 105

and 1.0 x 106 cells/mL by addition of freshmedium at each observation. Growth rateswere monitored to determine when adaptationwas complete.

If growth slowed enough to prevent at least50% fresh medium addition (1:2 split), thencells were gently centrifuged and resuspendedin fresh medium. Once growth rates hadstabilized at a doubling time of approximately30 to 40 hours, an aliquot of cells wascryopreserved and the remaining suspensionadapted cells were used for adaptation toserum-free media.

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Characteristics or functions required inserum-free media are shown in Table 1. Usingcomponents such as those listed in Table 1,many of the functions that serum provides canbe satisfied. These components are added tothe basal medium previously chosen. Cells arethen cultured immediately into the serum-freetrial medium. A similar process as describedfor suspension adaptation may be used toassess the growth-promoting properties of thetrial medium. During this process, samples arecollected and analyzed for elucidation ofcellular metabolism. High-performance liquidchromatography (HPLC) and biochemicalanalyzers (NOVA BioProfile 100, YSI 2700Select) can be used in this process.

When cells show high consumption rates of acomponent, one must consider increasing theconcentration of that component or addingsimilar components to prevent deficiencies.Components that show an increase inconcentration over time or very littleconsumption may be reduced or removed fromthe trial medium. If cells do not adapt well to

the trial medium, especially after following theabove adaptation procedure, a severedeficiency is probable, requiring a review of theserum replacement formulation. Figure 2shows HEK293 cells after adaptation toserum-free suspension culture.

Once growth rates have stabilized and cellshave adapted to the trial medium, the previousmetabolism studies should be repeated whilecells perform required processes. Because themodel medium being described here wasdeveloped for cells used to producerecombinant adenovirus vectors, the cellularmetabolism was analyzed from infection toharvest of progeny virus. Again, data areanalyzed as amounts consumed over time(consumption rate), and deficiencies ortoxicities are adjusted to remove limitingfactors. Two recombinant adenoviruses(QBI-AdenoGFP from Quantum Biotechnologies,Inc. Montreal, Quebec, Canada and Ad5-Luc,Crucell, Leiden, the Netherlands) were used toinoculate suspension cultures of HEK293 cellsin Thermo Scientific HyClone SFM4HEK293 and

PER.C6 cells in Thermo Scientific HyCloneCDM4Retino, respectively. Samples wereremoved every 24 hours during culture for virustitration, photographing, cell count, andgathering metabolic data. Titrations wereperformed by the Spearman-Kärber5 50%tissue culture infectious dose assay (TCID50) in96-well plates (Costar 3585). Figure 3 showscells expressing green-fluorescent protein (GFP)48 hours post-virus exposure with therecombinant adenovirus. Each individualcomponent requiring adjustment should besupplemented to the trial medium through agradient, and a growth promotion studyperformed. This will allow the researcher todetermine the optimum level of thatcomponent for the specific cell line.Components such as vitamins and lipids shouldbe tested over at least three passages andoften as many as ten, since slight adjustmentsmay not be noticeable in fewer passages.

As limiting factors are identified and resolved,one may begin to see significant improvementin both cell growth and productivity.

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Summary

By applying the described developmentprocess, media free of protein andanimal-derived components were produced forhigh-density suspension culture of HEK293 andretina-derived cells. Recombinant adenovirusproduction in HEK293 and PER.C6 cells inSFM4HEK293 and CDM4Retino was found tobe greater than 5x1010 infectious units per mL.Cell growth in these media was found to behighly consistant throughout a long-termculture. These media are free of animal derivedcomponents, protein-free and, in the case ofCDM4Retino, chemically defined.

Development of a serum-free or protein-freemedium could be a perpetual process as newcomponents are continually becomingavailable that are chemically defined,non-animal derived, and aid in the replacementof serum. By using the method describedabove, optimization was completed in anefficient and orderly manner. This processcould be effective for media development forother mammalian cell lines as well.

References1. Graham, F. L., Smiley, J., Russel, W.C., andNairn, R. (1977) Characteristics of a HumanCell Line Transformed by DNA from HumanAdenovirus Type 5. J Gen Virol 36: 59–72.

2. Froud, S. J. (1999) The Development,Benefits and Disadvantages of Serum-FreeMedia. Dev Biol Stand 99: 157–166.

3. Mather, J.P. (1998) Making InformedChoices: Medium, Serum, and Serum-FreeMedium. Methods Cell Biol 37: 19–30.

4. Merten, O.W. (1999) Safety Issues of AnimalProducts Used in Serum-Free Media. DevBiol Stand 99: 167–180.

5. Hierholzer, J.C., and Killington, R.A. (1996)in Virology Methods Manual (Mahy, B.,Kangro, H. eds.), pp. 35-46, Academic Press,London, San Diego, New York, Boston,Sydney, Tokyo, Toronto.

Performance data obtained from the finalprototype for retina-derived cells are alsoshown. A long-term passage study similar tothe HEK293 passage study was performedusing PER.C6 cells for nearly 180 days,reaching approximately 165 doublings, withan overall average doubling time of 26 hours,shown in figure 6. A 48-, 48-, and 72-hourpassage interval was found highly successfulwith the PER.C6 cells in this medium. Onceagain, the medium produced consistent andstable growth throughout the length of thestudy. Cell densities during the two-dayintervals typically reached between 1.0 x 106

and 1.5 x 106 cells/mL, while three-day intervalsresult in densities of 1.5 x 106 and higher.

As a measure of cell productivity in thismedium, PER.C6 cells were infected with arecombinant adenovirus. As in the previousHEK 293 study, virus yields in Thermo ScientificHyClone CDM4Retino were determined every24 hours beginning 24 hours post-virusexposure. Figure 7 shows titers obtained fromCDM4Retino, determined by TCID50 every24 hours. Recombinant adenovirus titersreached levels of greater than 6 x 1010

infectious units per mL.

Results and Discussion

Once final prototypes were developed, datawere collected to analyze growth andproductivity. Figure 4 shows a long-termpassage study conducted with HEK293 cellsto analyze consistency of growth over time.A passage schedule of alternating three- andfour-day intervals produced best results forthese cells. Cultures were seeded at 3.0 x 105

cells/mL throughout the study. Densities duringthe 3-day intervals reached 1.0 x 106 to 1.5 x106 cells/mL, while densities during 4-dayintervals typically reached between 1.5 x 106and 2.0 x 106. Cell growth of HEK293 cells inthe final prototype was found to be highlyconsistent through 40 passages and nearly 96doublings over a period of 130 days.Throughout this long-term culture period theaverage time of population doubling wasfound to be 32.5 hours.

Productivity of HEK 293 cells in this mediumwas also analyzed by infecting withrecombinant adenovirus and measuring virustiters every 24 hours. Figure 5 shows cellgrowth and virus production in a 10 L workingvolume, stirred-tank bioreactor using ThermoScientific HyClone SFM4HEK293. Virus titers inSFM4HEK293 reached levels of greater than 5x 1010 infectious units per mL.

Table 1. Many of the functions of serum are listed, along with potential replacementsto be used in serum-free media.

Function of Serum Possible ReplacementsProtein synthesis precursors Amino acids, small peptides, proteinsBuffering capacity Sodium bicarbonate, HEPES, albuminCarbon-chain energy sources Amino acids, small peptides, carbohydratesHormones and growth factors Recombinant growth factors (EGF, FGF, IGF, etc.)Membrane synthesis precursors Fatty acids, lipids, cholesterolMinerals and salts Sulfate, phosphate, trace metals (Zn, Se, etc.)Shear protectants, anti-aggregant Pluronics, dextran sulfate, heparin, albuminTransport and binding Albumin, transferrin, cyclodextrinsVitamins, anti-oxidant D-!tocopherol, riboflavin, pyridoxine, etc


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Production of Poliovirus Using Vero Cells Cultured inSFM4MegaVir with Nunc Cell Factories, T-Flasks,and Roller BottlesOverview

Researchers develop new products, to ensurethat the process remains the same frombench-scale to production-scale. Those whohave gone through the scaleup process realizethat simply putting more of the sameingredients in a larger container may notproduce the same results. Also, containerscale-up may not be feasible. For instance,if a researcher required a 100-fold increase invirus, the surface area required for a T-flaskwould need to be increased from 75 cm2 to7500 cm2. Imagine the size of a T-7500 flask(7500 cm2 or 8 square feet!). Many approachesexist for cell culture scale-up and theproduction of virus. Three cell culture vessels(Thermo Scientific Nunc Cell Factories, CorningT-75 flasks, and Corning roller bottles) arecompared in terms of why and how each isused, protocol differences, and virus productionperformance. Table 1 provides a summary ofthese characteristics.

Materials andMethods

Thermo Scientific HyClone SFM4MegaVir andHyQTase were used in this study to follow thistrend. SFM4MegaVir is a protein-free, animalderived component free (ADCF) medium de-signed for viral vaccine production. It has beenshown to sustain anchorage-dependent Vero,MDCK, MDBK, and COS-7 cell cultures overlong periods. Vaccine manufacturers employthese cell lines to produce viruses such as thepoliovirus used in this study. HyQTase is a non-mammalian Trypsin substitute which has theadded benefit of not requiring neutralization.

Vero cells (ATCC CCL-81, African GreenMonkey,adult kidney, epithelial) were adapted fromserum containing conditions to SFM4MegaVirand carried over 20 passages. Cells were thencultured in Corning T-75 cell culture flasks andexpanded into a Nunc Cell Factory 10-layer unit(CF10) and 850 cm2 roller bottles for viruspropogation studies. Surface area and mediumvolumes for each vessel type are outlined inTable 2. Cells were cultured at an initial densityof 8000 cells/cm2, in a 37°C, 5% CO2/95% airenvironment, and observed daily under amicroscope until at least 90% confluency was

Table 1. Outlined specific protocols and advantages for each vessel.

Vessel MarketApplication

Additional EquipmentRequired

Gas TransferMechanism

FluidMovement

T-Flask Research Incubator/Pipetting System Cap PipetteRoller Bottle Research &

IndustryIncubator/Pipetting SystemRoller Apparatus

Cap Pipette

Nunc CellFactory

Research &Industry

Incubator/PipettingSystem

Filter Pipette andGravityFeedvia tubing

achieved. Medium was then removed from thecell culture vessel, cells were rinsed withphosphate buffered saline solution (PBS), andHyQTase was applied at 6 to10 mL per 75 cm2

area. Cells were observed until all haddetached from the culture surface (typically15 to 20 minutes), then cell counts and viabilitywere performed, recorded, and analyzed fordoubling times. HyQTase was not removedfrom the culture. New vessels were seededfor scale-up at 8,000 cells/cm2.

Upon reaching 90% confluency the cultureswere inoculated with poliovirus at anapproximate MOI of 0.1. The culture was thenincubated at 37°C for 72 hours, followed byaseptic removal and storage of the supernatant.Cells remaining attached to the growth surfaceof the CF10 were removed using HyQTase, andadded to the supernatant. In order to lyse allcells, the supernatant was frozen at -70°C,then rapidly thawed at 37°C. This process wasrepeated twice to lyse cells and release virusparticles. Lysate was then collected andcentrifuged to pellet and remove cellulardebris. The supernatant containing virus wasused to prepare 10 log10 dilutions of eachsample. Titrations for each clarified samplewere performed in 96-well micro-titer plates

using a TCID50 assay.


Vero cells in this study reached confluency inall vessels tested with good surface attachment,normal morphology, and even distribution ofcells. All doubling times and peak cell densitiesat the time of passage were in normal rangefor protein-free cell culture conditions. Constantdoubling times (approximately 40 hours) andgood viability were maintained. No detrimentaleffects were observed from the use of HyQTase.

Cell handling was reduced since there is noneed to remove the HyQTase solution(via centrifugation) before reseeding theculture vessel. Scaling from a T-75 to a CF1 unitto a CF10 unit was accomplished in twopassages.The 85-fold increase of surfacearea/cell mass is similar to scaling upsuspension cultures from a 250 mL volumeshaker flask to a 15 L bioreactor in twopassages.

Virus Production

T-flasks are typically used for researchapplications, developing cell lines, etc., but arenot convenient for large-scale applicationssince considerable time is required to handle alarge number of vessels required. Roller bottlesare an improvement over T-flasks since agreater surface area is available compared tothe T-flasks while using less media.A disadvantage of the roller bottle method isthe requirement for an apparatus to rotate thecylinders, supplying nutrients and gasexchange to the entire surface.

An advantage of the Nunc Cell Factory is itssmall footprint and its scalability from R&Dthrough large-scale production (see Table 2). Ashort learning curve occurs as operators learnto dispense media, HyQTase, and PBS in andout of the vessel (see manufacturer'sinstructions for a detailed description). Asshown in Figure 1, the relative differencebetween viral titers and the different culturevessels is small. The consistent performance ofSM4Mega Vir in the culture vessels assuresthat as protocols are established, performanceremains the same throughout scaleup.The advantages in gaining surface areawithout consuming significant incubator spaceafforded by the Nunc Cell Factories make itideal for virus production scale-up.


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Table 2. Comparing surface area, reagent volume, total footprint, labor, and additionalequipment requirements for T-flasks, roller bottles, and Thermo Scientific Nunc CellFactories.

Criteria CF10 T-75 Roller BottleSurface Area (cm2) 6320 75 850Media Volume/Vessel (mL) 2000 50 200Units Required for 6320 cm2 area 1 85 8Total Media Required for6320 cm2 area (mL)

2000 4250 1600

Total Footprint (cm2) 670 2670* 4250**Total HyQTase (mL) 250 510*** 160****Total Volume PBS (mL) 250 850*** 160****Labor Hours to Harvest 0.5 2.0 0.5Virus Titer (TCID50/mL) 1.26 x 108 2.61 x 108 1.13 x 108

Total Virus Titer (TCID50) 2.52 x 1011 1.31 x 1010 2.26 x 1011

*Based on stacking T-75 flasks four high**Based on Bellco Technology Cell Production Roller Apparatus***Based on 6 mL HyQTase and 10 mL PBS/flask****Based on 20 mL HyQTase and 20 mL PBS/flask

Summary

Protein-free cell culture is becoming morecommon in biopharmaceutical production.The combination of Thermo Scientific HyCloneSFM4MegaVir and HyQTase suit this role andperform well in cell growth and virus produc-tion. SFM4MegaVir contains no animal derivedcomponents and HyQTase is a non-Trypsin,non-mammalian reagent that is ideal forbiopharmaceutical production methods.

Since SFM4MegaVir performs equally well inall systems tested, the choice of which vesselto use will depend on interests in time,convenience, and space savings. Roller bottlesmay outperform Cell Factories on virusproduction per mL of medium used, but totalvirus production, and footprint and laborsavings may outweigh this.

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A Novel Process forrProtein Production Using BEVSPence, Brandon L., Anderson, Matthew B., Barnett, Bill B., and Weiss, Stefan A.Art to Science, Vol. 19, No. 3; Logan, UT 2000When Yunker, et al. (1967) demonstrated thatinsect cell cultures could be adapted fromculture medium supplemented with silkwormhemolymph to medium containing 10% fetalbovine serum (FBS), 10% whole chicken-eggextract, and 1% bovine serum albumin, thereappeared to be potential for large-scale insectcell culture. Since that time, a number ofnon-proprietary FBS supplemented media suchas TNM-FH (Hink, 1970) and IPL-41(Weiss et al., 1981) have been successfullydeveloped and used in the cultivation of insectcells. Weiss, et al. (1980) further demonstratedthat FBS could be replaced in insect cellcultures by incorporating complex formulationsthat contained increased concentrations ofamino acids, trace elements, vitamins, growthfactors, peptides, and lipids. Weiss, et al.(1984) also documented the successfulreplication of Heliothis zea baculovirus ininsect cells propagated in serum-free/protein-free culture medium containing alipid-phospholipid complex.

The ability to culture insect cells in serum-freemedia grew in significance with the discoveryof the baculovirus expression vector system(BEVS) by Smith and Summers (1983). In areview of baculoviruses as vectors for geneexpression, Atkinson, et al. (1990) noted thatdevelopment of BEVS provided an efficientmechanism for expressing immuno-genically,

antigenically, and functionally correctrecombinant proteins when compared to theirauthentic counterparts. Improved serum-freemedia and optimized bioreactor configurationshave advanced BEVS technology as aproduction method for recombinant biologicals.

Weiss and Smith (1996) reported on thesuccessful large-scale production ofrecombinant hemagglutinin (HA) in BEVS forclinical trials. By using modern bioreactorsystems and metabolically designed media toestablish a peak culture environment, one canoptimize cell growth which improves the virusand recombinant protein yields.

The use of baculovirus to efficiently produce abiological product is primarily a batch processthat is terminated by a viral cytopathogeniceffect and consequent cell lysis. Different celllines respond differently to baculovirusinfection, and subsequently show variation inthe amounts of expressed recombinant protein.It is also likely in large scale serum-freecultures that a number of limiting factors existthat might inhibit the levels and/or functionalityof the expressed product. However, oneprinciple prevails, which is that healthy cellsare vital for high levels of expression andefficient co-translational and post-translationalprocessing of recombinant proteins (Jarvis et al.,1990 and Maeda, 1994). By identifying,optimizing, and controlling the critical process

variables, increased success in expressing highquality recombinant products will be realized.

Increased recombinant protein production inlarge-scale BEVS operations can be achievedby the optimization and careful monitoring ofculture gas flows, and controlling cytostaticmetabolite accumulation, nutrient depletion,and virus multiplicity of infection.The bioreactor used in this study is a 14 LCelligen Pluse bioreactor from New BrunswickScientific (Edison, NJ). The Celligen Plusbioreactor is designed to maximize gasexchange and oxygen mass transfer, andincorporates a four-gas control system to meetthe oxygen requirements for cell growth andviral infection (Julien, 1999). In the CelligenPlus bioreactor, the four-gas control system(air, O2, N2, and CO2) maintains the dissolvedoxygen (DO) and pH simultaneously byemploying any single gas or a combination ofthe four gases. Maintaining a 50% DO airsaturation throughout the culture is ideal. Athigh cell densities, this becomes a challenge.To maintain a DO of 50%, it is often necessaryto increase the airflow. However, the increasedairflow sometimes causes cell deposition abovethe medium level resulting in cell necrosis.

Nutrient depletion and cytostatic metaboliteaccumulation monitoring are essential inmaintaining viable and metabolically active

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ncells. Glucose is the most rapidly consumedcarbohydrate and is the major component ofinsect cell culture media (Barnett, 1998). Often,insect cell cultures are limited by the availabilityof glucose late in the growth or infectionphase. The depletion of glucose causesdiminished cellular performance. At the otherend of the metabolic pathway is theaccumulation of by-products such as lactate,ammonia, and peroxides. During the cultureprocess, this can be inhibitory to both cellgrowth and recombinant protein expression. Inthese studies, the consumption of glucose andthe production of lactate are monitored.

Another critical element that we investigatedfor large-scale insect cell culture is thebaculovirus infection protocol. To achievereproducible and optimal recombinant proteinyields, there must be an appropriate balancebetween the ratio of infectious particles tocells. By using a multiplicity of infection (MOI)that is between 0.1 to 2.0, the interference bydefective virus particles is minimized. However,this is not the only aspect of baculovirusinfection that plays a part in increasing theefficiency of recombinant protein production.A unique baculovirus infection protocol, asdescribed in this article, optimizes the cultureprior to, and during, baculovirus infection.

BioProcessing Configuration

The design of current bioprocessing systems isbecoming increasingly important. As theapplicability of BEVS to producing biologicalsincreases, attention to efficiency increases. It isimportant to ensure reproducibility.Figure 1 illustrates the bioprocessing systemused in this study. A 14 L Celligen Plusbioreactor is used in this example. Internally,the cells are suspended using a 45°

pitched-blade impeller that minimizes shearstress on the cells. Externally, through amultitude of ports on the top of the bioreactor,a 100 L BioProcessing Container (BPC) filledwith serum-free medium, a hollow fiber car-tridge with the accompanying retentate return,and a BPC used for product collection via thepermeate port of the hollow fiber cartridge areconnected. There is also an external additionsport that can be used for inoculation with cellsor baculovirus.

Three pumps control the medium feed, culturecirculation through the hollow fiber cartridge,the waste by-product, and recombinant proteincollection. Weiss, et al. (1989) used spin-filtertechnology to achieve continuous perfusion offresh medium and removal of cell-free spentmedium in high density Sf9 cultures. In thisconfiguration, cell separation from supernate isaccomplished using a 0.65 Rm hollow fibercartridge (A/G Technology Corporation inNeedham, MA). The hollow fiber deviceremoves cytostatic metabolites and autocrinewaste products during cell growth and prior tobaculovirus infection. It is also used to harvestthe secreted recombinant protein. The harvestedproduct goes into a sterile BPC which can befrozen in a bulk quantity until purification.

The culture medium is the metabolicallydesigned medium, Thermo Scientific HyCloneSFX-Insect (powdered medium denotedby MP). This medium helps achieveoptimization by providing key components notonly for viral synthesis, but for cell maintenanceand repair (Pence, 1999). SFX-Insect satisfiesmany aspects of insect cell culture andbaculovirus cultivation, including initiation ofstock cultures, production of buddedrecombinant baculovirus, and scale-up forBEVS expression in large-scale bioreactors.

SFX-Insect can accomplish this in a variety ofinsect cell lines.

Cell Growth

In our evaluation of potential growthperformance in the above configuration, wecultured Sf9 and High Fiveq (Boyce ThompsonInstitute for Plant Research in Ithaca, NY)cells in SFX-Insect and SFX-Insect MP,respectively. The seeding density was 0.5 x 106

cells/mL in a final culture volume of 10 L. Thebioreactor parameters at the time of cellinoculation were set as follows: DO: 50%;pH: 6.2; agitation: 65rpm; air flow: 0.7 L/min;temperature: 28°C. During batch mode growth(no medium or nutrient feed during process) inthe bioreactor, the peak Sf9 and High Five cellyield was ~7.0 x 106 cells/mL (120 hours postseeding) and 7.6 x 106 cells/mL (96 hours postseeding), respectively. After reaching their peakcell density in batch mode, the cultures werecut back to 2.5 x 106 cells/mL, completelyreplenished with medium (using hollow fiberseparation), and then initiated into a 50%medium perfusion rate (at this point theagitation was raised to 75 rpm). The peak Sf9and High Five cell yield was then elevated to~40.0 x 106 cells/mL and 28.0 x 106 cells/mL,respectively. Although cell numbers weregreater with Sf9 cultures, the representativewet biomass for the Sf9 and High Five cellswas ~120 g/L and ~180 g/L, respectively. Figure2 demonstrates the Sf9 and High Five cell yieldand biomass performance from the onset ofperfusion (batch mode growth not shown).Cell viability throughout both the batchand perfusion processes remained )90(Weiss, et al., 2000).

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Just prior to initiating perfusion in the Sf9culture, ~60% of the glucose had beenconsumed. Following the sub-culturing andcomplete medium replenishment, the glucoselevels were renewed and throughout theperfusion process never fell below 35%consumption, even though there was a nearlysix-fold increase in cell yield. We observed inHigh Five cultures that the demand forglucose was more significant than in the Sf9cultures. Just prior to initiating perfusion in theHigh Five culture, ~60% of the glucose hadbeen consumed. Following the same process asthe Sf9 culture to initiate and maintainperfusion, the glucose levels still dropped to)85% depletion at the completion of theperfusion cycle. Another observed differencebetween the two cell lines was that theaccumulation of lactate was more significant inthe High Five culture when compared to the Sf9culture. This was true during both batch andperfusion growth cycles. Lactate and glucoseconcentrations in the culture supernatantswere analyzed using a YSI model 2700 SelectBiochemistry analyzer (Yellow SpringsInstruments, Yellow Springs, OH).The glucose and lactate monitoring isdemonstrated in Figure 3.

Infection Protocol

To utilize the information gathered in growthtrials, we analyzed particular aspects of eachculture for items advantageous to baculovirusinfection. It was evident that the culturemedium exchange at a cell density of 2.5 x 106

cells/mL was beneficial because it occurredwhen the cells were firmly in log phase growthand were entering a period where the nutrient

supply would be greatly taxed. Duringbaculovirus infection, the rate of nutrientconsumption is increased by cellular demandfor repair mechanisms to stave off cytopathicdamage. Affording cells the opportunity tocontinue from the point of infection with afresh medium supply increased the averagecell viability during the baculovirus infection,particularly late in the infection process.

Specifically, the replenishment of glucose atthis junction plays an important role inmaximizing cell potential for growth, viability,and ultimately, recombinant protein expression.Aside from growth evaluation, we alsoassessed a variety of cell densities for infectionand a range of MOI. The balance betweenviable cells and infectious particles is criticalfor obtaining high levels of functionalrecombinant protein expression. At an MOIgreater than 5.0, the potential for defectiveinfectious particle interference significantlyincreases. Also, a cell density of 2.5 x 106

cells/mL was optimal for baculovirus infectionin this configuration. At this cell density, thecells were at or near their peak metabolicactivity, and thus were demonstrating a highpercentage of viability and productivity.

This baculovirus infection protocol involves amulti-step process for optimizing the cellconditions at the time of infection.After initiating the culture at 0.5 x 106 cells/mL,we allow ~48–72 hours of growth to reach thedesired infection density of 2.5 x 106 cells/mL.At this point the cells are separated from theculture fluids and replenished with freshmedium. Immediately following the mediumexchange, the baculovirus is added to the

culture. While the baculovirus is added, thebioreactor agitation is reduced by 40 to 50%.This reduced culture agitation continues 3 to 4hours from the time of infection. Decreasedagitation increases the baculovirus infectionsuccess. After 3 to 4 hours, the culture isreturned to the prior suspension rate(see Figure 4).

Recombinant Protein Expression

Following the infection, culture samples aretaken 12 to 24 hours apart. The samples areanalyzed for cell density, cell viability, andrecombinant protein expression. Recombinantprotein expression in BEVS is terminated whenthe cellular repair mechanisms can no longerkeep up with the infection induced damage tothe cellular biosynthetic machinery. Therecombinant cDNA replacing the polyhedrinprotein is expressed prior to cell lysis duringthe very late phase of baculovirus infection,beginning ~24 hours postinfection (PI) andextending through cell lysis at 96 to 120 hoursPI. The extended viability of baculovirusinfected cells in SFX-Insect may lead to morecomplete post-translational modification of therecombinant proteins during this very latephase of infection (Pence, 1999).

As previously cited by Jarvis (1990) and Maeda(1994), viable cells are necessary for the properlevels of post-translational modification tooccur on the recombinant protein expressed.With this in mind, cell viability may be thecritical determining factor for recombinantprotein harvests. Optimizations as describedhere regarding bioreactor configurations andunique infection protocols involving SFXInsect

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further the performance of insect cell cultures inexpressing fully processed recombinant proteins.

Sf9 and High Five cultures were evaluated forrecombinant protein expression using a humaninterleukin 6 (rhIL-6) BEVS construct(Invitrogen, Carlsbad, CA). The Sf9 and HighFive cells were cultured in SFX-Insect andSFX-Insect MP, respectively. In eachrecombinant protein expression trial using theCelligen Plus bioreactor, the aforementionedbaculovirus infection protocol was used. Inshaker flask conditions, the cells were culturedto 2.5 x 106 cells/mL and infected with theappropriate amount of baculovirus. The HighFive culture produced a peak expression levelof ~4.5 mg rhIL-6/L in the Celligen Plusbioreactor and ~1.5 mg rhIL-6/L in the shakerflask, each at 96 hours PI. The cell viability inthe Celligen Plus bioreactor and shaker flaskconditions at 96 hours PI was ~60% and ~40%,respectively. At 72 hours PI, the conditions inthe Celligen Plus bioreactor and shaker flaskwere ~75% viability and 3.2 mg rhIL-6/L, and~60% viability and 1.3 mg rhIL-6/L, respectively.The Sf9 culture produced a peak expressionlevel of ~4.0 mg rhIL-6/L in the Celligen Plusbioreactor and 1.8 mg rhIL-6/L in the shakerflask, each at 120 hours PI (see Figure 5). Theviability for Sf9 cells in the Celligen Plus biore-actor and shaker flask conditions at 120 hoursPI was ~70% and ~30%, respectively. In theCelligen Plus bioreactor with Sf9 cells at 96hours PI the viability was 75%, while the rhIL-6level of expression was 3.5 mg/L (see Figure 6).In the shaker conditions with Sf9 cells, theviability was 70% or greater, 72 hours PI witha rhIL-6 expression of 1.2 mg/L.

Initial unpublished testing on recombinant

proteins expressed by cultures, some withgreater than and some with less than 70% cellviability, show that post-translationalmodifications increase in cultures with highercell viabilities (Weiss, et al., 2000).

When assessing the final performance ofBEVS-expressed systems, levels of expressionat viabilities greater than 70% should becarefully analyzed, rather than final levels ofexpression that may disregard cell viability.

Conclusion

Dynamic bioprocessing configurations are cellculture in numerous biological andpharmaceutical processes. Increasing theoutput of functional recombinant proteinsexpressed in BEVS will further the utilization ofthis technology. Combining bioreactors capableof providing high performance cell cultureenvironments with medium metabolicallydesigned for high cell performance, yields asignificant increase in productivity.

Further demonstrated is that mediumremoval and replacement using hollow fibersenhances both cell growth and recombinantprotein expression. Identifying feedingstrategies, whether they be intermittent orconstant, will be critical for the scale-up phaseof insect cell culture systems. Insect cellculture configurations are designed to maintainmetabolically active cells longer to producemore fully processed rproteins at greater levels.

References

1. Atkinson, A.E., M.D. Weitzman, L. Obosi,D.J. Beadle, and L.A. King. 1990. Pestic. Sci.28:215.

2. Barnett, B.B. 1998. Art to Science 17(1):1.

3. Hink, W.F. 1970. Nature 195:788.

4. Jarvis, D.L., J.G. Flemming, G.R. Kovacs,M.D. Summers, and L.A. Guarino. 1990.Bio/Technology 8:950.

5. Julien, C. 1999. Genetic Engineering News19(3).

6. Maeda, S. 1994. pp. 1–22, in Insect CellBiotechnology, K. Maramorosch and A.H.McIntosh, Eds., CRC Press, Ann Arbor.

7. Pence, B.L., S.A. Weiss, and B.B. Barnett.1999. Art to Science 18(2):4.

8. Smith, G.E., M.D. Summers, and M.J. Fraser.1983. Journal of Molecular Cellular Biology.3(12):2156.

9. Weiss, S.A., T.L. Lester, S.S. Kalter, and R.L.Heberling. 1980. In Vitro 16:616.

10. Weiss, S.A., D. Peplow, G.C. Smith, andR.H. Godwin. 1984. In Vitro 20:271.

11. Weiss, S.A. and G.E. Smith. 1996.International Biotechnology Symposium.Sydney, Australia.

12. Weiss, S.A., G.C. Smith, S.S. Kalter, andJ.L. Vaughn. 1981. In Vitro 17(6):495.

13. Weiss, S.A., B.W. Belisle, A. DeGeiovanni,G. Godwin, J. Kohler, and M.D. Summers.1989. Continuous Cell Lines as Substratesfor Biologicals 70:271.

14. Weiss, S.A., B.L. Pence, M.B. Anderson,and B.B. Barnett. 2000. 15th AustralianBiotechnology Conference. Brisbane,Queensland, Australia.

15. Yunker, C.E., J.L. Vaughn, and J. Cory.1967. Science 155:1565.


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Growth Comparison Studies Between FBSand Other Serum Products

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Fetal Bovine Serum (FBS) has been the serumof choice for cell and tissue culture from thebeginning. While FBS is an excellent productfor many applications, it is subject to shortagesand consequently price fluctuations and availability difficulties. As an alternative,Thermo Scientific HyClone Bovine Calf Serumwas introduced to the market in various forms.These calf serum products provide advantagesin availability, price, and consistency of performance. In 1984, we were first to market with Thermo Scientific HyClone Iron-Supplemented Bovine Calf Serum. Sincethen, we have introduced other supplementedand processed calf serum products including

Thermo Scientific HyClone Alpha Calf Serum,Cosmic Calf Serum, FetalClone I, II, and III, andmost recently, Bovine Growth Serum. Todemonstrate the ability of these products toperform as well as FBS in many applications,we provide data comparing FBS to these products along with comparisons of non-U.S.sourced FBS to US sourced FBS. The following growth promotion studies were performed in our R&D department. Our non-US sourced FBS as well as our fetal bovineserum replacements were compared to a U.S.sourced FBS control for their ability to promoteculture growth in a variety of popular cell lines.All cultures were grown in T-25 cell culture

flasks using 10 mL of appropriate media sup-plemented with 10% serum and checked dailyfor confluency. Performance was normalized tothe FBS control by dividing the cell count fromeach FBS alternative condition by the cell countfrom the FBS control. The resulting ratios arepresented as relative yields in each study. Theratio, or relative yield, of FBS is always 1.0. Conditions that produce more cells than the control have values greater than 1.0.

Please call your Technical Sales Representativefor help in selecting the appropriate product foryour applications and for samples.

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Pancreatic cells Murine XSH-SY5Y Human neuroblastoma XHep 382 Human liver epithelial carcinoma XCOS African green monkey kidney fibroblast X XMRC-5 Human lung fibroblast XMv 1Lu Mink lung XHEL Human lung fibroblast XMDCK Canine kidney XCRFK Feline kidney cortex XK562 Human bone marrow XVero African green monkey kidney XAtT-20 Murine pituitary tumor XWl-38 Human lung fibroblast XACHN Human kidney renal cell carcinoma XEL-4 6.1 Murine thymoma XSAOS-2 Human bone osteosarcoma XU2-OS Human primary osteogenic sarcoma XHep 3B2 Human liver epithelial carcinoma X


Cell Lines Grown Successfully in FetalClone I, II, and IIICell Type Description Type of FetalClone

FetalClone I (SH30080) FetalClone II (SH30066) FetalClone III (SH30109)Sp2/0-Ag14 Murine myeloma X X XHEp-2 Human epithelial adenosarcoma X X XP3X63 Ag8.653 Murine myeloma X X XFOX-NY Murine myeloma X X XP388 Murine macrophage XNS-1 Hybrid Murine hybridoma X X XBHK Baby hamster kidney fibroblast X X XNamalwa Human histiocytic lymphoma XP3 Hybrid Murine hybridoma X X XU-937 Human histiocytic lymphoma X X XHeLa Human epithelial adenosarcoma X X XTh1-Helper 1 Human cord blood leukocyte XP3X63 Ag8.653 Hybrid Murine hybridoma X X XSp2/0-Ag14 Hybrid Murine hybridoma X X XMDBK Bovine kidney X X XNS-1 Murine myeloma X X XFOX-NY Hybrid Murine hybridoma X X XTh2 T-Helper 2 Human cord blood leukocyte XP3 Murine myeloma X X XNIH/3T3 Murine embryo fibroblast X X XPLB-985 Human histiocytic lymphoma X X786-O Renal cell carcinoma X XU9-lllB Human histiocytic lymphoma X XCHO-K1 Chinese hamster ovary X XL929 Murine areolar and adipose fibrosarcoma X XPC-12 Murine adrenal gland pheochromocytoma X XL(-tk) Murine L cell connective tissue XHEK-293 Human embryonic kidney X XJurkat T-lymphocyte Human thymoma X XDg44/CHO-K1 Chinese hamster ovary X XCHO-CCKR Chinese hamster ovary X9HTEo Human epithelial trachea XJEG3 Human choriocarcinoma X XC3H/10T1/2 Murine embryo fibroblast X X


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Heat Inactivation— Are You Wasting Your Time?Reprinted from Art To Science, Vol. 15, No. 1An issue that should be of interest to most cell culturists is the heatinactivation of serum products. It would be inconceivable to purchasethousands of dollars worth of expensive growth factors, vitamins, aminoacids, etc., and needlessly expose them to temperatures exceeding 56°Cfor at least 30 minutes. Nevertheless, this is exactly what takes place inmany cell culture laboratories. Many cell culturists routinely heatinactivate serum products without considering how this extremetreatment affects growth factors, vitamins, amino acids, etc. One ofthe most frequent inquiries that we receive on our Technical Hotline iswhether to heat inactivate serum. To address this concern and furtherbring cell culture from an art to a science, we investigated the effectsof heat inactivation on fetal bovine serum (FBS).

According to some estimates, at least 70 percent of all customers whoheat inactivate serum do so simply because it is included in theirprotocols or it has always been accepted as necessary. Recommendedtemperatures in the protocols range from 45°C to 62°C and times rangefrom 15 minutes to 60 minutes. The most common procedure involvessubjecting the serum to 56°C for 30 minutes. Many of these protocolsoriginated before the 1970s and have not been questioned. As a resultof improvements in the collection, processing, and understanding ofserum, many of the original reasons for heat inactivating serum are nolonger valid. Only a small percentage of customer who heat inactivatetheir serum have actually performed experiments to verify the efficacyof the process and determined whether heat inactivation is indeednecessary. Heat inactivation was once deemed necessary to destroyheat labile components, such as complement. However, it is apparentlyunnecessary to heat inactivate FBS to inactivate complement. Trigliaand Linscott1 assayed nine complement components, conglutinin andC3b inactivator in commercial FBS. The FBS contained approximately1-3 percent of adult levels of conglutinin, C1 and C6, and 5-50 percentof adult levels of the remaining components except C3, which wasundetectable. We have experienced similar results in our complementfixation assays; in 10 samples of FBS from different production lots, therewas no hemolysis even in undiluted serum. Additionally, serum samplessent to an independent laboratory for complement fixationassays yielded no red cell lysis in the lowest serum dilution tested (1:4).Furthermore, cell culture laboratories often warm their serum-containingmedia to 37°C prior to use, which in itself would inactivate heat-labilecomplement factors.

In addition to complement inactivation, heat inactivation was believedto inactivate adventitious microbial contaminants such as mycoplasmas.Years ago, serum was filtered through 0.45 Rm pore-size rated filtersand mycoplasmas were occasionally isolated from serum and mediaproducts. In response to this concern, we were the first serum supplier tofilter serum through three consecutive 0.1 Rm pore-size rated filters. Thecontinual improvement and validation of modern filtration systems haseliminated the need to rely on heat to inactivate mycoplasmas.

There are few published reports concerning the effects of heatinactivation on serum products, but our findings and customer commentsindicate that heat inactivation is not necessary for most cell cultureapplications.

Pinyopummintr et al.2 determined that it is not necessary to heatinactivate serum with high embryotrophic properties and that eliminatingheat inactivation does not affect bovine embryo development in vitro.It has been reported that the heat inactivation of FBS and bovine calfserum reduces their capacity to promote cell attachment.3 In tests for cellattachment activity with SV-BHK, BALB-3T3, CV-1, and FS-4 cells, heatinactivation had less effect on FBS than on bovine calf serum. Followingcell attachment, heat inactivation did not seem to affect cell growth. Ithas also been reported that heat inactivation is not necessary for serumused with insect cell cultures and in studies of baculovirus expression.4

We investigated the effects of heat inactivation on FBS and its abilityto support cell growth on a variety of cell lines using the most widelyaccepted protocol, which is described in Figure 1.

We compared the growth of 11 different cell lines in untreated FBS andheat-inactivated FBS (Figure 2, opposite). The growth of six of the celllines (HBAE, MDBK, Vero, human foreskin fibroblast, MRC-5, and Mv1Lu) was affected adversely by heat inactivation. The growth of threecell lines (FOX-NY hybrid, MDCK, and CHO-K1) was not affected byheat inactivation. Only two cell lines (Balb/3T3 and Sp2/0Ag14 hybrid)demonstrated slightly improved performance in heat inactivatedserum. Thus heat inactivation, when performed properly, offers littleor no advantages for cell growth and usually results in decreasedgrowth rates.

However, when performed improperly, heat inactivation will almostalways adversely affect serum products. The heating of serum productsresults in the formation of precipitates which are frequently mistaken formicrobial contaminants. Many cell culturists notice this cloudiness andwill further incubate the entire contents of the bottle at 37°C in anattempt to "culture" the microorganism. Unfortunately, this exacerbates

Figure 1. Protocol for heat inactivation of serum products.1. Thaw the serum and mix the contents of the bottle thoroughly.2. While the serum is thawing, prepare a control bottle containing

water. The control bottle should be stored along with the serumbottles to ensure identical beginning temperatures. This controlbottle will be used to monitor the temperature and should beidentical to the serum bottle (eg., PETG).

3. Place the bottle of serum and the control bottle in a 56°C water-bath containing sufficient water to immerse the bottle abovethe serum level. Suspend a thermometer or thermocouple inthe water bottle. The thermometer should not touch the sidesor bottom of the bottle.

4. Swirl the bottles every 10 minutes. for FBS and BCS and 5 minutes.for equine serum to ensure uniform heating of the serum.

5. Monitor the temperature of the control bottle closely and begintiming as the temperature reaches 56°C.

6. After 30 min. at 56°C, immediately cool the bottles of serum inan ice bath.


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the problem since prolonged incubation only results in the formationof additional precipitate. This precipitate resembles a microbialcontaminant to the extent that even experienced cell culturists andmicrobiologists often cannot distinguish between the two. Extensivetesting (electron microscopy, plating on bacterial growth media, Gramstaining, etc.) is required to confirm the presence of a precipitate.

These tests waste considerable time and resources simply to convincecell culturists that the serum is not contaminated with bacteria.Precipitates are also more likely to form if serum is heat inactivated attemperatures above 56°C or for more than 30 minutes or if the contentsof the bottle are not properly mixed.


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MRC-5, MDBK, and Sp2/0 Ag14 hybrid cells in serum that has beenheated at 56°C for 30, 75, 120, and 1,080 minutes. The growth of HBAE,MRC-5, and MDBK cells is affected adversely with each increase inexposure time. The HBAE cell line is very sensitive to heat inactivatedserum and failed to grow in FBS heat inactivated for 1,080 minutes.The growth of MRC-5 and MDBK cells was also impaired significantly,although to a lesser degree than the HBAE cells. Again, as in theexposure to higher temperatures, the growth of Sp2/0 Ag14 hybrids didnot seem to be affected by increased exposure times.

Several factors can influence the total time that serum is exposed to hightemperatures. Glass and plastic (PETG) differ in their heat capacities,which directly affects heating rates. To reduce breakage and facilitatestorage, plastic bottles have largely replaced borosilicate glass bottles.These plastic bottles increase the time required for the contents to reach

Although 56°C is the most common temperature for heat inactivation,some protocols list higher temperatures, which further compromisethe growth-promoting ability of serum, in addition to resulting in theformation of additional precipitates. The growth of MRC-5 and MDBKcells decreases dramatically in FBS that has been heat inactivated at65°C for 30 minutes (Figure 3). FBS heat inactivated at 65°C failsto support the growth of HBAE cells, even though the growth ofSp2/0 Ag14 hybrid cells is not affected.

Serum products are often exposed to high temperatures for more than 30minutes, either intentionally or unintentionally, e.g., serum is mistakenlyleft in the water bath overnight. Extended heat inactivation alsoincreases the formation of precipitates and decreases the growthpromotion capacity of the serum. Figure 4 shows the growth of HBAE,


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since the bottles will have a tendency to float. Floating can be preventedby placing commercially available lead weights around the necks of thebottles. Figure 6 illustrates the heating rates in water baths in whichlevels were equal to either the 300 mL or the 500 mL graduation of astandard plastic bottle. At the 500 mL graduation, the serum required40 minutes to reach 56°C. At the 300 mL graduation, the serum required60 minutes to reach 56°C thus increasing the total exposure time toelevated temperatures.

In summary, most cell culture applications do not require heatinactivation of serum. In most cases, heat inactivation does not improvethe growth promotion capability of the serum and may actually haveadverse effects. In the few instances in which heat inactivation improvedperformance, the improvement was minimal. Moreover, heat inactivationcauses an increase in the formation of precipitates which are frequentlymistaken for a microbial contaminant. This creates unnecessaryconcerns and also consumes valuable resources on behalf of the useras well as the supplier.

Customers who currently heat inactivate their serum should determineif this procedure is really necessary with their particular cell lines orculture systems. Serum is often inadvertently heated for longer periodsof time and at higher temperatures than recommended in traditionalprotocols. If heat inactivation is necessary, it should be strictlymonitored and performed using a procedure that is repeatable.

References1. Triglia, R.P., Linscott, W.D. 1980. Titers of Nine ComplementComponents, Conglutinin and C3b-Inactivator in Adult and FetalBovine Sera. Mol. Immunol. 17:741-748.

2. Pinyopummintr, T., and Bavister, B.D. 1994. Development of BovineEmbryos in a Cell-Free Medium—Effects of Type of Serum, Timingof its Inclusion and Heat Inactivation. Theriogenology. 41:1241-1249.

3. Giard, D.J. 1987. Routine Heat Inactivation of Serum Reduces itsCapacity to Promote Cell Attachment. In Vitro Cellular &Developmental Biology. 23:691-697.

4. Invitrogen. 1995. EXPRESSIONS. Vol. 2 Issue 2. p 11.

56°C by approximately 30 percent. This insulating effect also increasesthe amount of time required for the contents of plastic bottles to coolfollowing heat inactivation. The cooling rate also depends on whetherthe serum is placed into a refrigerator, freezer, or ice bath following heatinactivation (Figure 5). Heat inactivated serum required 30 minutes tocool to a temperature of 10°C in an ice bath versus 330 minutes whensimply placed into a refrigerator. Dividing the serum into aliquotsfacilitates heating and cooling. The water level in the heat inactivationbath will also affect heating rates of the serum. It is often inconvenientto fill the water bath such that it equals the level of serum in the bottle


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Viral Safety in Serum for Cell Culture UseReprinted from Art To Science, Vol. 16 No. 2

Figure 1. Preliminary rawmaterial risk assessment worksheet

Consideration Response RangeI. Geographic Origin* 1. Obtained From Animal

Originating CountriesFromWhich Bovine Serum Importinto the U.S. is—

Permitted without safety testing = 1(U.S., Canada, New Zealand, etc.)Permitted with safety testing = 2(Australia, Central America, etc.)Not permitted(non-"BSE" countries) = 5** "BSE" countries = 10

2. Documentation of Traceability Complete, authentic certificate of origin:Yes = 1No = 4

Number of companies involved in supplychain from collection toend user:NONE = 0ONE = 1TWO = 3THREE ORMORE = 5

II. Animal Donor1. Age Fetus = 1

Adolescent = 2Adult = 4Newborn = 5

2. Species Different from recipient = 1Same as recipient = 5Domesticated, common = 1Exotic, uncommon = 10

3. Anatomical Origin of Material Blood, Milk = 1Organs = 2CNS, CNS associated glands = 4

Consideration Response Range4. Ancestry No known ancestry linked to "BSE":

countries = 1Known ancestry linked to "BSE":countries = 5

5. Status as Virus Reservoir Viral infections well known = 1Viral infections not well known = 4Viral infections reported andmonitored = 1Viral infections not reported = 4

III. Animal Husbandry1. Feeding Method Open = 1

Intense = 4

2. Feed Type Natural = 1(grass, hay, grain)Formula-fed without renderedsupplements = 1Rendered supplements = 3

3. Health Care Veterinary care is generally good and available= 1Veterinary care is inconsistent and not easilyavailable = 4

IV. Harvesting1. Location Closed herd donor = 1

Abattoir = 32. Method Aseptic (venipuncture, cardiac

puncture) = 1Open (exposed to uncontrolledenvironment) = 4

3. Personnel Ante-/Post-mortem veterinaryexamination = 1No Ante-/Post-mortem veterinaryexamination = 3

Collection personnel task dedicated, trained =1Collection personnel multi-tasked,untrained = 3

V. Post-harvesting Handling1. Time Rapidly achieve suitable

storage state = 1Slow in achieving suitablestorage state = 3

2. Method Aseptic handling = 1Non-aseptic handling = 3

VI. Final Preparation of Materials1. Adventitious AgentReduction Several validated reduction processes

(filtration, centrifugation, etc.) = 1At least one validated reduction process = 2No validated reduction process = 4

2. Adventitious AgentInactivation At least one validated inactivation process = 0

No validated inactivation process = 4

3. Adventitious AgentConcentration Process

No concentrating processes = 0At least one concentrating process = 4

Now sold under the Thermo Scientific brand, HyClone products were cre-ated nearly 40 years ago by a virologist, Dr. Rex Spendlove, because hewas not satisfied with the quality of commercially available animal serafor his virology work. We continue to improve serum quality throughcollection, processing, and filtration innovation. For example, serumendotoxin and hemoglobin are indicators of the care exercised duringblood collection and processing. Through the use of a patented,disposable, closed-system cardiac puncture collection system and rapidprocessing, serum hemoglobin and endotoxin levels are significantlyreduced. We are the first to use serial 100 nm pore-size filters and laterserial 40 nm pore-size filters.

With the belief there is virus present in all commercial-size batches ofanimal sera1 and that safe use is based on informed consumersmanaging the reduction of viral load, this article addresses serumtreatments designed to reduce adventitious agents yet preservegrowth- promoting characteristics, while also being commerciallyfeasible and reliable. A method to assess risk is also discussed.


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Consideration Response Range

4. Testing for Relevant AdventitiousAgents (Viruses which areknown to, or are likely to,contaminate raw material)

Tests available and results reported = 1Tests difficult or not available = 4

VII. Supplement Use

1. Cell Culture Cells of different species = 1Cells of same species or knownto be susceptible = 5

Low concentration of material(P1% Serum) = 1High concentration of material ()10% Serum)= 5

2. Time of Additionto Product

Prior to purification procedures designed toreduce nucleic acids = 0Directly to finished product = 5

Assessment of Risk for use of Animal-DerivedMaterial

A rawmaterial risk assessment tool2 has been developed for ThermoScientific HyClone products based on geographic origin, animal donor,animal husbandry, material harvest, post harvest handling, final preparation,and material use. Both rawmaterial risk (Figure 1) and recipient risk (Figure2) are considered. It is designed as a discussion tool to direct in-house rawmaterial reviews. The numbers ascribed in the Response Range columnmay be changed based on your estimate of relative risk. In fact, the higherthe number, the greater the risk. Entire responses may be eliminated due tounacceptably high risk. A total score of less than 40, in this example, isconsidered minimal risk requiring no additional pre-use processing beyondsterile filtration. Risk is moderate if the score is 40 to 100. If a careful reviewof the assessment does not lead to a lower total score by changing vendors,geographic origins, animal species, etc., additional pre-use processingshould be considered. A total score greater than 100 indicates high risk andadditional pre-use processing beyond sterile filtrations is required.

Regulatory Control

Government agencies worldwide are promulgating regulations tominimize the risk from the use of animal-derived materials. Regulationsusually call for evidence that the material is safe for use based on animalsource, geographic origin, treatment, and/or testing. Four regulatorydocuments published by the European Economic Community (EEC)illustrate the efforts agencies are taking.

1. EEC Regulatory Document: Note for Guidance (1992) Guideline forMinimizing the Risk of Transmitting Agents Causing SpongiformEncephalopathy via Medicinal Products3. This note for guidance coversmanufacturing, animals as source materials, age of animals, parts ofanimals used as starting materials, cellular substrates, and proceduresto remove or inactivate agents causing spongiform encephalopathies.The treatments recommended (autoclaving, addition of 1N sodiumhydroxide, addition of 2% sodium hypochlorite, organic solvent extrac-tion, or protein removal by precipitation) are harsh and woulddestroy the growth-promoting characteristics of animal sera for cellculture use. Therefore, strict geographic origin evidenced bytreaceability is currently the only viable management tool for BSE.

Figure 2. Recipient preliminary risk assessment

Consideration Response RangeI. Recipient1. Species Different from donor = 1

Same as donor = 52. State of Health Healthy = 1

Compromised = 43. Age Adolescent, Adult = 1

Very young, very old = 4

II. Administration1. Dose Single, small = 1

Large, continuous = 42. Method Transdermal = 1

Oral = 2Injection = 4Intraneural = 10

3. Council Directive 92/118/EEC of 17 December 1992: Laying Down theAnimal Health and Public Health Requirements Governing Trade inImports Into the Community of Products Not Subject to the SaidRequirements Laid Down in Specific Community Rules Referred to inAnnex A1 to Directive 92/6621/EEC and, as regards to pathogens, toDirective 90/425/EEC5: This directive covers the animal health andpublic health requirements governing trade in certain products. Thisis the "Balai Directive" or broom sweeping directive.

4. Draft Commission Decision of 1993: Laying Down the Animal HealthRequirements and Veterinary Certification for the Import from ThirdCountries of Blood Products of Animals Not Intended for HumanConsumption and Amending Commission Decision 94/278/EC,V1/9440/93 Revision 106. This decision is the further definition ofthe specific annex chapter dealing with blood and blood products ofanimal origin from the "Balai Directive." It is currently in draft-revision10, and it requires that blood products for pharmaceutical or technicaluse imported from third countries where foot-and-mouth disease

2. EEC Regulatory Document: Note for Guidance (1991) Validation ofVirus Removal and Inactivation Procedures 4. This document reviewsthe sources of viral contamination, the choice of viruses for validation,the design and implications of validation studies, and the limitationsof validation.

Figure 3. Virus recovery after virus-spiked bottles of serum areexposed to various doses of gamma radiation. Recoveries ofBVD, IBR, P13 , REO, and PPV are compared with untreatedcontrols of the same lots of spiked serum.

Gamma Irradiation—Virus Recovery MatrixMean of 3 DeterminationsDose (Mrad) Log 10 Residual TCID50/mL

BVD IBR P13 REO PPVControl 6.11 7.11 6.72 6.72 7.170.5 4.72 5.50 5.56 6.61 7.131.0 3.67 3.89 5.22 6.28 6.792.0 2.28 * 2.56 4.00 6.332.5 * * * * 6.083.0 * * * * 5.714.0 * * * * 5.255.0 * * * * 4.296.0 * * * * 3.29* Not Detected


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10, and it requires that blood products for pharmaceutical or technicaluse imported from third countries where foot-and-mouth disease(FMD) and/or bluetongue (BT) are known to exist be treated in one ofthree ways:

1) Heat to 65°C for at least 3 hours, or2) Change to pH 5 for 3 hours, or3) Gamma irradiation at 2.5 Mrads

Regulatory control of animal-derived cell culture supplements is lesscomplicated in the United States. The United States Code of FederalRegulations (21 CFR 864) defines animal sera and media for cell cultureuse as Class I Medical Devices. In December 1993 and again in May1996, the United States Food and Drug Administration (FDA) sent lettersto regulated manufacturers with recommendations for the use of bovinederived products for human use. They stressed the importance ofmaintaining records of material origin, specifically showing that animalmaterial from a designated BSE country was not used. In order toprovide recommendation to customers regarding the efficacy of theproposed EEC treatment requirements, a study of each procedurewas prepared.

Gamma Irradiation—Serum

In our initial study7, the inactivation of bovine viral diarrhea (BVD) virus,infectious bovine rhinotracheitis (IBR) virus, parainfluenza 3 (PI3) virus,bovine REO (REO) virus, and porcine parvovirus (PPV) was compared atdoses from 0 to 6.0 Mrad. These viruses were chosen because they arerepresentative of viruses commonly found in bovine serum or are modelviruses which closely resemble viruses of interest. For example, PPV isthe model for FMD disease virus. Serum shown not to be inhibitory tothe viruses was used. Minimum log viral recovery was 6.0 TCID50/mL.The assay level of detectability was 0.5 TCID50/mL. Standard 500mL sizepolyethylene terephthalate G copolymer (PETG) bottles of serum wereinoculated with virus, and the serum frozen and irradiated in productionscale load configurations. The studies were performed in triplicate. It isimportant to remember the virus titers used in these experimentalconditions are extreme worst case. In nature, virus titers would neverreach these levels.

The results are summarized in Figure 3. A dose of 2.5 Mrad (25 kGy)effectively destroyed 6 logs of BVD, IBR, PI3, and REO virus; but even 6.0Mrad (60 kGy) inactivated only about 4 logs of PPV. In a later study, wedemonstrated a 4 log reduction in BVD titer in two liter containers offrozen serum when irradiated at 2.0 Mrad (20 kGy). This agrees with ourearlier study in 500 mL bottles.

With a retrovirus, feline leukemia virus (FeLV), we demonstrated that adose of 2.0 Mrad (20kGy) could inactivate 3.0–4.0 logs of virus in 500 mLPETG bottles of frozen serum, 2.5–3.0 logs of virus in one liter PETGbottles, and 2.0–3.0 logs of virus in two liter high density polyethylene(HDPE) bottles.

Three lots of fetal bovine serum (FBS), inoculated with high titer BT viruswere irradiated at 2.5 Mrad (25 kGy), the minimum dose required by theEEC draft directive for blood product import. In standard 500 mL PETGbottles, 3.5–4.0 logs of virus were destroyed.

Biochemical analysis of serum irradiated at 2.5–3.5 Mrad (25–35 kGy)indicated that the effects were minimal. The results of this study aresummarized in Figure 4. The concentrations of some vitamins, alkalinephosphatase, and lactate dehydrogenase were decreased by half.

Growth promotion based on ten lots of irradiated FBS ranged from 94percent of control to 101 percent of control. Cell lines tested wereFOX-NY Hybrid, MRC-5, CHO-K1, and VERO.

One of the most important characteristics of treatment by gammairradiation is that it is a final container treatment or terminal sterilization.The sterility assurance level (SAL) of irradiation post sterile filling is 10-6.Sterile filtration alone provides only a 10-3 SAL.

Heat Treatment

Heat treatment at 65°C for three hours caused the PETG bottles to deformand the serum to cloud and gel. Because the serum gelled, many of thebiochemical assay results may not be reliable. However, more biochemicalvalues were altered using this treatment than with either gammairradiation or acid treatment. Components most affected were hormones,vitamins, enzymes, cholesterol, and IgG. (Figure5) Growth promotion aspercent of untreated control ranged from 0 to 53 for FOX-NY Hybrid,MRC-5, CHO-K1, and VERO. As expected, heat treatment was effectivein reducing BT virus less than detectable. Because of the effect heattreatment has on the PETG bottles and the need for industrial scaleprocessing, heat treatment would need to be performed on pre-filtrationpools of serum. The SAL for this product would therefore be that ofaseptic filling (10-3). Heating to the temperatures required by the draftEEC directive was found to be the most damaging to serum for cellculture use of the treatments recommended.

Acid Treatment

Reducing the pH of the animal derived material to 5.0 for 3 hours isanother treatment recommended by the EEC. The biochemical profile ofthree lots of FBS treated with 5N hydrochloric acid to lower serum pH to5.0 for 3 hours and then treated with 5N sodium hydroxide to returnserum pH to normal were compared with untreated controls. Measuredbiochemical effects included a small decrease in riboflavin, biotin, folicacid, and alkaline phosphatase. Lactate dehydrogenase was decreasedby three fourths. Sodium and chloride were increased due to the additionof the acid and the base. Iron content was decreased. (Figure 6)

Average growth promotion ranged from 83 percent to 106 percent ofcontrol for FOX-NY hybrid, MRC-5, CHO-K1, and VERO cells. Acidtreatment reduced the level of recoverable BT virus in spiked lots of FBSfrom 6.0 logs to less than 0.5 logs TCID50/mL but had little effect onother viruses tested (Table 5). Acid treatment is a pre-filtration treatment.The SAL of this product is that of aseptic filling (10-3).


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Figure 4. Three lots of FBS inoculated with high titer BT virus were irradiated at 2.5 Mrad (25 kGy). Biochemical analysis of this serumindicated that the effects were minimal.

Test Units 11112568Orginal

I 2568Irradiated

11112594Orginal

I 2594Irradiated

11112620Orginal

I 2620Irradiated

Endotoxin EU/mL P0.06 0.125 P0.06 P0.06 P0.06 0.125Hemoglobin mg/dl 7 3 7 3 10 5Progesterone ng/dl P10 13 4.1 15 P10 12Insulin RU/mL 6.6 6.5 9.3 6.3 8.5 8.8PTH pg/mL 46 56 33.2 P33.2 149 66Vitamin D 1,25 pg/mL 64 39 44 27 * 41pH 7.51 7.53 7.46 7.49 7.47 7.39Osmolality mosm/kg 304 302 299 302 309 307Thiamine B1 ng/mL 172 52 95 38 93 33Riboflavin B2 ng/mL 215 131 227 186 223 185Pyridoxine B6 ng/mL 95 37 81 40 66 43Niacin ng/mL 1.6 1 0.92 0.9 1.1 1Biotin Vitamin H pg/mL 37,083 20,667 43,542 22,611 33,611 21,000Retinol ng/mL 170 P100 130 P100 190 P100Beta Carotene Rg/mL 0.11 0.11 P0.1 0.11 0.16 0.11Total Vitamin E Rg/mL P1.0 P1.0 2 P1.0 P1.0 P1.0Cobalamine B12 pg/mL 356 317 231 258 379 263Folic Acid mg/mL 36 32 44 29 79 38Vitamin C mg/dl P0.05 P0.05 0.8 P0.05 0.8 P0.05Gamma Globulin %tp 0.5 P0.1 P0.1 P0.1 P0.5 P0.1Total Protein gm% 3.3 3.4 3.4 3.7 3.7 3.8Albumin gm% 2.4 2.5 2.6 2.6 2.8 2.7Blood Urea Nitrogen md/dl 16 17 15 16 17 17Uric Acid mg/dl 1.8 1.6 3.4 2 2.7 2.3Creatine mg/dl 2.2 2.3 2.7 2.6 3 2.8Total Bilirubin mg/dl 0.3 0.2 0.3 0.2 0.3 0.3Sodium meq/L 136 134 136 136 139 139Potassium meq/L 10.4 10.4 10 10.2 10.5 10.4Calcium mg/dl 13.8 13.4 13.6 13.2 14.8 13.6Chloride meq/dl 97 99 99 99 101 102Inorganic Phosphorus mg/dl 10.2 10.9 9.3 10.4 10.7 10.9Glucose mg/dl 86 79 89 84 112 103Alkaline Phosphatase IU/L 206 140 259 178 246 175Lactate Dehydrogenase IU/L 590 392 709 487 771 530SGPT IU/L 5 4 8 2 7 5SGOT IU/L 9 64 90 62 114 79Cholesterol mg/dl 35 38 32 33 33 35HDL mg/dl 9 18 11 20 10 18LDL mg/dl 14 7 10 2 12 6Triglycerides mg/dl 62 64 54 56 54 55Phospholipids mg/dl 42 45 44 42 40 42Copper Rg/dl 0.14 0.18 0.13 0.1 0.16 0.13Magnesium Rg/mL 25.3 28.7 24.1 24 23.8 23.6Selenium ng/mL 9 13 28 23 17 19Iron Rg/dl 160 153 176 171 173 164TIBC Rg/dl 242 213 226 241 317 252Percent Saturation % 66 72 78 71 55 65IgG mg/mL 0.108 )0.06 0.132 0.11 0.22 0.19

FOX-NY Hybrid % 96 80 90 92 114 94MRC-5 % 101 109 104 78 101 107CHO-K1 % 95 96 113 99 N/A 91VERO % N/A 89 N/A 95 N/A 102Bluetongue Virus Titer TCID50/mL 6.00 2.00 6.33 2.50 6.00 2.67*Results not available/detectable


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Figure 5. Three lots of FBS were heat treated which caused the most alteration of biochemical values compared to gamma irradiationor acid treatment.


H 2568Heat

11112594Orginal

H 2594Heat

11112620Orginal

H 2620Heat

Endotoxin EU/mL P0.06 0.125 P0.06 P0.06 P0.06 P0.06Hemoglobin mg/dl 7 P1 7 P1 10 P1Progesterone ng/dl P10 P10 4.1 P10 P10 11Insulin RU/mL 6.6 2.5 9.3 3.0 8.5 3.2PTH pg/mL 46 531 33.2 1,129 149 6,275Vitamin D 1,25 pg/mL 64 12 44 P10 * 19pH 7.51 7.44 7.46 7.43 7.47 7.32Osmolality mosm/kg 304 302 299 301 309 308Thiamine B1 ng/mL 172 159 95 152 93 136Riboflavin B2 ng/mL 215 152 227 154 223 156Pyridoxine B6 ng/mL 95 62 81 64 66 67Niacin ng/mL 1.6 1 0.92 1 1.1 1Biotin Vitamin H pg/mL 37,083 22,778 43,542 18,000 33,611 19,667Retinol ng/mL 170 P100 130 P100 190 P100Beta Carotene Rg/mL 0.11 0.11 P0.1 0.11 0.16 0.11Total Vitamin E Rg/mL P1.0 P1.0 2 P1.0 P1.0 P1.0Cobalamine B12 pg/mL 356 203 231 193 379 212Folic Acid mg/mL 36 27 44 42 79 55Vitamin C mg/dl P0.05 P0.05 0.8 P0.05 0.8 P0.05Gamma Globulin %tp 0.5 * P0.1 * P0.5 *Total Protein gm% 3.3 3.4 3.4 3.7 3.7 3.9Albumin gm% 2.4 3.5 2.6 3.7 2.8 4.0Blood Urea Nitrogen md/dl 16 17 15 16 17 16Uric Acid mg/dl 1.8 1.7 3.4 2.3 2.7 2.6Creatine mg/dl 2.2 2.3 2.7 2.7 3.0 3.0Total Bilirubin mg/dl 0.3 0.3 0.3 0.3 0.3 0.4Sodium meq/L 136 135 136 136 139 138Potassium meq/L 10.4 10.5 10 10.2 10.5 10.4Calcium mg/dl 13.8 13.6 13.6 13.6 14.8 13.8Chloride meq/dl 97 99 99 99 101 101Inorganic Phosphorus mg/dl 10.2 11.1 9.3 10.4 10.7 10.8Glucose mg/dl 86 73 89 79 112 97Alkaline Phosphatase IU/L 206 0 259 0 246 0Lactate Dehydrogenase IU/L 590 0 709 0 771 0SGPT IU/L 5 2 8 0 7 1SGOT IU/L 9 8 90 14 114 23Cholesterol mg/dl 35 40 32 37 33 37HDL mg/dl 9 36 11 33 10 34LDL mg/dl 14 * 10 * 12 *Triglycerides mg/dl 62 66 54 58 54 60Phospholipids mg/dl 42 47 44 43 40 45Copper Rg/dl 0.14 0.38 0.13 0.24 0.16 0.13Magnesium Rg/mL 25.3 29.1 24.1 26.1 23.8 23.6Selenium ng/mL 9 12 28 18 17 22Iron Rg/dl 160 171 176 179 173 183TIBC Rg/dl 242 * 226 * 317 247Percent Saturation % 66 * 78 * 55 74IgG mg/mL 0.108 P0.06 0.132 P0.06 0.22 P0.06

FOX-NY Hybrid % 96 70 90 28 114 60MRC-5 % 101 0 104 0 101 0CHO-K1 % 95 0 113 0 N/A 0VERO % N/A 33 N/A 42 N/A 34Bluetongue Virus Titer TCID50/mL 6.00 P0.50 6.33 P0.50 6.00 P0.50*Results not available/detectable


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Figure 6. Three lots of FBS were acid treated by reducing the pH.


A 2568Acid

11112594Orginal

A 2594Acid

11112620Orginal

A 2620Acid

Endotoxin EU/mL P0.06 0.125 P0.06 P0.06 P0.06 P0.06Hemoglobin mg/dl 7 4 7 3 10 6Progesterone ng/dl P10 P10 4.1 P10 P10 P10Insulin RU/mL 6.6 7.4 9.3 6.0 8.5 6.0PTH pg/mL 46 39.8 33.0 60 149 40Vitamin D 1,25 pg/mL 64 36 44 28 * 55pH 7.51 7.53 7.46 7.48 7.47 7.44Osmolality mosm/kg 304 353 299 349 309 357Thiamine B1 ng/mL 172 131 95 125 93 125Riboflavin B2 ng/mL 215 161 227 180 223 174Pyridoxine B6 ng/mL 95 73 81 61 66 75Niacin ng/mL 1.6 1 0.92 1 1.1 1Biotin Vitamin H pg/mL 37,083 21,111 43,542 21,444 33,611 21,444Retinol ng/mL 170 P100 130 P100 190 P100Beta Carotene Rg/mL 0.11 0.16 P0.1 0.16 0.16 0.16Total Vitamin E Rg/mL P1.0 P1.0 2 P1.0 P1.0 P1.0Cobalamine B12 pg/mL 356 304 231 250 379 292Folic Acid mg/mL 36 22 44 31 79 44Vitamin C mg/dl P0.05 P0.05 0.8 P0.05 0.8 P0.05Gamma Globulin %tp 0.5 P0.1 P0.1 P0.1 P0.5 P0.1Total Protein gm% 3.3 3.4 3.4 3.7 3.7 3.8Albumin gm% 2.4 2.4 2.6 2.7 2.8 2.7Blood Urea Nitrogen md/dl 16 16 15 16 17 17Uric Acid mg/dl 1.8 1.7 3.4 2.2 2.7 2.4Creatine mg/dl 2.2 2.2 2.7 2.7 3 2.9Total Bilirubin mg/dl 0.3 0.3 0.3 0.3 0.3 0.4Sodium meq/L 136 164 136 163 139 165Potassium meq/L 10.4 10.4 10 10.1 10.5 10.3Calcium mg/dl 13.8 13.2 13.6 13.2 14.8 13.6Chloride meq/dl 97 133 99 131 101 135Inorganic Phosphorus mg/dl 10.2 10.8 9.3 10.4 10.7 10.7Glucose mg/dl 86 78 89 85 112 105Alkaline Phosphatase IU/L 206 140 259 193 246 187Lactate Dehydrogenase IU/L 590 138 709 172 771 190SGPT IU/L 5 6 8 5 7 4SGOT IU/L 9 149 90 53 114 69Cholesterol mg/dl 35 38 32 34 33 34HDL mg/dl 9 12 11 13 10 13LDL mg/dl 14 13 10 10 12 10Triglycerides mg/dl 62 64 54 56 54 57Phospholipids mg/dl 42 49 44 43 40 46Copper Rg/dl 0.14 0.1 0.13 0.12 0.16 0.18Magnesium Rg/mL 25.3 23.8 24.1 22.8 23.8 27.3Selenium ng/mL 9 12 28 16 17 23Iron Rg/dl 160 126 176 117 173 138TIBC Rg/dl 242 214 226 181 317 218Percent Saturation % 66 59 78 65 55 63IgG mg/mL 0.108 0.1 0.132 0.11 0.22 0.2

FOX-NY Hybrid % 96 102 90 112 114 104MRC-5 % 101 114 104 100 101 98CHO-K1 % 95 73 113 87 N/A 88VERO % N/A 90 N/A 103 N/A 92Bluetongue Virus Titer TCID50/mL 6.00 P0.50 6.33 P0.50 6.00 P0.50*Results not available/detectable


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Total Sterility Assurance

Many factors affect the total sterility assurance level of an animal-derived material like FBS. These factors should be considered for impactindividually and in combination. Viral load depends on the virus titerfound in nature, the material of origin, the extent of virus, cellassociation, the presence of antibodies against the virus, fetalseroconversion/tolerance to virus infection, filtration effects, storageconditions, and additional inactivation processes.

The safe use of animal-derived materials in the manufacture of productsfor human and animal use depends on an understanding of viral load,methods to reduce viral load, and the final application.

Recommendation

For manufacture of human and/or animal medicinals where the minimalrisk is essential, the following specifications for all animal serumproducts are recommended1. Low hemoglobin levels (P10 mg/dl).2. Low endotoxin levels (P10 EU/mL).3. Reliable traceability to countries without BSE and/or FMD.4. Serial filtration using 40 nm pore-size filters.5. Terminal sterilization with )2.5 Mrad (25 kGy) gamma irradiationusing validated procedures.

Compliance with these recommendations is state-of-the-art riskmanagement.

References1. BVD Virus, Contamination of Fetal Bovine Serum and Infection ofCell Cultures, 1986. Art to Science 5:3:1.

2. Brown, F., Lubiniecki, AA. Viral Safety and Evaluation of ViralClearance from Biopharmaceutical Products. Dev Biol Stand.88:283-290.

3. EEC regulatory document: Note for Guidance (1992) Guideline forMinimizing the Risk of Transmitting Agents Causing SpongiformEncephalopathy via Medicinal Products. Biologicals 18:155-158

4. EEC regulatory document: Note for Guidance (1991) Validation ofVirus Removal and Inactivation Procedures. Biologicals 19:247-251.

5. Council directive 92/118/EEC of 17 December 1992: Laying Down theAnimal Health and Public Health Requirements Governing Trade inImports into the Community of Products Not Subject to the SaidRequirements Laid Down in Specific Community Rules Referred to inAnnex A (1) to Directive 89/662/EEC and, as Regards to Pathogens,to Directive 90/425/EEC, Official Journal L, 62, 49-68, 15.3.93.

6. Draft commission decision of 1993: Laying Down the Animal HealthRequirements and Veterinary Certification for the Import Form ThirdCountries of Blood Products of Animals Not Intended for HumanConsumption and Amending Commission Decision 94/278/EC,VI/9440/93 Revision 10.

7. Hanson G, Wilkinson R, Black J, 1993. Gamma Radiation and VirusInactivation: New Findings of Old Theories. Art to Science; 12 : 2 : 1.

Figure 7: Three lots of virus-spiked equine serum treated by pHadjustment to 5.0 for 3 hours compared with untreated virusspiked controls for BVD, IBR, P13 , and PPV.

Acid Treatment—Equine Serum Log 10 Residual TCID50/mLLot V Control Treated

BVD 33112378 6.50 6.5033112414 7.00 6.5033112415 6.70 6.70

IBR 33112378 6.50 5.5033112414 7.50 6.3333112415 7.00 6.33

P13 33112378 7.50 6.7033112414 7.33 6.3333112415 7.50 6.70

PPV 33112378 7.33 6.0033112414 7.33 6.7033112415 7.33 6.33


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Single-Use Bioreactor (S.U.B.) for Vero Cell Cultureon Microcarriers

IntroductionMany vaccine or virus-related products utilizekidney-derived, adherent cells duringmanufacturing. Microcarrier cultures canprovide a large surface area for growth ofattachment-dependant cells in a small overallfootprint. Since microcarriers are maintained insuspension, they lend themselves to use instir-tanks or other types of bioreactors. TheThermo Scientific HyClone Single-UseBioreactor (S.U.B.) is designed as analternative to conventional stirred-tankbioreactors currently utilized in animal cellculture consisting of an outer support containerand disposable S.U.B. BioProcess Container(BPC). The disposable BPC designincorporates an impeller system, gas spargerand ports for integration of sensor probessimilar to their stainless-steel counterparts.With the gamma irradiated S.U.B. BPC, there isno need for cleaning or sterilizing betweenruns, providing for quick turnaround time withreduced capital and utility costs.

A current trend in the bioprocess industry is theincreased interest in using animal-derivedcomponent free (ADCF) materials in theirprocesses. In the case of adherent cell culture,microcarriers have often utilized collagen orother animal-derived material to promote cellattachment and spreading. The ThermoScientific HyClone line of microcarriers,HyQSpheres, are available with collagen orother attachment factors, but also includeoptions that are ADCF. The Plastic PlusHyQSphere is a solid, polystyrene beadwith an electrostatic charge which promotescell attachment.

In this study Vero cells were successfullycultured in a disposable, stirred-tank bioreactoron microcarriers using a serum-free medium in

an ADCF process. Through optimization ofparameters, such as bead concentration andcell seeding density, this system will be highlybeneficial to virus production processes byalleviating many concerns regardingcontamination, viral clearance and safety whileproviding the capability to rapidly transitionprocesses.

Materials andMethods

MaterialsVero cells (ATCC, CCL-81) were adapted toThermo Scientific HyCone SFM4MegaVir(Cat V SH30552), a serum-free, protein-free,ADCF medium developed to support virusproduction in a variety of cell lines. Materialsused in this study are described in Table 1.

MethodsBioreactor Setup and Microcarrier Preparation

The S.U.B. system was set up according to themanufacturer’s guidelines (consult User GuideUG003) and equipped with a pH/DO controlsystem (Applikon ADI 1010 Bio Controller andADI 1025 Bio Console). Direct air flow to thesparger was set to 0.1 lpm and the overlay gasflow setting was 0.2 lpm. SFM4MegaVir (20 L)was added aseptically by sterile tubingwelding of the media BPC to the addition lineof the S.U.B. Plastic Plus HyQSpheres (250 g)were autoclaved in PBS, washed twice thenresuspended in fresh culture medium. Themedium was allowed to warm to 37°C at anagitation rate of 30 rpm before themicrocarriers were pushed into the bioreactorvia positive pressure (bead density may requireprocess-specific optimization). Additionalculture medium was used to "chase" themicrocarriers into the bioreactor in order toflush any beads from the addition line intothe vessel.

Bioreactor SeedingThe bioreactor was seeded (via positivepressure) with a viable cell population densityof 5.0 x 108 cells in 25 L, or 2 x 104 cells/mL.The remaining volume of culture medium wasused to flush any remaining cells through theline and to bring the intermediate workingvolume to 25 L. Agitation speed was increasedto 50 rpm, providing enough agitation to

prevent settling of the microcarriers and pro-mote homogeneity throughout the workingvolume.

Culture MonitoringSamples were collected aseptically using aLuer lock syringe connected to the samplingport of the S.U.B. Samples were taken every 24hours after seeding to observe cell attachmenton beads, determine cell population density(stained nuclei counting) and monitor theculture biochemistry. On day five of the culture,the working volume was increased to 50 L tosupport the increasing cell density. Thisreduced the cell and microcarrier concentrationby one-half. Other methods, such as haltingagitation to allow settling of microcarriers andremoving half of the working volume, can beused to feed the cells without affecting theconcentration of cells and microcarriers.


Bioreactor Control and OperationCulture temperature was controlled duringthe run at 37°C. The pH of the culture wasmaintained by addition of CO2 only, andremained within allowable limits for the lengthof the culture. Additionally, the dissolvedoxygen (DO) level was maintained at thedesired level of 50% by addition of air throughthe integrated gas sparging systemof the S.U.B. Agitation speed was easilycontrolled during the run with the built-inmixing control. The bioreactor system wasmicroscopically evaluated and remained freeof contamination for the duration of theexperiment.

Microcarrier Culture EvaluationSeeding of the S.U.B. is best accomplishedthrough gravity feeding or positive pressure, aspassing the seed culture through a peristalticpump decreases cell viability and can cause aninitial lag in cell growth. During the attachmentphase, it is important to slow agitation to allowcells to attach and spread on the growthsurface, especially under serum-freeconditions. The lowest acceptable agitationrate capable of maintaining the microcarriers insuspension while allowing cell attachment wasdetermined to be 50 rpm. Microscopic


166

Conclusions

Key factors in a successful microcarrier cultureinclude: 1) the ability of the cells to attach andspread onto the microcarrier surface, 2) theability to provide adequate dissolved oxygen(DO) to the complete culture, and 3) the ability

Ordering Information:HardwarePart Number Description Additional InformationSV50171.01 50 L Single-Use Bioreactor, US

version (120 VAC) Includes: 304 stainless steel outer supportcontainer with swivel caster platform,variable speed agitation controller, motor,drive assembly with shaft, PID temperaturecontroller, RTD sensor, integrated resistiveheating element, probe shelf, and standardtool set

SV50172.01 250 L Single-Use Bioreactor, USversion (120 VAC)

SV50171.02 50 L Single-Use Bioreactor, EUversion (240 VAC)

SV50172.02 250 L Single-Use Bioreactor, EUversion (240 VAC)

ConsumablePart Number Description Additional InformationSH30715.01 50 L BPC for S.U.B.

(gamma-sterilized)For use with tubing welder and QuickConnects

SH30716.01 250 L BPC for S.U.B.(gamma-sterilized)


For use with tubing welder andTriclamps


Liquid MediumSFM4MegaVir All products are 0.2 Rm sterile filtered. Store at 2-8°CPart Number Volume Delivery SystemSH30552.01 500 mL PETE BottleSH30522.02 1000 mL PETE BottleSH30552.03 5 L BPC: Hanging Pillow BagSH30552.04 10 L BPC: Hanging Pillow BagSH30552.05 20 L BPC: Hanging Pillow BagSH30552.06 50 L BPC: 3-D BagSH30552.07 100 L BPC: 3-D BagSH30552.08 200 L BPC: 3-D BagSH30552.09 500 L BPC: 3-D Bag

MicrocarriersHyQSpheres Plastic Plus P 102-LP 102-L is a cross-linked polystyrene microcarrier (uncoated) with a nominal specificgravity of 1.02 and bead diameter of 125-212 Rm.Part Number PackagingSH30056.01 10 gSH30056.02 50 gSH30056.03 100 gSH30056.04 500 gSH30056.05 1000 g

observation of the microcarriers at 18 hourspost-seeding revealed that cells had attachedand spread on the surface of the beads andnearly half of the culture had undergonemitosis. Figure 1 shows the daily Vero cellpopulation density daily over a 15-day period.

The decrease in cell population seen on dayfive occurred when the working volume wasincreased to 50 L. Cells reached the peakdensity of 5.0 x 105 cells/mL on day 10 of theculture and were observed to be confluent. Thefinal microcarrier concentration of 5 g/Lappeared to not be optimized in this evaluationand, as a result, the surface area available tocell growth was limited. (Note:Microcarrierconcentrations of up to 20 g/L have been usedsuccessfully with the same combination ofmicrocarriers, cells and culture medium.)However, by calculating the number of cells percm2, the peak population density of 2.5 x 105

cells/cm2 is consistent with the density of aconfluent Vero cell culture in traditionalvessels. The average rate of growth over theentire culture period was approximately 36 hr.,a rate slightly lower than Vero cells cultured inSFM4MegaVir in static culture (28 to 30 hoursper doubling). However, this may be attributedto the low cell seeding density, an increasedattachment phase with microcarrier cultures,or the fact that these parameters were notoptimized for this experiment.

The culture biochemistry (Figure 1) wascomparable to other cultures of Vero cells inthis culture medium. Lactate reached nearly2 g/L, with a peak concentration atapproximately day 12. This is consistent withthe utilization of glucose during the logarithmicgrowth phase, predominantly between daysfour and 10. Inversely, glutamine wasconsumed rapidly during early culture throughday five. These utilization/production rates areindicative of a normal metabolic pattern withthis culture combination.

The culture was maintained for approximately15 days, exhibiting normal cell metabolism andgrowth kinetic within acceptable limits. A peakcell population density of approximately 2.5 x105 cells/cm2 was achieved by day 10, typicalof a confluent culture. Throughout this culture,the pH and DO were adequately maintained bysparging of CO2 and air only.

to maintain a generally homogenous suspensionthroughout the working volumewhile minimizingthe shear forces acting upon the cells. TheSingle-Use Bioreactor in combination withSFM4MegaVir and Plastic Plus HyQSpheressupported these required conditions to providea successful microcarrier culture.


167

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Table 2: Operating Parameters for50 L S.U.B. Microcarrier CultureParameter S.U.B. SettingFinal OperatingLiquid Volume

50 L

Seeding Cell/Volume Ratio

2 x 104 cells/mL

Agitation Rate 50 rpmTemperature 37 _0.1°CpH 6.8-7.1Dissolved Oxygen 40-60% air saturationAir OverlaySparge Rate

0.2 lpm

Air DirectSparge Rate

0.1 lpm

Table 1. Materials ListMaterial and Description Part Number Product LiteratureBioreactorHardware

Single-UseBioreactor (50 L)

SV50171 SM0500, UG003, DataSheet 026

Bioreactor BPC Single-UseBioreactor (5 L)

SH30715.01

Media(serum-free)

SFM4MegaVir SH30552 SM0417

Microcarrier Plastic PlusHyQSphere

SV30056 SM0454

Cell Line Vero cells CCL-81SalineSolution

D-PBS (PhosphateBuffered Saline)

SH30028 SM0424

DissociationReagent

HyQTase(non-mammalian)

SV30030.01


168

Formulation

Basal Medium Eagle with Earle’s Balanced SaltsLiquid and Dry Powdered Media

Part Number SH30159

Description mg/L mmol/LCaCl2 (anhydrous) 200 1.8021KCl 400 5.3655MgSO4 (anhydrous) 97.67 0.8112NaCl 6800 116.3586NaH2PO4·H2O 140 1.0145L-Arginine HCl 21 0.0996L-Cystine 2HCl 15.65 0.0499L-Glutamine 292 1.9979L-Histidine 8 0.0515L-Isoleucine 26 0.1982L-Leucine 26 0.1982L-Lysine HCl 36.47 0.1996L-Methionine 7.5 0.0502L-Phenylalanine 16.5 0.0998L-Threonine 24 0.2014L-Tryptophan 4 0.0195L-Tyrosine 2Na·2H2O 25.95 0.0993L-Valine 23.5 0.2005d-Biotin 1 0.004D-Ca Pantothenate 1 0.002Choline Chloride 1 0.0071Folic Acid 1 0.0022Myo-Inositol 2 0.0111Niacinamide 1 0.0081Pyridoxal HCl 1 0.0049Riboflavin 0.1 0.0002Thiamine HCl 1 0.0029D-Glucose 1000 5.5506Phenol Red (Sodium) 10 0.0265Add NaHCO3 2200 26.1873

Liquid Media

Part Number SH30157

Description mg/L mmol/L

CaCl2 (anhydrous) 200 1.8021KCl 400 5.3655MgSO4 (anhydrous) 97.67 0.8112NaCl 6800 116.3586NaH2PO4·H2O 140 1.0145L-Arginine HCl 21 0.0996L-Cystine 2HCl 15.65 0.0499L-Glutamine 292 1.9979L-Histidine 8 0.0515L-Isoleucine 26 0.1982L-Leucine 26 0.1982L-Lysine HCl 36.47 0.1996L-Methionine 7.5 0.0502L-Phenylalanine 16.5 0.0998L-Threonine 24 0.2014L-Tryptophan 4 0.0195L-Tyrosine 2Na·2H2O 25.95 0.0993L-Valine 23.5 0.2005d-Biotin 1 0.004D-Ca Pantothenate 1 0.002Choline Chloride 1 0.0071Folic Acid 1 0.0022Myo-Inositol 2 0.0111Niacinamide 1 0.0081Pyridoxal HCl 1 0.0049Riboflavin 0.1 0.0002Thiamine HCl 1 0.0029D-Glucose 1000 5.5506Phenol Red (Sodium) 10 0.0265NaHCO3 2200 26.1873

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Dry Powdered Media

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169

Form

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Formulation

Dulbecco’s Modified Eagle’s Medium/Nutrient MixtureF12 Ham’sDry Powdered Media

Part Number SH30004 SH30069

Description mg/L mmol/L mg/L mmol/L

CaCl2 (anhydrous) 116.61 1.0507 116.61 1.0507

CuSO4·5H2O 0.0013 0.00001 0.0013 0.00001

Fe(NO3)3·9H2O 0.05 0.0001 0.05 0.0001

FeSO4·7H2O 0.42 0.0015 0.42 0.0015

KCl 311.8 4.1824 311.8 4.1824

MgCl2 (anhydrous) 28.61 0.3005 28.61 0.3005

MgSO4 (anhydrous) 48.84 0.4056 48.84 0.4056

NaCl 6999.5 119.7724 6999.5 119.7724

Na2HPO4 (anhydrous) 71.02 0.5003 71.02 0.5002

NaH2PO4·H2O 62.5 0.4529 62.5 0.4529

ZnSO4·7H2O 0.43 0.0015 0.43 0.0015

L-Alanine 4.55 0.05 4.46 0.05

L-Arginine HCl 147.5 0.7002 147.5 0.7001

L-Asparagine H2O 7.5 0.0499 7.5 0.0499

L-Aspartic Acid 6.65 0.0499 6.65 0.0499

L-Cysteine HCI·H2O 17.56 0.0999 17.56 0.0999

L-Cystine 2HCl 31.29 0.0999 31.29 0.0998

L-Glutamic Acid 7.35 0.0499 7.35 0.0499

L-Glutamine 365 2.4974 365 2.4974

Glycine 18.75 0.2498 18.75 0.2497

L-Histidine HCl·H2O 31.48 0.1502 31.48 0.1501

L-Isoleucine 54.47 0.4153 54.47 0.4152

L-Leucine 59.05 0.4502 59.05 0.4501

L-Lysine HCl 91.26 0.4996 91.25 0.4995

L-Methionine 17.24 0.1155 17.24 0.1155

L-Phenylalanine 35.48 0.2148 35.48 0.2147

L-Proline 17.25 0.1498 17.25 0.1498

L-Serine 26.25 0.2498 26.25 0.2497

L-Threonine 53.45 0.4487 53.45 0.4487

L-Tryptophan 9.02 0.0442 9.02 0.0441

L-Tyrosine 2Na·2H2O 55.79 0.2136 55.79 0.2135

L-Valine 52.85 0.4511 52.85 0.4511

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Formulation

Dulbecco’s Modified Eagle’s Medium/Nutrient MixtureF12 Ham’sDry Powdered Media (continued)



d-Biotin 0.0036 0.00002 0.0037 0.00002

D-Ca Pantothenate 2.24 0.0047 2.24 0.0047

Choline Chloride 8.98 0.0643 8.98 0.0643

Folic Acid 2.65 0.006 2.65 0.006

Myo-Inositol 12.6 0.0699 12.6 0.0699

Niacinamide 2.02 0.0165 2.02 0.0165

Pyridoxine HCl 2.03 0.0099 2.03 0.0098

Riboflavin 0.22 0.0006 0.22 0.0005

Thiamine HCl 2.17 0.0064 2.17 0.0064

Vitamin B12 0.68 0.0005 0.68 0.0005

D-Glucose 3151 17.49 3151 17.49

HEPES 3575 15.002 0 0

Hypoxanthine 2Na 2.7 0.015 2.7 0.0149

Linoleic Acid 0.05 0.0002 0.05 0.0001

Lipoic Acid 0.1 0.0005 0.105 0.0005

Phenol Red (Sodium) 8.6 0.0229 8.6 0.0228

Putrescine 2HCl 0.08 0.0005 0.08 0.0005

Sodium Pyruvate 55 0.4999 55 0.4998

Thymidine 0.37 0.0015 0.37 0.0015

Add NaHCO3 1200 14.284 1200 14.284

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171

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Formulation

Dulbecco’s Modified Eagle’s Medium/Nutrient MixtureHam’s F12Liquid Media

Part Number SH30023 SH30261 SH30126 SH30271

Description mg/L mmol/L mg/L mmol/L mg/L mmol/L mg/L mmol/L

CaCl2 (anhydrous) 116.61 1.0507 116.61 1.0507 116.69 1.0514 116.61 1.0507

CuSO4·5H2O 0.0013 0.00001 0.0013 0.00001 0.0013 0.00001 0.0013 0.00001

Fe(NO3)3·9H2O 0.05 0.0001 0.05 0.0001 0.05 0.0001 0.05 0.0001

FeSO4·7H2O 0.42 0.0015 0.42 0.0015 0.42 0.0015 0.42 0.0015

KCl 311.8 4.1824 311.8 4.1824 312 4.18524 311.8 4.1824

MgCl2 (anhydrous) 28.61 0.3005 0 0 28.63 0.3007 28.61 0.3005

MgSO4 (anhydrous) 48.84 0.4056 84.95 0.7056 48.87 0.4059 48.84 0.4056

NaCl 6999.5 119.7724 7000 119.781 7004 119.85 6999.5 119.7724

Na2HPO4 (anhydrous) 71.02 0.5003 71.02 0.5003 71.07 0.5006 71.02 0.5002

NaH2PO4·H2O 62.5 0.4529 62.5 0.4529 62.5 0.4532 62.5 0.4529

ZnSO4·7H2O 0.43 0.0015 0.43 0.0015 0.43 0.0015 0.43 0.0015

L-Alanine 4.45 0.05 4.45 0.05 4.46 0.05 4.46 0.05

L-Arginine HCl 147.5 0.7002 147.51 0.7002 147.61 0.7007 147.5 0.7001

L-Asparagine H2O 7.5 0.0499 7.501 0.05 7.5 0.05 7.5 0.0499

L-Aspartic Acid 6.65 0.0499 6.65 0.05 6.65 0.05 6.65 0.0499

L-Cysteine HCI·H2O 17.56 0.0999 17.56 0.1 17.57 0.1 17.56 0.0999

L-Cystine 2HCl 31.29 0.0999 31.29 0.0999 31.31 0.0999 31.29 0.0998

L-Glutamic Acid 7.35 0.0499 7.35 0.05 7.36 0.05 7.35 0.0499

L-Glutamine 365 2.4974 365 2.4974 0 0 365 2.4974

Glycine 18.75 0.2498 18.75 0.2498 18.76 0.2498 18.75 0.2497

L-Histidine HCl·H2O 31.48 0.1502 31.48 0.1502 31.50 0.1502 31.48 0.1501

L-Isoleucine 54.47 0.4153 54.47 0.4153 54.51 0.4155 54.47 0.4152

L-Leucine 59.05 0.4502 59.05 0.4502 59.09 0.4505 59.05 0.4501

L-Lysine HCl 91.26 0.4996 91.26 0.4996 91.32 0.4999 91.25 0.4995

L-Methionine 17.24 0.1155 17.24 0.1155 17.25 0.1155 17.24 0.1155

L-Phenylalanine 35.48 0.2148 35.48 0.2148 35.51 0.2148 35.48 0.2147

L-Proline 17.25 0.1498 17.25 0.1498 17.26 0.1498 17.25 0.1498

L-Serine 26.25 0.2498 26.25 0.2498 26.27 0.2498 26.25 0.2497

L-Threonine 53.45 0.4487 53.55 0.4495 53.49 0.449 53.45 0.4487

L-Tryptophan 9.02 0.0442 9.02 0.0442 9.03 0.0442 9.02 0.0441

L-Tyrosine 2Na·2H2O 55.79 0.2136 55.81 0.2137 55.83 0.2136 55.79 0.2135

L-Valine 52.85 0.4511 52.85 0.4511 52.88 0.4514 52.85 0.4511

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Formulation

Dulbecco’s Modified Eagle’s Medium/Nutrient MixtureHam’s F12

Liquid Media (continued)



d-Biotin 0.0036 0.00002 0.0036 0.00002 0.0037 0.00002 0.0037 0.00002

D-Ca Pantothenate 2.24 0.0047 2.24 0.0047 2.24 0.0047 2.24 0.0047

Choline Chloride 8.98 0.0643 8.98 0.0643 8.99 0.0643 8.98 0.0643

Folic Acid 2.65 0.006 2.65 0.006 2.65 0.006 2.65 0.006

Myo-Inositol 12.6 0.0699 12.6 0.0699 12.6 0.0699 12.6 0.0699

Niacinamide 2.02 0.0165 2.02 0.0165 2.02 0.0165 2.02 0.0165

Pyridoxal HCl 0 0 0 0 2 0.0098 0 0

Pyridoxine HCl 2.03 0.0099 2.03 0.0099 0.03 0.0002 2.03 0.0098

Riboflavin 0.22 0.0006 0.22 0.0006 0.22 0.0006 0.22 0.0005

Thiamine HCl 2.17 0.0064 2.17 0.0064 2.17 0.0064 2.17 0.0064

Vitamin B12 0.68 0.0005 0.68 0.0005 0.68 0.0005 0.68 0.0005

D-Glucose 3151 17.49 3151 17.49 3153 17.50 3151 17.49

HEPES 3575 15.0021 3574 15 3577 15.01 0 0

Hypoxanthine 2Na 2.7 0.015 2.39 0.0132 2.7 0.015 2.7 0.0149

Linoleic Acid 0.05 0.0002 0.04 0.0002 0.05 0.0002 0.05 0.0001

Lipoic Acid 0.10 0.0005 0.105 0.0005 0.105 0.0005 0.105 0.0005

Phenol Red (Sodium) 8.6 0.0229 8.1 0.0215 8.6 0.0229 8.6 0.0228

Putrescine 2HCl 0.08 0.0005 0.08 0.0005 0.08 0.0005 0.08 0.0005

Sodium Pyruvate 55 0.4999 110 0.9996 55 0.5 55 0.4998

Thymidine 0.37 0.0015 0.37 0.0015 0.37 0.0015 0.37 0.0015

NaHCO3 1200 14.284 2438 29.0204 1200 14.284 1200 14.284

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Formulation

Dulbecco’s Modified Eagle’s Medium/Nutrient MixtureHam’s F12Liquid Media (continued)

Part Number SH30272


CaCl2 (anhydrous) 116.61 1.0507

CuSO4·5H2O 0.0013 0.00001

Fe(NO3)3·9H2O 0.05 0.0001

FeSO4·7H2O 0.42 0.0015

KCl 311.8 4.1824

MgCl2 (anhydrous) 28.61 0.3005

MgSO4 (anhydrous) 48.84 0.4056

NaCl 6999.5 119.7724

NaH2PO4 (anhydrous) 71.02 0.5003

NaH2PO4·H2O 62.5 0.4529

ZnSO4·7H2O 0.43 0.0015

L-Alanine 4.46 0.05

L-Arginine HCl 147.5 0.7002

L-Asparagine H2O 7.5 0.0499

L-Aspartic Acid 6.65 0.0499

L-Cysteine HCI·H2O 17.56 0.0999

L-Cystine 2HCl 31.29 0.0998

L-Glutamic Acid 7.35 0.0499

L-Glutamine 365 2.4974

Glycine 18.75 0.2498

L-Histidine HCl·H2O 31.48 0.1502

L-Isoleucine 54.47 0.4152

L-Leucine 59.05 0.4502

L-Lysine HCl 91.25 0.4996

L-Methionine 17.24 0.1155

L-Phenylalanine 35.48 0.2148

L-Proline 17.25 0.1498

L-Serine 26.25 0.2498

L-Threonine 53.45 0.4487

L-Tryptophan 9.02 0.0442

L-Tyrosine 2Na·2H2O 55.79 0.2136

L-Valine 52.85 0.4511

Part Number SH30272


d-Biotin 0.0037 0.00002

D-Ca Pantothenate 2.24 0.0047

Choline Chloride 8.98 0.0643

Folic Acid 2.65 0.006

Myo-Inositol 12.6 0.0699

Niacinamide 2.02 0.0165

Pyridoxine HCl 2.03 0.0099

Riboflavin 0.22 0.0006

Thiamine HCl 2.17 0.0064

Vitamin B12 0.68 0.0005

D-Glucose 3151 17.49

Hypoxanthine 2Na 2.7 0.015

Linoleic Acid 0.05 0.0002

Lipoic Acid 0.105 0.0005

Putrescine 2HCl 0.08 0.0005

Sodium Pyruvate 55 0.4998

Thymidine 0.37 0.0015

NaHCO3 1200 14.284

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174

Formulation

Dulbecco’s Modified Eagle’s MediumHigh Glucose—Dry Powdered Media

Part Number SH30003 SH30045 SH30053

Description mg/L mmol/L mg/L mmol/L mg/L mmol/L

CaCl2 (anhydrous) 200 1.8021 200 1.8021 200 1.8021

Fe(NO3)3·9H2O 0.1 0.0002 0.1 0.0002 0.1 0.0002

KCl 400 5.3655 400 5.3655 400 5.3655

MgSO4 (anhydrous) 97.67 0.8112 97.67 0.8112 97.67 0.8112

NaCl 6400 109.514 6400 109.514 6400 109.514

NaH2PO4·H2O 125 0.9059 125 0.9059 125 0.9059

L-Arginine HCl 84 0.3987 84 0.3987 84 0.3987

L-Cystine 2HCl 62.57 0.1998 62.57 0.1998 62.57 0.1998

L-Glutamine 584 3.9959 584 3.9959 0 0

Glycine 30 0.3996 30 0.3996 30 0.3996

L-Histidine HCl·H2O 42 0.2004 42 0.2004 42 0.2004

L-Isoleucine 104.8 0.7989 104.8 0.7989 104.8 0.7989

L-Leucine 104.8 0.7989 104.8 0.7989 104.8 0.7989

L-Lysine HCl 146.2 0.8004 146.2 0.8004 146.2 0.8004

L-Methionine 30 0.2011 30 0.2011 30 0.2011

L-Phenylalanine 66 0.3995 66 0.3995 66 0.3995

L-Serine 42 0.3997 42 0.3997 42 0.3997

L-Threonine 95.2 0.7992 95.2 0.7992 95.2 0.7992

L-Tryptophan 16 0.0783 16 0.0783 16 0.0783

L-Tyrosine 2Na·2H2O 103.79 0.3974 103.79 0.3974 103.79 0.3974

L-Valine 93.6 0.7989 93.6 0.7989 93.6 0.799

D-Ca Pantothenate 4 0.0084 4 0.0084 4 0.0084

Choline Chloride 4 0.0286 4 0.0286 4 0.0286

Folic Acid 4 0.0091 4 0.0091 4 0.0091

Myo-Inositol 7 0.0389 7 0.0389 7 0.0389

Niacinamide 4 0.0328 4 0.0328 4 0.0328

Pyridoxine HCl 4 0.0195 4 0.0195 4 0.0195

Riboflavin 0.4 0.0011 0.4 0.0011 0.4 0.0011

Thiamine HCl 4 0.0119 4 0.0119 4 0.0119

D-Glucose 4500 24.9778 4500 24.9778 4500 24.9778

Phenol Red (Sodium) 15.9 0.0422 15.9 0.0422 15.9 0.0422

Sodium Pyruvate 0 0 110 0.9996 0 0

Add NaHCO3 3700 44.0424 3700 44.0424 3700 44.0423

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Formulation

Dulbecco’s Modified Eagle’s MediumHigh Glucose—Dry Powdered Media (continued)



CaCl2 (anhydrous) 0 0 200 1.8021

Fe(NO3)3·9H2O 0.1 0.0002 0.1 0.0002

KCl 400 5.3655 400 5.3655

MgSO4 (anhydrous) 0 0 97.67 0.8112

NaCl 6400 109.514 6400 109.514

NaH2PO4·H2O 125 0.9059 125 0.9059

L-Arginine HCl 84 0.3987 84 0.3987

L-Cystine 2HCl 62.57 0.1998 62.57 0.1998

L-Glutamine 0 0 584 3.9959

Glycine 30 0.3996 30 0.3996

L-Histidine HCl·H2O 42 0.2004 42 0.2004

L-Isoleucine 104.8 0.7989 104.8 0.7989

L-Leucine 104.8 0.7989 104.8 0.7989

L-Lysine HCl 146.2 0.8004 146.2 0.8004

L-Methionine 30 0.2011 30 0.2011

L-Phenylalanine 66 0.3995 66 0.3995

L-Serine 42 0.3997 42 0.3997

L-Threonine 95.2 0.7992 95.2 0.7992

L-Tryptophan 16 0.0783 16 0.0783

L-Tyrosine 2Na·2H2O 103.79 0.3974 103.79 0.3974

L-Valine 93.6 0.799 93.6 0.799

D-Ca Pantothenate 0 0 4 0.0084

D-Na Pantothenic Acid 4 0.0166 0 0

Choline Chloride 4 0.0286 4 0.0286

Folic Acid 4 0.0091 4 0.0091

Myo-Inositol 7 0.0389 7 0.0389

Niacinamide 4 0.0328 4 0.0328

Pyridoxine HCl 4 0.0195 4 0.0195

Riboflavin 0.4 0.0011 0.4 0.0011

Thiamine HCl 4 0.0119 4 0.0119

D-Glucose 4500 24.9778 4500 24.9778

Phenol Red (Sodium) 15.9 0.0422 0 0

Add NaHCO3 3700 44.0424 3700 44.0424

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176

Formulation

Dulbecco’s Modified Eagle’s MediumHigh Glucose—Liquid Media

Part Number SH30022 SH30081 SH30284 SH30243 SH30249

Description mg/L mmol/L mg/L mmol/L mg/L mmol/L mg/L mmol/L mg/L mmol/L

CaCl2 (anhydrous) 200 1.8021 200 1.8021 200 1.8021 200 1.8021 200 1.8021

Fe(NO3)3·9H2O 0.1 0.0002 0.1 0.0002 0.1 0.0002 0.1 0.0002 0.1 0.0002

KCl 400 5.3655 400 5.3655 400 5.3655 400 5.3655 400 5.3655

MgSO4 (anhydrous) 97.67 0.8112 97.67 0.8112 97.67 0.8112 97.67 0.8112 97.67 0.8112

NaCl 6400 109.514 6400 109.514 6400 109.514 6400 109.514 4750 81.2799

NaH2PO4·H2O 125 0.9059 125 0.9059 125 0.9059 125 0.9059 125 0.9059

L-Arginine HCl 84 0.3987 84 0.3987 84 0.3987 84 0.3987 84 0.3987

L-Cystine 2HCl 62.57 0.1998 62.57 0.1998 62.57 0.1998 62.57 0.1998 62.57 0.1998

L-Glutamine 584 3.9959 0 0 584 3.9959 584 3.9959 584 3.9959

Glycine 30 0.3996 30 0.3996 30 0.3996 30 0.3996 30 0.3996

L-Histidine HCl·H2O 42 0.2004 42 0.2004 42 0.2004 42 0.2004 42 0.2004

L-Isoleucine 104.8 0.7989 104.8 0.7989 104.8 0.7989 104.8 0.7989 104.8 0.7989

L-Leucine 104.8 0.7989 104.8 0.7989 104.8 0.7989 104.8 0.7989 104.8 0.7989

L-Lysine HCl 146.2 0.8004 146.2 0.8004 146.2 0.8004 146.2 0.8004 146.2 0.8004

L-Methionine 30 0.2011 30 0.2011 30 0.2011 30 0.2011 30 0.2011

L-Phenylalanine 66 0.3995 66 0.3995 66 0.3995 66 0.3995 66 0.3995

L-Serine 42 0.3997 42 0.3997 42 0.3997 42 0.3997 42 0.3997

L-Threonine 95.2 0.7992 95.2 0.7992 95.2 0.7992 95.2 0.7992 95.2 0.7992

L-Tryptophan 16 0.0783 16 0.0783 16 0.0783 16 0.0783 16 0.0783

L-Tyrosine 2Na·2H2O 103.79 0.3974 103.79 0.3974 103.79 0.3974 103.79 0.3974 103.79 0.3974

L-Valine 93.6 0.799 93.6 0.799 93.6 0.799 93.6 0.799 93.6 0.799

D-Ca Pantothenate 4 0.0084 4 0.0084 4 0.0084 4 0.0084 4 0.0084

Choline Chloride 4 0.0286 4 0.0286 4 0.0286 4 0.0286 4 0.0286

Folic Acid 4 0.0091 4 0.0091 4 0.0091 4 0.0091 4 0.0091

Myo-Inositol 7 0.0389 7 0.0389 7 0.0389 7 0.0389 7 0.0389

Niacinamide 4 0.0328 4 0.0328 4 0.0328 4 0.0328 4 0.0328

Pyridoxine HCl 4 0.0195 4 0.0195 4 0.0195 4 0.0195 4 0.0195

Riboflavin 0.4 0.0011 0.4 0.0011 0.4 0.0011 0.4 0.0011 0.4 0.0011

Thiamine HCl 4 0.0119 4 0.0119 4 0.0119 4 0.0119 4 0.0119

D-Glucose 4500 24.9778 4500 24.9778 4500 24.9778 4500 24.9778 4500 24.9778

HEPES 0 0 0 0 0 0 0 0 5958 25.002

Phenol Red (Sodium) 15.9 0.0422 15.9 0.0422 0 0 15.9 0.0422 15.9 0.0422

Sodium Pyruvate 0 0 0 0 0 0 110 0.9996 0 0

NaHCO3 3700 44.0424 3700 44.0424 3700 44.0424 3700 44.0424 3700 44.0424

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Formulation

Dulbecco’s Modified Eagle’s MediumHigh Glucose—Liquid Media (continued)



CaCl2 (anhydrous) 200 1.8021 0 0 200 1.8021 200 1.8021

Fe(NO3)3·9H2O 0.1 0.0002 0.1 0.0002 0.1 0.0002 0.1 0.0002

KCl 400 5.3655 400 5.3655 400 5.3655 400 5.3655

MgSO4 (anhydrous) 97.67 0.8112 0 0 97.67 0.8112 97.67 0.8112

NaCl 6400 109.514 6400 109.514 6400 109.514 6400 109.514

NaH2PO4·H2O 125 0.9059 125 0.9059 125 0.9059 125 0.9059

L-Arginine HCl 84 0.3987 84 0.3987 84 0.3987 84 0.3987

L-Cystine 2HCl 62.57 0.1998 62.57 0.1998 62.57 0.1998 62.57 0.1998

L-Glutamine 0 0 0 0 0 0 0 0

Glycine 30 0.3996 30 0.3996 30 0.3996 30 0.3996

L-Histidine HCl·H2O 42 0.2004 42 0.2004 42 0.2004 42 0.2004

L-Isoleucine 104.8 0.7989 104.8 0.7989 104.8 0.7989 104.8 0.7989

L-Leucine 104.8 0.7989 104.8 0.7989 104.8 0.7989 104.8 0.7989

L-Lysine HCl 146.2 0.8004 146.2 0.8004 146.2 0.8004 146.2 0.8004

L-Methionine 30 0.2011 30 0.2011 30 0.2011 30 0.2011

L-Phenylalanine 66 0.3995 66 0.3995 66 0.3995 66 0.3995

L-Serine 42 0.3997 42 0.3997 42 0.3997 42 0.3997

L-Threonine 95.2 0.7992 95.2 0.7992 95.2 0.7992 95.2 0.7992

L-Tryptophan 16 0.0783 16 0.0783 16 0.0783 16 0.0783

L-Tyrosine 2Na·2H2O 103.79 0.3974 103.79 0.3974 103.79 0.3974 103.79 0.3974

L-Valine 93.6 0.799 93.6 0.799 93.6 0.799 93.6 0.799

D-Ca Pantothenate 4 0.0084 0 0 4 0.0084 4 0.0084

D-Na Pantothenic Acid 0 0 4 0.0166 0 0 0 0

Choline Chloride 4 0.0286 4 0.0286 4 0.0286 4 0.0286

Folic Acid 4 0.0091 4 0.0091 4 0.0091 4 0.0091

Myo-Inositol 7 0.0389 7 0.0389 7 0.0389 7 0.0389

Niacinamide 4 0.0328 4 0.0328 4 0.0328 4 0.0328

Pyridoxine HCl 4 0.0195 4 0.0195 4 0.0195 4 0.0195

Riboflavin 0.4 0.0011 0.4 0.0011 0.4 0.0011 0.4 0.0011

Thiamine HCl 4 0.0119 4 0.0119 4 0.0119 4 0.0119

D-Glucose 4500 24.9778 4500 24.9778 4500 24.9778 4500 24.9778

HEPES 0 0 0 0 0 0 0 0

Phenol Red (Sodium) 15.9 0.0422 15.9 0.0422 0 0 0 0

Sodium Pyruvate 110 0.9996 0 0 0 0 110 0.9996

NaHCO3 3700 44.0424 3700 44.0424 3700 44.0424 3700 44.0424

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Formulation

Dulbecco’s Modified Eagle’s MediumHigh Glucose—Liquid Media (continued)



CaCl2 (anhydrous) 200 1.8021 200 1.8021 200 1.8021

Fe(NO3)3·9H2O 0.1 0.0002 0.1 0.0002 0.1 0.0002

KCl 400 5.3655 400 5.3655 400 5.3655

MgSO4 (anhydrous) 97.67 0.8112 97.67 0.8112 97.67 0.8112

NaCl 6400 109.514 6400 109.514 6400 109.514

NaH2PO4·H2O 125 0.9059 0 0 125 0.9059

L-Arginine HCl 84 0.3987 84 0.3987 84 0.3987

L-Cystine 2HCl 0 0 62.57 0.1998 62.57 0.1998

Glycine 30 0.3996 30 0.3996 30 0.3996

L-Histidine HCl·H2O 42 0.2004 42 0.2004 42 0.2004

L-Isoleucine 104.8 0.7989 104.8 0.7989 104.8 0.7989

L-Leucine 104.8 0.7989 104.8 0.7989 104.8 0.7989

L-Lysine HCl 146.2 0.8004 146.2 0.8004 146.2 0.8004

L-Methionine 0 0 30 0.2011 30 0.2011

L-Phenylalanine 66 0.3995 66 0.3995 66 0.3995

L-Serine 42 0.3997 42 0.3997 42 0.3997

L-Threonine 95.2 0.7992 95.2 0.7992 95.2 0.7992

L-Tryptophan 16 0.0783 16 0.0783 16 0.0783

L-Tyrosine 2Na·2H2O 103.79 0.3974 103.79 0.3974 103.79 0.3974

L-Valine 93.6 0.799 93.6 0.799 93.6 0.799



Folic Acid 4 0.0091 4 0.0091 4 0.0091

Myo-Inositol 7 0.0389 7 0.0389 7 0.0389

Niacinamide 4 0.0328 4 0.0328 4 0.0328

Pyridoxine HCl 4 0.0195 4 0.0195 4 0.0195

Riboflavin 0.4 0.0011 0.4 0.0011 0.4 0.0011

Thiamine HCl 4 0.0119 4 0.0119 4 0.0119

D-Glucose 4500 24.9778 4500 24.9778 4500 24.9778

HEPES 0 0 0 0 5958 25.002


Sodium Pyruvate 110 0.9996 110 0.9996 110 0.9996

NaHCO3 3700 44.0424 3700 44.0424 3700 44.0424

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Formulation

Dulbecco’s Modified Eagle’s MediumLow Glucose—Dry Powdered Media

SH30002 SH30044

Part Number


CaCl2 (anhydrous) 200 1.8021 200 1.8021

Fe(NO3)3·9H2O 0.1 0.0002 0.1 0.0002

KCl 400 5.3655 400 5.3655

MgSO4 (anhydrous) 97.46 0.8095 97.67 0.8112

NaCl 6400 109.514 6400 109.514

NaH2PO4·H2O 125 0.9059 125 0.9059

L-Arginine HCl 84 0.3987 84 0.3987

L-Cystine 2HCl 62.57 0.1998 62.57 0.1998

L-Glutamine 584 3.9959 584 3.9959

Glycine 30 0.3996 30 0.3996


L-Isoleucine 104.8 0.7989 104.8 0.7989

L-Leucine 104.8 0.799 104.8 0.799

L-Lysine HCl 146.2 0.8004 146.2 0.8004

L-Methionine 30 0.2011 30 0.2011


L-Serine 42 0.3997 42 0.3997

L-Threonine 95.2 0.7992 95.2 0.7992

L-Tryptophan 16 0.0783 16 0.0783

L-Tyrosine 2Na·2H2O 103.79 0.3974 103.79 0.3974

L-Valine 93.6 0.799 93.6 0.799

D-Ca Pantothenate 4 0.0084 4 0.0084


Folic Acid 4 0.0091 4 0.0091

Myo-Inositol 7 0.0389 7 0.0389

Niacinamide 4 0.0328 4 0.0328


Riboflavin 0.4 0.0011 0.4 0.0011

Thiamine HCl 4 0.0119 4 0.0119

D-Glucose 1000 5.5506 1000 5.5506


Sodium Pyruvate 110 0.9996 110 0.9996

Add NaHCO3 3700 44.0424 3700 44.0424

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180

Formulation

Dulbecco’s Modified Eagle’s MediumLow Glucose—Liquid Media

Part Number SH30021


CaCl2 (anhydrous) 200 1.8021

Fe(NO3)3·9H2O 0.1 0.0002

KCl 400 5.3655


NaCl 6400 109.514

NaH2PO4·H2O 125 0.9059

L-Arginine HCl 84 0.3987



Glycine 30 0.3996

L-Histidine HCl·H2O 42 0.2004


L-Leucine 104.8 0.799


L-Methionine 30 0.2011

L-Phenylalanine 66 0.3995

L-Serine 42 0.3997


L-Tryptophan 16 0.0783

L-Tyrosine 2Na·2H2O 103.79 0.3974

L-Valine 93.6 0.799

D-Ca Pantothenate 4 0.0084

Choline Chloride 4 0.0286

Folic Acid 4 0.0091

Myo-Inositol 7 0.0389

Niacinamide 4 0.0328

Pyridoxine HCl 4 0.0195


Thiamine HCl 4 0.0119

D-Glucose 1000 5.5506

Phenol Red (Sodium) 15.9 0.0422


NaHCO3 3700 44.0424

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Form

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Formulation

Ham’s Nutrient Mixture F10Dry Powdered Media

Part Number SH30009



CuSO4·5H2O 0.0025 0.00001

FeSO4·7H2O 0.83 0.003

KCl 285 3.8229

KH2PO4 83 0.6099


NaCl 7400 126.6256

Na2HPO4 (anhydrous) 153.58 1.0819

ZnSO4·7H2O 0.03 0.0001

L-Alanine 8.91 0.1


L-Asparagine H2O 15 0.0999


L-Cysteine HCI·H2O 25 0.2063


L-Glutamine 146.2 1.0003

Glycine 7.51 0.1



L-Leucine 13.1 0.0999




L-Proline 11.5 0.0999

L-Serine 10.5 0.0999




L-Valine 3.5 0.0299

Part Number SH30009


d-Biotin 0.02 0.0001











Vitamin B12 1.36 0.001


D-Glucose 1100 6.1057



Add NaHCO3 1200 14.284

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182

Formulation

Ham’s Nutrient Mixture F10Liquid Media

Part Number SH30025



CuSO4·5H2O 0.0025 0.00001

FeSO4·7H2O 0.83 0.003

KCl 285 3.8229

KH2PO4 83 0.6099


NaCl 7400 126.6256


ZnSO4·7H2O 0.03 0.0001

L-Alanine 8.91 0.1







Glycine 7.51 0.1



L-Leucine 13.1 0.0999




L-Proline 11.5 0.0999

L-Serine 10.5 0.0999




L-Valine 3.5 0.0299

Part Number SH30025


d-Biotin 0.02 0.0001











Vitamin B12 1.36 0.001


D-Glucose 1100 6.1057



NaHCO3 1200 14.284

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183

Form

ulations

Formulation

Ham’s Nutrient Mixture F12Dry Powdered Media



CaCl2 (anhydrous) 33.22 0.2993 33.22 0.2993 33.22 0.2993

CuSO4·5H2O 0.0025 0.00001 0.0025 0.00001 0.0025 0.00001

FeSO4·7H2O 0.83 0.003 0.83 0.003 0.83 0.003

KCl 223.65 3 223.65 3 223.65 3

MgCl2 (anhydrous) 57.22 0.601 57.22 0.601 57.22 0.601

NaCl 7599 130.0308 7599 130.0308 7599 130.0308

Na2HPO4 (anhydrous) 142.04 1.0006 142.04 1.0006 142.04 1.0006

ZnSO4·7H2O 0.86 0.003 0.86 0.003 0.86 0.003

L-Alanine 8.91 0.1 8.91 0.1 8.91 0.1

L-Arginine HCl 210.7 1.0002 210.7 1.0002 210.7 1.0002

L-Asparagine H2O 15.01 0.1 15.01 0.1 15.01 0.1

L-Aspartic Acid 13.3 0.0999 13.3 0.0999 13.3 0.0999

L-Cysteine HCI·H2O 35.12 0.2 35.12 0.2 35.12 0.2

L-Glutamic Acid 14.7 0.0999 14.7 0.0999 14.7 0.0999

L-Glutamine 146.2 1.0003 0 0 146.2 1.0003

Glycine 7.51 0.1 7.51 0.1 7.51 0.1

L-Histidine HCl·H2O 20.96 0.1 20.96 0.1 20.96 0.1

L-Isoleucine 3.94 0.03 3.94 0.03 3.94 0.03

L-Leucine 13.12 0.1 13.12 0.1 13.12 0.1

L-Lysine HCl 36.54 0.2001 36.54 0.2001 36.54 0.2001

L-Methionine 4.48 0.03 4.48 0.03 4.48 0.03

L-Phenylalanine 4.96 0.03 4.96 0.03 4.96 0.03

L-Proline 34.53 0.2999 34.53 0.2999 34.53 0.2999

L-Serine 10.51 0.1 10.51 0.1 10.51 0.1

L-Threonine 11.91 0.1 11.91 0.1 11.91 0.1

L-Tryptophan 2.04 0.01 2.04 0.01 2.04 0.01

L-Tyrosine 2Na·2H2O 7.78 0.0298 7.78 0.0298 7.78 0.0298

L-Valine 11.71 0.1 11.71 0.1 11.71 0.1

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184

Formulation

Ham’s Nutrient Mixture F12Dry Powdered Media (continued)



d-Biotin 0.00733 0.00003 0.00733 0.00003 0.00733 0.00003

D-Ca Pantothenate 0.24 0.0005 0.24 0.0005 0.24 0.0005

Choline Chloride 13.96 0.1 13.96 0.1 13.96 0.1

Folic Acid 1.3 0.0029 1.3 0.0029 1.3 0.0029

Hypoxanthine 2Na 5.4 0.03 5.4 0.03 5.4 0.03

Myo-Inositol 18.02 0.1 18.02 0.1 18.02 0.1

Niacinamide 0.04 0.0003 0.04 0.0003 0.04 0.0003

Pyridoxine HCl 0.06 0.0003 0.06 0.0003 0.06 0.0003

Riboflavin 0.04 0.0001 0.04 0.0001 0.04 0.0001

Thiamine HCl 0.34 0.001 0.34 0.001 0.34 0.001

Thymidine 0.73 0.003 0.73 0.003 0.73 0.003

Vitamin B12 1.36 0.001 1.36 0.001 1.36 0.001

Lipoic Acid 0.2 0.001 0.2 0.001 0.2 0.001

Linoleic Acid 0.09 0.0003 0.09 0.0003 0.09 0.0003

D-Glucose 1801.6 10 1801.6 10 1801.6 10


Sodium Pyruvate 110 0.9996 110 0.9996 110 0.9996

Putrescine 2HCI 0.16 0.001 0.16 0.001 0.16 0.001

Add NaHCO3 1176 13.9983 1176 13.9983 1176 13.9983

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Form

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Formulation

Ham’s Nutrient Mixture F12Liquid Media

Part Number SH30026 SH30526 Kaighn’s


CaCl2 (anhydrous) 33.22 0.2993 102 0.9191

CuSO4·5H2O 0.0025 0.00001 0.002 0.000008

FeSO4·7H2O 0.83 0.003 0.80 0.003

KCl 223.65 3 285 3.8229

KH2PO4 0 0 59 0.4335

MgCl2 (anhydrous) 57.22 0.601 49.7 0.522

MgSO4 (anhydrous) 0 0 192 1.5947

NaCl 7599 130.0308 7530 128.8501

Na2HPO4 (anhydrous) 142.04 1.0006 115.5 0.8136

ZnSO4·7H2O 0.86 0.003 0.14 0.0005

L-Alanine 8.91 0.1 18 0.202

L-Arginine HCl 210.7 1.0002 422 2.0032

L-Asparagine H2O 15.01 0.1 30 0.1998

L-Aspartic Acid 13.3 0.0999 26.6 0.1998

L-Cysteine HCI·H2O 35.12 0.2 70 0.3985

L-Glutamic Acid 14.7 0.0999 29 0.1971

L-Glutamine 146.2 1.0003 292 1.9979

Glycine 7.51 0.1 15 0.1998

L-Histidine HCl·H2O 20.96 0.1 45.8 0.2185

L-Isoleucine 3.94 0.03 7.88 0.0601

L-Leucine 13.12 0.1 26.2 0.1997

L-Lysine HCl 36.54 0.2001 73 0.3997

L-Methionine 4.48 0.03 8.96 0.06

L-Phenylalanine 4.96 0.03 9.92 0.0601

L-Proline 34.53 0.2999 69 0.5993

L-Serine 10.51 0.1 21 0.1998

L-Threonine 11.91 0.1 23 0.1931

L-Tryptophan 2.04 0.01 4.1 0.0201

L-Tyrosine 2Na·2H2O 7.78 0.0298 13.5 0.0517

L-Valine 11.71 0.1 23 0.1963

Part Number SH30026 SH30526 Kaighn's

SH30312


d-Biotin 0.00733 0.00003 0.07 0.0003


Choline Chloride 13.96 0.1 14 0.1003

Folic Acid 1.3 0.0029 1.3 0.0029

Hypoxanthine Na 5.4 0.03 4 0.025

Myo-Inositol 18.02 0.1 18 0.0999

Niacinamide 0.04 0.0003 0.04 0.0003

Pyridoxine HCl 0.06 0.0003 0.06 0.0003

Riboflavin 0.04 0.0001 0.04 0.0001

Thiamine HCl 0.34 0.001 0.30 0.0001

Thymidine 0.73 0.003 0.70 0.003

Vitamin B12 1.36 0.001 1.4 0.001

Lipoic Acid 0.20 0.001 0.21 0.001

Linoleic Acid 0.09 0.0003 0 0

D-Glucose 1801.6 10 1260 6.9938

Phenol Red (Sodium) 1.3 0.0035 3 0.008

Sodium Pyruvate 110 0.9996 220 1.9993

Putrescine 2HCI 0.16 0.001 0.32 0.002

NaHCO3 1176 13.9983 2500 29.7584

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186

Formulation

Iscove’s Modified Dulbecco’s MediumDry Powdered Media



CaCl2 (anhydrous) 165 1.4868 165 1.4868

KCl 330 4.4266 330 4.4266

KNO3 0.08 0.0008 0.08 0.0008

MgSO4 (anhydrous) 97.67 0.8112 97.67 0.8112

NaCl 4505 77.0876 4505 77.0876

Na2SE3 (anhydrous) 0.01 0.0001 0.01 0.0001

NaH2PO4·H2O 125 0.9059 125 0.9059

L-Alanine 25 0.2806 25 0.2806

L-Arginine HCl 84 0.3987 84 0.3987

L-Asparagine H2O 28.4 0.1892 28.4 0.1892

L-Aspartic Acid 30 0.2254 30 0.2254

L-Cystine 2HCl 91.24 0.2913 91.24 0.2913

L-Glutamic Acid 75 0.5098 75 0.5098

L-Glutamine 584 3.9959 0 0

Glycine 30 0.3996 30 0.3996


L-Isoleucine 105 0.8004 105 0.8004

L-Leucine 105 0.8005 105 0.8005

L-Lysine HCl 146 0.7993 146 0.7993

L-Methionine 30 0.2011 30 0.2011


L-Proline 40 0.3474 40 0.3474

L-Serine 42 0.3997 42 0.3997

L-Threonine 95 0.7975 95 0.7975

L-Tryptophan 16 0.0783 16 0.0783

L-Tyrosine 2Na·2H2O 103.79 0.3974 103.79 0.3974

L-Valine 94 0.8024 94 0.8024



d-Biotin 0.01 0.0001 0.01 0.0001



Folic Acid 4 0.0091 4 0.0091

Myo-Inositol 7.2 0.04 7.2 0.04

Niacinamide 4 0.0328 4 0.0328

Pyridoxal HCl 4 0.0196 4 0.0196

Riboflavin 0.4 0.0011 0.4 0.0011

Thiamine HCl 4 0.0119 4 0.0119

Vitamin B12 0.013 0.00001 0.013 0.00001

D-Glucose 4500 24.9778 4500 24.9778

HEPES 5958 25.0021 5958 25.0021


Sodium Pyruvate 110 0.9996 110 0.9996

Add NaHCO3 3024 35.9957 3024 35.9957

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Form

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Formulation

Iscove’s Modified Dulbecco’s MediumLiquid Media



CaCl2 (anhydrous) 165 1.4868 165 1.4868

KCl 330 4.4266 330 4.4266

KNO3 0.08 0.0008 0.08 0.0008

MgSO4 (anhydrous) 97.67 0.8112 97.67 0.8112

NaCl 4505 77.0876 4505 77.0876

Na2SE3 (anhydrous) 0.01 0.0001 0.01 0.0001

NaH2PO4·H2O 125 0.9059 125 0.9059

L-Alanine 25 0.2806 25 0.2806

L-Arginine HCl 84 0.3987 84 0.3987

L-Asparagine H2O 28.4 0.1892 28.4 0.1892

L-Aspartic Acid 30 0.2254 30 0.2254

L-Cystine 2HCl 91.24 0.2913 91.24 0.2913

L-Glutamic Acid 75 0.5098 75 0.5098

L-Glutamine 584 3.9959 0 0

Glycine 30 0.3996 30 0.3996


L-Isoleucine 105 0.8004 105 0.8004

L-Leucine 105 0.8005 105 0.8005

L-Lysine HCl 146 0.7993 146 0.7993

L-Methionine 30 0.2011 30 0.2011


L-Proline 40 0.3474 40 0.3474

L-Serine 42 0.3997 42 0.3997

L-Threonine 95 0.7975 95 0.7975

L-Tryptophan 16 0.0783 16 0.0783

L-Tyrosine 2Na·2H2O 103.79 0.3974 103.79 0.3974

L-Valine 94 0.8024 94 0.8024



d-Biotin 0.01 0.0001 0.01 0.0001



Folic Acid 4 0.0091 4 0.0091

Myo-Inositol 7.2 0.04 7.2 0.04

Niacinamide 4 0.0328 4 0.0328

Pyridoxal HCl 4 0.0196 4 0.0196

Riboflavin 0.4 0.0011 0.4 0.0011

Thiamine HCl 4 0.0119 4 0.0119

Vitamin B12 0.013 0.00001 0.013 0.00001

D-Glucose 4500 24.9778 4500 24.9778

HEPES 5958 25.0021 5958 25.0021


Sodium Pyruvate 110 0.9996 110 0.9996

NaHCO3 3024 35.9957 3024 35.9957

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188

Formulation

Leibovitz L-15 MediumDry Powdered and Liquid Media

Part Number SH30048



KCl 400 5.3655

KH2PO4 60 0.4409



NaCl 8000 136.8925

Na2HPO4 (anhydrous) 190 1.3384

L-Alanine 225 2.5255





Glycine 200 2.6642


L-Isoleucine 125 0.9529

L-Leucine 125 0.95

L-Lysine HCl 75 0.51



L-Serine 200 1.9031

L-Threonine 300 2.5185


L-Tyrosine 300 1.6556

L-Valine 100 0.8536



Folic Acid 1 0.0023





Thiamine PO4Cl 2H2O 1.1 0.0026

D (+) Galactose 900 4.9956



Part Number SH30525



KCl 400 5.3655

KH2PO4 60 0.4409



NaCl 8000 136.8925

Na2HPO4 (anhydrous) 190 1.3384






Glycine 200 2.6642



L-Leucine 125 0.95




L-Serine 200 1.9031



L-Tyrosine 300 1.6556

L-Valine 100 0.8536



Folic Acid 1 0.0023





Thiamine PO4Cl 2H2O 1.1 0.0026

D (+) Galactose 900 4.9956



Dry Powdered Media Liquid MediaInorga

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189

Form

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Formulation

Medium 199 Earle’sDry Powdered Media



CaCl2 (anhydrous) 200 1.8021 200 1.8021 200 1.8021

C2H3O2Na 0 0 0 0 50 0.6095

Fe(NO3)3·9H2O 0.72 0.0018 0.72 0.0018 0.72 0.0018

KCl 400 5.3655 400 5.3655 400 5.3655

MgSO4 (anhydrous) 97.67 0.8112 97.67 0.8112 97.67 0.8112

NaCl 6800 116.3587 6800 116.3587 6800 116.3587

NaC2H3O·3H2O 83 0.6099 83 0.6099 0 0

NaH2PO4·H2O 140 1.015 140 1.015 140 1.015

L-Alanine 25 0.2806 25 0.2806 25 0.2806

L-Arginine HCl 70 0.3323 70 0.3323 70 0.3323

L-Aspartic Acid 30 0.2254 30 0.2254 30 0.2254

L-Cysteine HCI·H2O 0.11 0.0006 0.11 0.0006 0.10 0.0006

L-Cystine 2HCl 26 0.083 26 0.083 12 0.0383

L-Glutamic Acid 66.82 0.4542 66.9 0.4547 75 0.5098

L-Glutamine 100 0.6842 100 0.6842 100 0.6842

Glycine 50 0.666 50 0.666 50 0.666


Hydroxy-L-Proline 10 0.0763 10 0.0763 10 0.0763

L-Isoleucine 20 0.1525 20 0.1525 20 0.1525

L-Leucine 60 0.4574 60 0.4574 60 0.4574

L-Lysine HCl 70 0.3832 70 0.3832 70 0.3832

L-Methionine 15 0.1005 15 0.1005 15 0.1005

L-Phenylalanine 25 0.1513 25 0.1513 25 0.1513

L-Proline 40 0.3474 40 0.3474 40 0.3474

L-Serine 25 0.2379 25 0.2379 25 0.2379

L-Threonine 30 0.2518 30 0.2518 30 0.2518

L-Tryptophan 10 0.049 10 0.049 10 0.049

L-Tyrosine 2Na·2H2O 57.66 0.2208 57.66 0.2208 45 0.1723

L-Valine 25 0.2134 25 0.2134 25 0.2134

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190

Formulation

Medium 199 Earle’sDry Powdered Media (continued)



L-Ascorbic Acid 0.05 0.0003 0.05 0.0003 0.05 0.0003

d-Biotin 0.01 0.00004 0.01 0.00004 0.01 0.00004



Folic Acid 0.01 0.00002 0.01 0.00002 0.01 0.00002

Myo-Inositol 0.05 0.0003 0.05 0.0003 0.05 0.0003

Niacin 0.03 0.0002 0.02 0.0002 0.02 0.0002

Niacinamide 0.03 0.0002 0.02 0.0002 0.02 0.0002

Pyridoxal HCl 0.03 0.0001 0.02 0.0001 0.02 0.0001

Pyridoxine HCl 0.03 0.0001 0.02 0.0001 0.02 0.0001

Riboflavin 0.01 0.00003 0.01 0.00003 0.01 0.00003

Thiamine HCl 0.01 0.00003 0.01 0.00003 0.01 0.00003

Vitamin D2-Ergocalciferol 0.1 0.0003 0.1 0.0003 0.1 0.0003

Menadione 0.01 0.0001 0.01 0.0001 0.01 0.0001

Vitamin A Acetate 0.14 0.0004 0.14 0.0004 0.1 0.0003

DL-Alpha Tocoph.-PO4-2Na 0.01 0.00002 0.01 0.00002 0.01 0.00002

Adenine Sulfate 2H2O 10 0.0247 10 0.0247 10 0.0247

ATP 2Na 1 0.0017 1 0.0017 1 0.0017

AMP 0.2 0.0005 0.2 0.0005 0.2 0.0005

2 -Deoxy- D-ribose 0.5 0.0037 0.5 0.0037 0.5 0.0037

Guanine HCl 0.3 0.0016 0.3 0.0016 0.3 0.0016

Hypoxanthine 2Na 0.4 0.0022 0.4 0.0022 0.4 0.0022

D-Ribose 0.5 0.0033 0.5 0.0033 0.5 0.0033

D-Glucose 1000 5.5506 1000 5.5506 1000 5.5506

Thymine 0.3 0.0024 0.3 0.0024 0.3 0.0024

Uracil 0.3 0.0027 0.3 0.0027 0.3 0.0027

Xanthine Na 0.34 0.0018 0.34 0.0018 0.34 0.0018

L-Glutathione (reduced) 0.05 0.0002 0.05 0.0002 0.05 0.0002

Phenol Red (Sodium) 21.3 0.0566 0 0 20 0.0531

PABA 0.05 0.0004 0.05 0.0004 0.05 0.0004

Cholesterol 0.2 0.0005 0.2 0.0005 0.2 0.0005

Tween 80 18.82 N/A 18.79 N/A 18.80 N/A

Add NaHCO3 2200 26.1874 2200 26.1874 2200 26.1874

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191

Form

ulations

Formulation

Medium 199 Earle’sLiquid Media



CaCl2 (anhydrous) 200 1.8021 140 1.2615 140 1.2615

Fe(NO3)3·9H2O 0.72 0.0018 0.72 0.0018 0.72 0.0018

KCl 400 5.3655 400 5.3655 400 5.3655

KH2PO4 (anhydrous) 0 0 60 0.441 60 0.441

NaCl 6800 116.3587 8000 136.8925 8000 136.8925

NaC2H3O2·3H2O 83 0.6099 50 0.3674 50 0.3674

NaH2PO4·H2O 140 1.015 0 0 0 0

MgSO4 (anhydrous) 97.67 0.8112 97.7 0.8115 97.7 0.8115

Na2HPO4·H2O 0 0 48 0.3381 48 0.3381

L-Alanine 25 0.2806 25 0.2806 25 0.2806

L-Arginine HCl 70 0.3323 70 0.3323 70 0.3323

L-Aspartic Acid 30 0.2254 30 0.2254 30 0.2254

L-Cysteine HCI·H2O 0.11 0.0006 0.11 0.0006 0.11 0.0006

L-Cystine 2HCl 26 0.083 26 0.083 26 0.083

L-Glutamic Acid 66.82 0.4542 75 0.5098 75 0.5098

L-Glutamine 100 0.6842 100 0.6842 100 0.6842

Glycine 50 0.666 50 0.666 50 0.666

L-Histidine HCI·H2O 21.88 0.1044 21.88 0.1044 21.88 0.1044

Hydroxy-L-Proline 10 0.0763 10 0.0763 10 0.0763

L-Isoleucine 20 0.1525 20 0.1525 20 0.1525

L-Leucine 60 0.4574 60 0.4574 60 0.4574

L-Lysine HCl 70 0.3832 70 0.3832 70 0.3832

L-Methionine 15 0.1005 15 0.1005 15 0.1005

L-Phenylalanine 25 0.1513 25 0.1513 25 0.1513

L-Proline 40 0.3474 40 0.3474 40 0.3474

L-Serine 25 0.2379 25 0.2379 25 0.2379

L-Threonine 30 0.2518 30 0.2518 30 0.2518

L-Tryptophan 10 0.049 10 0.049 10 0.049

L-Tyrosine 2Na·2H2O 57.66 0.2208 57.88 0.2216 57.88 0.2216

L-Valine 25 0.2134 25 0.2134 25 0.2134

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192

Formulation

Medium 199 Earle’sLiquid Media (continued)



L-Ascorbic Acid 0.05 0.0003 0.05 0.0003 0.05 0.0003

d-Biotin 0.01 0.00004 0.01 0.00004 0.01 0.00004



Folic Acid 0.01 0.00002 0.01 0.00002 0.01 0.00002

Myo-Inositol 0.05 0.0003 0.05 0.0003 0.05 0.0003

Niacin 0.03 0.0002 0.03 0.0002 0.03 0.0002

Niacinamide 0.03 0.0002 0.03 0.0002 0.03 0.0002

Pyridoxal HCl 0.03 0.0001 0.03 0.0001 0.03 0.0001

Pyridoxine HCl 0.03 0.0001 0.03 0.0001 0.03 0.0001

Riboflavin 0.01 0.00003 0.01 0.00003 0.01 0.00003

Thiamine HCl 0.01 0.00003 0.01 0.00003 0.01 0.00003

DL-Alpha Tocoph.-PO4-2Na 0.01 0.00002 0.01 0.00002 0.01 0.00002

Vitamin D2-Ergocalciferol 0.1 0.0003 0.1 0.0003 0.1 0.0003

Menadione 0.01 0.0001 0.01 0.0001 0.01 0.0001

Vitamin A Acetate 0.14 0.0004 0.14 0.0004 0.14 0.0004

Adenine Sulfate 2H2O 10 0.0247 10 0.0247 10 0.0247

ATP 2Na 1 0.0017 1 0.0017 1 0.0017

AMP 0.2 0.0005 0.2 0.0005 0.2 0.0005

2 -Deoxy- D-ribose 0.5 0.0037 0.5 0.0037 0.5 0.0037

Guanine HCl 0.3 0.0016 0.3 0.0016 0.3 0.0016

Hypoxanthine 2Na 0.40 0.0022 0.3 0.0017 0.3 0.0017

D-Glucose 1000 5.5506 1000 5.5506 1000 5.5506

D-Ribose 0.5 0.0033 0.5 0.0033 0.5 0.0033

Thymine 0.3 0.0024 0.3 0.0024 0.3 0.0024

Uracil 0.3 0.0027 0.3 0.0027 0.3 0.0027

Xanthine Na 0.34 0.0016 0.3 0.0016 0.3 0.0016

L-Glutathione (reduced) 0.05 0.0002 0.05 0.0002 0.05 0.0002

Phenol Red (Sodium) 21.3 0.0566 20 0.0531 20 0.0531

PABA 0.05 0.0004 0.05 0.0004 0.05 0.0004

Cholesterol 0.2 0.0005 0.2 0.0005 0.2 0.0005

Tween 80 18.82 N/A 18.8 N/A 18.8 N/A

NaHCO3 2200 26.1874 350 4.1662 350 4.1662

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193

Form

ulations

Formulation

McCoy’s 5A MediumDry Powdered Media

Part Number SH30049



KCl 400 5.3655


NaCl 6460 110.5407

NaH2PO4·H2O 580 4.2032

L-Alanine 13.36 0.15

L-Arginine HCl 42.14 0.2

L-Asparagine H2O 45.03 0.2999





Glycine 7.51 0.1

L-Histidine 20.96 0.1

Hydroxy L-Proline 19.67 0.15


L-Leucine 39.36 0.3




L-Proline 17.27 0.15

L-Serine 26.28 0.25




L-Valine 17.57 0.15

Part Number SH30049


d-Biotin 0.2 0.0008



Folic Acid 10 0.0227

L-Ascorbic Acid 0.5 0.0028


Niacin 0.5 0.004


Para-Aminobenzoic Acid (PABA) 1 0.0073

Pyridoxal HCl 0.5 0.0025




Vitamin B12 2 0.0015

Bacto Peptone G 600 N/A

D-Glucose 3000 16.6519

L-Glutathione (reduced) 0.5 0.0016

Phenol Red (Sodium) 11 0.0292

Add NaHCO3 2200 26.1874

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194

Formulation

McCoy’s 5A MediumLiquid Media


Description mg/L mmol/L mg/L mmol/L mg/L mmol/LCaCl2 (anhydrous) 100 0.9011 100 0.9011 100 0.9011KCl 400 5.3655 400 5.3655 400 5.3655MgSO4 (anhydrous) 97.67 0.8112 97.67 0.8112 97.67 0.8112NaCl 6460 110.5407 6460 110.5407 6460 110.5407NaH2PO4·H2O 580 4.2032 580 4.2032 580 4.2032L-Alanine 13.36 0.15 13.36 0.15 13.36 0.15L-Arginine HCl 42.14 0.2 42.14 0.2 42.14 0.2L-Asparagine H2O 45.03 0.2999 45.03 0.2999 45.03 0.2999L-Aspartic Acid 19.97 0.15 19.97 0.15 19.97 0.15L-Cysteine HCI·H2O 35.14 0.2 35.14 0.20 35.14 0.20Hydroxy-L-Proline 19.67 0.15 19.67 0.15 19.67 0.15L-Glutamic Acid 22.07 0.15 22.07 0.15 22.07 0.15L-Glutamine 219.15 1.4995 219.15 1.4995 219.15 1.4995Glycine 7.51 0.1 7.51 0.1 7.51 0.1L-Histidine 20.96 0.1 20.96 0.1 20.96 0.1L-Isoleucine 39.36 0.3 39.36 0.3 39.36 0.3L-Leucine 39.36 0.3 39.36 0.3 39.36 0.3L-Lysine HCl 36.54 0.2 36.54 0.2 36.54 0.2L-Methionine 14.92 0.1 14.92 0.1 14.92 0.1L-Phenylalanine 16.52 0.1 16.52 0.1 16.52 0.1L-Proline 17.27 0.15 17.27 0.15 17.27 0.15L-Serine 26.28 0.25 26.28 0.25 26.28 0.25L-Threonine 17.87 0.15 17.87 0.15 17.87 0.15L-Tryptophan 3.06 0.015 3.06 0.015 3.06 0.015L-Tyrosine 2Na·2H2O 26.1 0.0999 26.1 0.0999 26.1 0.0999L-Valine 17.57 0.15 17.57 0.15 17.57 0.15d-Biotin 0.2 0.0008 0.2 0.0008 0.2 0.0008D-Ca Pantothenate 0.2 0.0004 0.2 0.0004 0.2 0.0004Choline Chloride 5 0.0358 5 0.0358 5 0.0358Folic Acid 10 0.0227 10 0.0227 10 0.0227L-Ascorbic Acid 0.5 0.0028 0.5 0.0028 0.5 0.0028Myo-Inositol 36 0.1998 36 0.1998 36 0.1998Niacin 0.5 0.004 0.5 0.004 0.5 0.004Niacinamide 0.5 0.004 0.5 0.004 0.5 0.004Para-Aminobenzoic Acid (PABA) 1 0.0073 1 0.0073 1 0.0073Pyridoxal HCl 0.5 0.0025 0.5 0.0025 0.5 0.0025Pyridoxine HCl 0.5 0.0024 0.5 0.0024 0.5 0.0024Riboflavin 0.2 0.0005 0.2 0.0005 0.2 0.0005Thiamine HCl 0.2 0.0006 0.2 0.0006 0.2 0.0006Vitamin B12 2 0.0015 2 0.0015 2 0.0015Bacto Peptone G 600 N/A 600 N/A 600 N/AD-Glucose 3000 16.6519 3000 16.6519 3000 16.6519HEPES 0 0 5598 25 0 0L-Glutathione (reduced) 0.5 0.0016 0.5 0.0016 0.5 0.0016Phenol Red (Sodium) 11 0.0292 11 0.0292 0 0NaHCO3 2200 26.1874 2200 26.1874 2200 26.1874

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195

Form

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Formulation

Minimum Essential MediumDry Powdered Media


(AlphaMod.)


CaCl2 (anhydrous) 200 1.8021 200 1.8021 200 1.8021

KCl 400 5.3655 400 5.3655 400 5.3655

MgSO4 (anhydrous) 97.67 0.8112 97.67 0.8112 97.67 0.8112

NaCl 6800 116.359 6800 116.3587 6800 116.3587

NaH2PO4·H2O 140 1.0146 140 1.0146 140 1.0146

L-Alanine 0 0 0 0 25 0.28

L-Arginine HCl 126.98 0.6028 126.98 0.6028 126.98 0.6028

L-Asparagine H2O 0 0 0 0 50 0.333

L-Aspartic Acid 0 0 0 0 30 0.2254

L-Cysteine HCI·H2O 0 0 0 0 100 0.5693

L-Cystine 2HCl 31.28 0.0999 31.28 0.0999 31.28 0.0999

L-Glutamic Acid 0 0 0 0 75 0.5098

L-Glutamine 0 0 292 1.9979 292 1.9979

Glycine 0 0 0 0 50 0.666


L-Isoleucine 52 0.3964 52 0.3964 52.5 0.4002

L-Leucine 52 0.3964 52 0.3964 52.4 0.3995

L-Lysine HCl 72.47 0.3968 72.47 0.3968 72.47 0.3968

L-Methionine 15 0.1005 15 0.1005 15 0.1005

L-Phenylalanine 32 0.1937 32 0.1937 32 0.1937

L-Proline 0 0 0 0 40 0.3474

L-Serine 0 0 0 0 25 0.2379

L-Threonine 48 0.403 48 0.403 48 0.403

L-Tryptophan 10 0.049 10 0.049 10 0.049

L-Tyrosine 2Na·2H2O 51.9 0.1987 51.9 0.1987 51.9 0.1987

L-Valine 46 0.3927 46 0.3927 46 0.3927

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196

Formulation

Minimum Essential MediumDry Powdered Media (continued)

Part Number SH30054 SH30171 SH30219(Alpha Mod.)


L-Ascorbic Acid 0 0 0 0 50 0.2839

d-Biotin 0 0 0 0 0.1 0.0004



Folic Acid 1 0.0023 1 0.0023 1 0.0023

Myo-Inositol 2 0.0111 1 0.0056 2 0.0111

Niacinamide 1 0.0082 1 0.0082 1 0.0082

Pyridoxal HCl 1 0.0049 1 0.0049 1 0.0049

Riboflavin 0.1 0.0003 0.1 0.0003 0.1 0.0003

Thiamine HCl 1 0.003 1 0.003 1 0.003

Vitamin B12 0 0 0 0 1.36 0.001

D-Glucose 1000 5.5506 1000 5.5506 1000 5.5506

Lipioc Acid 0 0 0 0 0.2 0.001

Phenol Red (Sodium) 11 0.0292 0 0 11 0.0292

Sodium Pyruvate 0 0 0 0 110 0.9996

Add NaHCO3 2200 26.1874 2200 26.1874 2200 26.1874

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Part Number SH30193 SH30327 SH30007 SH30269Glasgow’s


CaCl2 (anhydrous) 140 1.26 200 1.8021 200 1.8021 200 1.8021

Fe(NO3)3·9H2O 0 0 0 0 0 0 0.1 0.0002

KCl 400 5.3655 400 5.3655 400 5.3655 400 5.3655

MgSO4 (anhydrous) 97.67 0.8112 97.67 0.8112 97.67 0.8112 97.67 0.8112

NaCl 8000 136.8925 6800 116.3587 6800 116.3587 6400 109.514

KH2PO4 60 0.4409 0 0 0 0 0 0

NaH2PO4·H2O 0 0 140 1.0146 140 1.0146 124 0.8986

Na2HPO4 47.58 0.3352 0 0 0 0 0 0

L-Alanine 0 0 0 0 25 0.28 0 0

L-Arginine HCl 126.98 0.6028 126 0.5981 126.98 0.6028 42 0.1994

L-Asparagine H2O 0 0 0 0 50 0.333 0 0

L-Aspartic Acid 0 0 0 0 30 0.2254 0 0

L-Cysteine HCI·H2O 0 0 0 0 100 0.5693 0 0

L-Cystine 2HCl 31.28 0.0999 31.30 0.0999 31.28 0.0999 31.3 0.0999

L-Glutamic Acid 0 0 0 0 75 0.5098 0 0

L-Glutamine 292 1.9979 0 0 292 1.9979 292 1.9979

Glycine 0 0 0 0 50 0.666 0 0

L-Histidine HCl·H2O 41.88 0.1998 42 0.2004 41.88 0.1998 21 0.1002

L-Isoleucine 52 0.3964 52 0.3964 52.5 0.4002 52.4 0.3995

L-Leucine 52 0.3964 52 0.3964 52.4 0.3995 52.4 0.3995

L-Lysine HCl 72.47 0.3968 72.5 0.3969 72.47 0.3968 73.1 0.4002

L-Methionine 15 0.1005 15 0.1005 15 0.1005 15 1.0053

L-Phenylalanine 32 0.1937 32 0.1937 32 0.1937 33 0.1998

L-Proline 0 0 0 0 40 0.3474 0 0

L-Serine 0 0 0 0 25 0.2379 0 0

L-Threonine 48 0.403 48 0.403 48 0.403 47.6 0.3996

L-Tryptophan 10 0.049 10 0.049 10 0.049 8 0.0392

L-Tyrosine 2Na·2H2O 51.9 0.1987 0 0 51.9 0.1987 60.42 0.2313

L-Tyrosine 0 0 36 0.1987 0 0 0 0

L-Valine 46 0.3927 46 0.3927 46 0.3927 46.8 0.3995

197

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Formulation


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198

Formulation


Part Number SH30193 SH30327 SH30007 SH30269Glasgow’s


L-Ascorbic Acid 0 0 0 0 50 0.2839 0 0

d-Biotin 0 0 0.02 0.0001 0.1 0.0004 0 0

D-Ca Pantothenate 1 0.0021 1 0.0021 1 0.0021 2 0.0042

Choline Chloride 1 0.0072 0 0 1 0.0072 2 0.0143

Folic Acid 1 0.0023 1 0.0023 1 0.0023 2 0.0045

Myo-Inositol 2 0.0111 2 0.0111 2 0.0111 3.6 0.02

Niacinamide 1 0.0082 1 0.0082 1 0.0082 2 0.0164

Pyridoxal HCl 1 0.0049 1 0.0049 1 0.0049 2 0.0098

Riboflavin 0.1 0.0003 0.1 0.0003 0.1 0.0003 0.2 0.0005

Thiamine HCl 1 0.003 1 0.003 1 0.003 2 0.0059

Vitamin B12 0 0 0 0 1.36 0.001 0 0

Adenosine 0 0 0 0 10 0.0374 0 0

Cytidine 0 0 0 0 10 0.0411 0 0

Choline Bitartrate 0 0 1.8 0.007 0 0 0 0

D-Glucose 1000 5.5506 1000 5.5506 1000 5.5506 4500 24.9778

Guanosine 0 0 0 0 10 0.0353 0 0

Lipioc Acid 0 0 0 0 0.2 0.001 0 0

Phenol Red (Sodium) 11 0.0292 6.4 0.017 11 0.0292 16 0.0425

Sodium Pyruvate 0 0 0 0 110 0.9996 0 0

Thymidine 0 0 0 0 10 0.0413 0 0

2’-Deoxyadenosine H2O 0 0 0 0 10 0.0371 0 0

2’-Deoxycytidine HCI 0 0 0 0 11 0.0417 0 0

2’-Deoxyguanosine 0 0 0 0 10 0.0351 0 0

Sodium Succinate 6H2O 0 0 100 0.3702 0 0 0 0

Succinic Acid 0 0 75 0.6351 0 0 0 0

Uridine 0 0 0 0 10 0.041 0 0

Add NaHCO3 2200 26.1874 2200 26.1874 2200 26.1874 2200 26.1874

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199

Form

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Formulation

Minimum Essential MediumLiquid Media

Part Number SH30024 SH30235Suspension

SH30244


CaCl2 (anhydrous) 200 1.8021 0 0 200 1.8021

KCl 400 5.3655 400 5.3655 400 5.3655

MgSO4 (anhydrous) 97.67 0.8112 0 0 97.67 0.8112

MgCl2.6H2O 0 0 0 0 0 0

MgSO4·7H2O 0 0 0 0 0 0

NaCl 6800 116.3587 6800 116.3587 6800 116.359

NaH2PO4·H2O 140 1.0146 1400 10.15 140 1.0146

Ferric Chloride ·6H2O 0 0 0 0 0 0

ZnSO4·7H2O 0 0 0 0 0 0

Linoleic Acid 0 0 0 0 0 0

L-Alanine 0 0 0 0 0 0

L-Arginine HCl 126.98 0.6028 126 0.60 126.98 0.6028

L-Asparagine H2O 0 0 0 0 0 0

L-Aspartic Acid 0 0 0 0 0 0

L-Cysteine HCI·H2O 0 0 0 0 0 0

L-Cystine 2HCl 31.28 0.0999 31 0.0999 31.28 0.0999

L-Glutamic Acid 0 0 0 0 0 0

L-Glutamine 292 1.9979 292 1.9979 0 0

Glycine 0 0 0 0 0 0

L-Histidine HCl·H2O 41.88 0.1998 42 0.2004 41.88 0.1998

L-Isoleucine 52 0.3964 52 0.3964 52 0.3964

L-Leucine 52 0.3964 52 0.3964 52 0.3964

L-Lysine HCl 72.47 0.3968 73 0.3997 72.47 0.3968

L-Methionine 15 0.1005 15 0.1005 15 0.1005

L-Phenylalanine 32 0.1937 32 0.1937 32 0.1937

L-Proline 0 0 0 0 0 0

L-Serine 0 0 0 0 0 0

L-Threonine 48 0.403 48 0.403 48 0.403

L-Tryptophan 10 0.049 10 0.049 10 0.049

L-Tyrosine 2Na·2H2O 51.9 0.1987 52 0.199 51.9 0.1987

L-Valine 46 0.3927 46 0.3927 46 0.3927

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200

Formulation

Minimum Essential MediumLiquid Media (continued)

Part Number SH30024 SH30235Suspension

SH30244




Folic Acid 1 0.0023 1 0.0023 1 0.0023

Myo-Inositol 2 0.0111 2 0.0111 2 0.0111

Niacinamide 1 0.0082 1 0.0082 1 0.0082

Pyridoxal HCl 1 0.0049 1 0.0049 1 0.0049

Riboflavin 0.1 0.0003 0.1 0.0003 0.1 0.0003

Thiamine HCl 1 0.003 1 0.003 1 0.003

Cytidine 0 0 0 0 0 0

D-Glucose 1000 5.5506 1000 5.5506 1000 5.5506

Phenol Red (Sodium) 11 0.0292 10 0.0266 11 0.0292

NaHCO3 2200 26.1874 2200 26.1874 2200 26.1874

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Form

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Formulation



SH30568(Alpha Mod.)

SH30603Suspension


CaCl2 (anhydrous) 200 1.8021 200 1.8021 200 1.8021 0 0 0 0

CaCl2.2H2O 0 0 0 0 0 0 265 1.8026 0 0

KCl 400 5.3655 400 5.3655 400 5.3655 400 5.3655 400 5.3655

MgSO4 (anhydrous) 0 0 0 0 97.67 0.8112 0 0 0 0

MgCl2.6H2O 183 0.9001 183 0.9001 0 0 0 0 0 0

MgSO4·7H2O 25 0.1014 25 0.1014 0 0 200 0.8114 0 0

NaCl 6800 116.3587 6800 116.3587 6800 116.3587 6800 116.3587 6800 116.3587

NaH2PO4·H2O 150 1.087 150 1.087 140 1.0146 140 1.0146 140 1.0146

Ferric Chloride ·6H2O 0.54 0.002 0.54 0.002 0 0 0 0 0 0

ZnSO4·7H2O 0.14 0.0005 0.14 0.0005 0 0 0 0 0 0

Linoleic Acid 0.09 0.0003 0.09 0.0003 0 0 0 0 0 0

L-Alanine 0 0 0 0 25 0.28 25 0.2806 0 0

L-Arginine HCl 127 0.6029 127 0.6029 126.98 0.6028 126.4 0.06 126.98 0.6028

L-Asparagine H2O 60 0.3997 60 0.3997 50 0.333 50 0.333 0 0

L-Aspartic Acid 0 0 0 0 30 0.2254 30 0.2254 0 0

L-Cysteine HCI·H2O 0 0 0 0 100 0.5693 100 0.5693 0 0

L-Cystine 2HCl 31.28 0.0999 31.28 0.0999 31.28 0.0999 24 0.0999 31.28 0.0999

L-Glutamic Acid 0 0 0 0 75 0.5098 75 0.5098 0 0

L-Glutamine 292 1.9979 292 1.9979 292 1.9979 0 0 0 0

Glycine 0 0 0 0 50 0.666 50 0.666 0 0

L-Histidine HCl·H2O 42 0.2004 42 0.2004 41.88 0.1998 42 0.2004 41.88 0.1998

L-Isoleucine 52 0.3964 52 0.3964 52.5 0.4002 52.4 0.3995 52 0.3964

L-Leucine 131.2 1.0002 131.2 1.0002 52.4 0.3995 52.4 0.3995 52 0.3964

L-Lysine HCl 72.6 0.3975 72.6 0.3975 72.47 0.3968 73 0.3997 72.47 0.3968

L-Methionine 15 0.1005 15 0.1005 15 0.1005 15 0.1005 15 0.1005

L-Phenylalanine 32 0.1937 32 0.1937 32 0.1937 33 0.1998 32 0.1937

L-Proline 0 0 0 0 40 0.3474 40 0.3474 0 0

L-Serine 42 0.3997 42 0.3997 25 0.2379 25 0.2379 0 0

L-Threonine 48 0.403 48 0.403 48 0.403 47.6 0.3996 48 0.403

L-Tryptophan 10 0.049 10 0.049 10 0.049 10.2 0.0499 10 0.049

L-Tyrosine 2Na·2H2O 51.89 0.1987 51.89 0.1987 51.9 0.1987 0 0 51.9 0.1987

L-Tyrosine 0 0 0 0 0 0 36.2 0.1998 0 0

L-Valine 46 0.3927 46 0.3927 46 0.3927 46.8 0.3995 46 0.3927

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Formulation



SH30568(Alpha Mod.)

SH30603Suspension


L-Ascorbic Acid 0 0 0 0 50 0.2839 50 0.2839 0 0

d-Biotin 0.1 0.0004 0.1 0.0004 0.1 0.0004 0.1 0.0004 0 0

D-Ca Pantothenate 1 0.0021 1 0.0021 1 0.0021 1 0.0021 1 0.0021

Choline Chloride 56 0.4011 56 0.4011 1 0.0072 1 0.0072 1 0.0072

Folic Acid 2.2 0.005 2.2 0.005 1 0.0023 1 0.0023 1 0.0023

Myo-Inositol 36 0.1998 36 0.1998 2 0.0111 2 0.0111 2 0.0111

Niacinamide 1 0.008 1 0.008 1 0.0082 1 0.0082 1 0.0082

Pyridoxal HCl 0 0 0 0 1 0.0049 0 0 1 0.0049

Pyridoxine HCl 1 0.0049 1 0.0049 0 0 1 0.0049 0 0

Riboflavin 0.1 0.0003 0.1 0.0003 0.1 0.0003 0.1 0.0003 0.1 0.0003

Thiamine HCl 1 0.003 1 0.003 1 0.003 1 0.003 1 0.003

Vitamin B12 1.36 0.001 1.36 0.001 1.36 0.001 1.33 0.001 0 0

Putrescine·2HCl 0.16 0.001 0.16 0.001 0 0 0 0 0 0

Adenosine 0 0 0 0 10 0.0374 0 0 0 0

Cytidine 0 0 0 0 10 0.0411 0 0 0 0

D-Glucose 2000 11.10 2000 11.10 1000 5.5506 1000 5.5506 1000 5.5506

Guanosine 0 0 0 0 10 0.0353 0 0 0 0

Lipioc Acid 0.2 0.001 0.2 0.001 0.2 0.001 0.2 0.001 0 0

Phenol Red (Sodium) 0 0 10 0.0266 11 0.0292 10 0.0266 11 0.0292

Sodium Pyruvate 110 0.9996 110 0.9996 110 0.9996 110 0.9996 0 0

Thymidine 0 0 0 0 10 0.0413 0 0 0 0

2’-Deoxyadenosine H2O 0 0 0 0 10 0.0371 0 0 0 0

2’-Deoxycytidine HCI 0 0 0 0 11 0.0417 0 0 0 0

2’-Deoxyguanosine 0 0 0 0 10 0.0351 0 0 0 0

Uridine 0 0 0 0 10 0.041 0 0 0 0

NaHCO3 2200 26.1874 2200 26.1874 2200 26.1874 2200 26.1874 2200 26.1874

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Form

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Formulation

RPMI 1640 MediumDry Powdered Media



Ca(NO3)2·4H2O 100 0.4235 100 0.4235 100 0.4235 100 0.4235

KCl 400 5.3655 400 5.3655 400 5.3655 400 5.3655

MgSO4 (anhydrous) 48.84 0.4056 48.84 0.4056 48.84 0.4056 48.84 0.4056

NaCl 6000 102.6694 6000 102.6694 6000 102.6694 6000 102.6694

Na2HPO4 (anhydrous) 800 5.6354 0 0 800 5.6354 800 5.6354

NaH2PO4·H2O 0 0 778.4 5.641 0 0 0 0

Sodium Succinate 6H2O 0 0 0 0 0 0 0 0

Succinic Acid 0 0 0 0 0 0 0 0

L-Arginine 200 1.15 0 0 200 1.15 200 1.15

L-Arginine HCl 0 0 242 1.15 0 0 0 0

L-Asparagine 50 0.378 50 0.378 50 0.378 50 0.378

L-Aspartic Acid 20 0.1503 20 0.1503 20 0.1503 20 0.1503

L-Cystine 2HCl 65.15 0.208 65.15 0.208 65.15 0.208 65.15 0.208

L-Glutamic Acid 20 0.1359 20 0.1359 20 0.1359 20 0.1359

L-Glutamine 300 2.0527 300 2.0527 0 0 300 2.0527

Glycine 10 0.1332 10 0.1332 10 0.1332 10 0.1332

L-Histidine FB 15 0.0967 0 0 15 0.0967 15 0.0967

L-Histidine HCl·H2O 0 0 20.3 0.0968 0 0 0 0

L-Hydroxyproline 20 0.1525 30 0.2288 20 0.1525 20 0.1525

L-Isoleucine 50 0.3812 50 0.3812 50 0.3812 50 0.3812

L-Leucine 50 0.3812 50 0.3812 50 0.3812 50 0.3812

L-Lysine HCl 40 0.219 40 0.219 40 0.219 40 0.219

L-Methionine 15 0.1005 15 0.1005 15 0.1005 15 0.1005

L-Phenylalanine 15 0.0908 15 0.0908 15 0.0908 15 0.0908

L-Proline 20 0.1737 20 0.1737 20 0.1737 20 0.1737

L-Serine 30 0.2855 30 0.2855 30 0.2855 30 0.2855

L-Threonine 20 0.1679 20 0.1679 20 0.1679 20 0.1679

L-Tryptophan 5 0.0245 5 0.0245 5 0.0245 5 0.0245

L-Tyrosine 2Na·2H2O 28.83 0.1104 28.83 0.1104 28.83 0.1104 28.83 0.1104

L-Valine 20 0.1707 20 0.1707 20 0.1707 20 0.1707

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Formulation

RPMI 1640 MediumDry Powdered Media (continued)



d-Biotin 0.2 0.0008 0.2 0.0008 0.2 0.0008 0.2 0.0008

D-Ca Pantothenate 0.25 0.0005 0.25 0.0005 0.25 0.0005 0.25 0.0005

Choline Bitartrate 0 0 0 0 0 0 0 0

Choline Chloride 3 0.0215 3 0.0215 3 0.0215 3 0.0215

Folic Acid 1 0.0023 1 0.0023 1 0.0023 1 0.0023

Myo-Inositol 35 0.1943 35 0.1943 35 0.1943 35 0.1943

Niacinamide 1 0.0082 1 0.0082 1 0.0082 1 0.0082

Pyridoxine HCl 1 0.0049 1 0.0049 1 0.0049 1 0.0049

Riboflavin 0.2 0.0005 0.2 0.0005 0.2 0.0005 0.2 0.0005

Thiamine HCl 1 0.003 1 0.003 1 0.003 1 0.003

Vitamin B12 0.005 0.00004 0.005 0.000004 0.005 0.000004 0.005 0.000004

D-Glucose 2000 11.1012 2000 11.1012 2000 11.1012 2000 11.1012

Para-Aminobenzoic Acid (PABA) 1 0.0073 1 0.0073 1 0.0073 1 0.0073

Glutathione (Reduced) 1 0.0033 1 0.0033 1 0.0033 1 0.0033

Phenol Red (Sodium) 5.3 0.0141 5.3 0.0141 5.3 0.0141 0 0

Add NaHCO3 2000 23.8067 2000 23.8067 2000 23.8067 2000 23.8067

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Form

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Formulation

RPMI 1640 MediumLiquid Media



Ca(NO3)2·4H2O 100 0.4235 100 0.4235 100 0.4235

KCl 400 5.3655 400 5.3655 400 5.3655

MgSO4 (anhydrous) 48.84 0.4056 48.84 0.4056 48.84 0.4056

NaCl 6000 102.6694 6000 102.6694 6000 102.6694

NaH2PO4 (anhydrous) 800 5.6354 800 5.6354 800 5.6354

L-Arginine 200 1.15 200 1.15 200 1.15

L-Asparagine 50 0.378 50 0.378 50 0.378

L-Aspartic Acid 20 0.1503 20 0.1503 20 0.1503

L-Cystine 2HCl 65.15 0.208 65.15 0.208 65.15 0.208

L-Glutamic Acid 20 0.1359 20 0.1359 20 0.1359

L-Glutamine 300 2.0527 0 0 0 0

Glycine 10 0.1332 10 0.1332 10 0.1332

L-Histidine FB 15 0.0967 15 0.0967 15 0.0967

L-Hydroxyproline 20 0.1525 20 0.1525 20 0.1525

L-Isoleucine 50 0.3812 50 0.3812 50 0.3812

L-Leucine 50 0.3812 50 0.3812 50 0.3812

L-Lysine HCl 40 0.219 40 0.219 40 0.219

L-Methionine 15 0.1005 15 0.1005 15 0.1005

L-Phenylalanine 15 0.0908 15 0.0908 15 0.0908

L-Proline 20 0.1737 20 0.1737 20 0.1737

L-Serine 30 0.2855 30 0.2855 30 0.2855

L-Threonine 20 0.1679 20 0.1679 20 0.1679

L-Tryptophan 5 0.0245 5 0.0245 5 0.0245

L-Tyrosine 2Na·2H2O 28.83 0.1104 28.83 0.1104 28.83 0.1104

L-Valine 20 0.1707 20 0.1707 20 0.1707

d-Biotin 0.2 0.0008 0.2 0.0008 0.2 0.0008



Folic Acid 1 0.0023 1 0.0023 1 0.0023

Myo-Inositol 35 0.1943 35 0.1943 35 0.1943

Niacinamide 1 0.0082 1 0.0082 1 0.0082

Pyridoxine HCl 1 0.0049 1 0.0049 1 0.0049

Riboflavin 0.2 0.0005 0.2 0.0005 0.2 0.0005

Thiamine HCl 1 0.003 1 0.003 1 0.003

Vitamin B12 0.005 0.000004 0.005 0.000004 0.005 0.000004

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206

Formulation

RPMI 1640 MediumLiquid Media (continued)



D-Glucose 2000 11.1012 2000 11.1012 2000 11.1012Para-AminobenzoicAcid (PABA)

1 0.0073 1 0.0073 1 0.0073

Glutathione (Reduced) 1 0.0033 1 0.0033 1 0.0033


HEPES 0 0 0 0 5958 25.0021

NaHCO3 2000 23.8067 2000 23.8067 2000 23.8067

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Form

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Formulation

RPMI 1640 MediumLiquid Media (continued)



Ca(NO3)2·4H2O 100 0.4235 100 0.4235

KCl 400 5.3655 400 5.3655

MgSO4 (anhydrous) 48.84 0.4056 48.84 0.4056

NaCl 6000 102.6694 6000 102.6694

Na2HPO4 (anhydrous) 800 5.6354 800 5.6354

L-Arginine 200 1.15 200 1.15

L-Asparagine 50 0.378 50 0.378

L-Aspartic Acid 20 0.1503 20 0.1503

L-Cystine 2HCl 65.15 0.208 65.15 0.208

L-Glutamic Acid 20 0.1359 20 0.1359

L-Glutamine 300 2.0527 0 0

Glycine 10 0.1332 10 0.1332

L-Histidine FB 15 0.0967 15 0.0967

L-Hydroxyproline 20 0.1525 20 0.1525

L-Isoleucine 50 0.3812 50 0.3812

L-Leucine 50 0.3812 50 0.3812

L-Lysine HCl 40 0.219 40 0.219

L-Methionine 15 0.1005 15 0.1005


L-Proline 20 0.1737 20 0.1737

L-Serine 30 0.2855 30 0.2855

L-Threonine 20 0.1679 20 0.1679

L-Tryptophan 5 0.0245 5 0.0245

L-Tyrosine 2Na·2H2O 28.83 0.1104 28.83 0.1104

L-Valine 20 0.1707 20 0.1707



d-Biotin 0.2 0.0008 0.2 0.0008



Folic Acid 1 0.0023 1 0.0023

Myo-Inositol 35 0.1943 35 0.1943

Niacinamide 1 0.0082 1 0.0082


Riboflavin 0.2 0.0005 0.2 0.0005

Thiamine HCl 1 0.003 1 0.003

Vitamin B12 0.005 0.000004 0.005 0.000004

D-Glucose 2000 11.1012 2000 11.1012Para-AminobenzoicAcid (PABA) 1 0.0073 1 0.0073

Glutathione (Reduced) 1 0.0033 1 0.0033


HEPES 5958 25.0021 0 0

NaHCO3 2000 23.8067 2000 23.8067

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208

Formulations

William’s E MediumDry Powdered Media

Part Number SH30094



CuSO4·5H2O 0.0001 0.0000004

Fe(NO3)3·9H2O 0.0001 0.0000002

KCl 400 5.3655


MnCl2·4H2O 0.0001 0.0000005

NaCl 6800 116.3587

NaH2PO4·H2O 140 1.0146

ZnSO4·7H2O 0.0002 0.0000007

DL-Alpha Tocoph ·PO4 2NA 0.0078 0.00001

L-Alanine 90 1.0102

L-Arginine 50 0.287

L-Ascorbic Acid 2 0.0114


L-Aspartic Acid 30 0.2254



L-Glutamic Acid 50 0.3398

L-Glutamine 292.3 2

Glycine 50 0.666

L-Histidine 20.27 0.0967


L-Leucine 75 0.5718




L-Proline 30 0.2606

L-Serine 10 0.0952




L-Valine 50 0.4268

Part Number SH30094


d-Biotin 0.5 0.002



Folic Acid 1 0.0023

Menadione 0.01 0.0001



Pyridoxal HCl 1 0.0049


Thiamine HCl 1 0.003

Vitamin A Acetate 0.1 0.0003

Vitamin B12 0.2 0.0001

Vitamin D2-Ergocalciferol 0.1 0.0003

D-Glucose 2000 11.1012

L-Glutathione (reduced) 0.05 0.0002

Methyl Linoleate 0.03 0.0001



Add NaHCO3 2.2 0.0262

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Form

ulations

Formulation

Nutrient Supplements

Part Number SH30599

Description mg/L mmol/LNaCl 8500 145.4483D-Ca Pantothenate 100 0.2098Choline Chloride 100 0.7162Folic Acid 100 0.2266Myo-Inositol 200 1.1101Niacinamide 100 0.8188Pyridoxine HCl 100 0.4863Riboflavin 10 0.0266Thiamine HCl 100 0.2965

MEM Vitamins (100X)

Part Number SH30238




L-Aspartic Acid 1330 9.9925

L-Glutamic Acid 1470 9.9912

Glycine 750 9.9907

L-Proline 1150 9.9887

L-Serine 1050 9.9914

MEM NEAA (100X)

Part Number SH30598



L-Cystine 2HCl 1560 4.9805



L-Leucine 2625 20.0122






L-Tyrosine 2Na·2H2O 2160 8.2698

L-Valine 2340 19.9744

MEM Amino Acids (50X)


210

Formulation

Salts Solutions and Buffers



CaCl2 (anhydrous) 100 0.9011 0 0 0 0 0 0 1000 9.0106

KCl 200 2.6828 200 2.6828 200 2.6828 2000 26.8276 2000 26.8276

KH2PO4 200 1.4696 200 1.4696 200 1.4696 2000 14.6962 2000 14.6962

MgCl2.6H2O 100 0.4919 0 0 0 0 0 0 1000 4.9188

NaCl 8000 136.8925 8000 136.8925 8000 136.8925 80000 1368.9254 80000 1368.9254

Na2HPO4 (anhydrous) 1150 8.1009 1150 8.1009 1150 8.1009 0 0 11500 81.0087

Na2HPO4·7H2O 0 0 0 0 0 0 21600 80.576 0 0



CaCl2 (anhydrous) 0 0 0 0 200 1.8021 200 1.8021 140 1.2615

KCl 0 0 0 0 400 5.3655 400 5.3655 400 5.3655

KH2PO4 144 1.0581 1440 10.5812 0 0 0 0 60 0.4409

MgSO4 (anhydrous) 0 0 0 0 97.67 0.8112 97.67 0.8112 97.67 0.8112

NaCl 9000 154.0041 90000 1540.0411 6800 116.3587 6800 116.3587 8000 136.8925

Na2HPO4 795 5.6002 7950 56.0017 0 0 0 0 47.68 0.3359

Na2HPO4·7H2O 0 0 0 0 140 1.0146 140 1.0146 0 0

D-Glucose 0 0 0 0 1000 5.5506 1000 5.5506 1000 5.5506

Phenol Red (Sodium) 0 0 0 0 11 0.0292 11 0.0292 11 0.0292

NaHCO3 0 0 0 0 2200 26.1874 0 0 350 4.1662


211

Form

ulations

Formulation

Salts Solutions and Buffers

Part Number SH30015

Description mg/L mmol/LCaCl2 (anhydrous) 140 1.2615KCl 400 5.3655KH2PO4 60 0.4409MgCl2.6H2O 0 0MgSO4 (anhydrous) 97.67 0.8112NaCl 8000 136.8925Na2HPO4 (anhydrous) 47.68 0.3359D-Glucose 1000 5.5506Phenol Red (Sodium) 11 0.0292NaHCO3 350 4.1662

Part Number SH30031


MgSO4 (anhydrous) 0 0

CaCl2 (anhydrous) 0 0

KCl 400 5.3655

KH2PO4 60 0.4409

NaCl 8000 136.8925


D-Glucose 1000 5.5506

Phenol Red (Sodium) 11 0.0292

NaHCO3 350 4.1662



MgSO4 (anhydrous) 0 0 97.67 0.8112 0 0 0 0

CaCl2 (anhydrous) 0 0 140 1.2615 0 0 0 0

KCl 400 5.3655 400 5.3655 400 5.3655 400 5.3655

KH2PO4 60 0.4409 60 0.4409 60 0.4409 60 0.4409

NaCl 8000 136.8925 8000 136.8925 8000 136.8925 8000 136.8925

Na2HPO4 (anhydrous) 47.68 0.3359 47.68 0.3359 47.68 0.3359 47.68 0.3359

D-Glucose 1000 5.5506 1000 5.5506 1000 5.5506 1000 5.5506

Phenol Red (Sodium) 11 0.0292 0 0 0 0 0 0

NaHCO3 0 0 350 4.1662 0 0 350 4.1662


212

Standard BPC Packaging Options for Liquid Media/Buffers

3-D SquareCx5-14; 4 ports, top dispense

Line 1 1⁄4" Male Luer Lock (48")Line 2 1⁄4" Female Luer Lock (48")Line 3 3⁄8" QC Insert (48")Line 4 3⁄8" QC Body (48")

Part Number SizeSH2O663.01 50 LSH2O663.02 100 LSH2O663.03 200 LSH2O663.04 500 L

2-D, PillowCX5-14; 2 ports, face dispense

Line 1 1⁄4" QC Insert (36")Line 2 1⁄4" QC Body (36")

2-D, PillowCX5-14; 2 ports, face dispense

Line 1 1⁄4" Male Luer Lock (36")Line 2 1⁄4" Female Luer Lock (36")

Part Number SizeSH2O702.01 5 LSH2O702.02 10 LSH2O702.03 20 L

Part Number SizeSH2O698.01 10 LSH2O698.02 20 L

Product Overview• Manufactured with Thermo Scientific HyClone CX5-14 film• End fittings are capped or plugged and protected with a resealable polybag• Tubing lines are C-Flexe and provided with clamps• Gamma irradiated• Customizable through tubing assemblies


213

Sta

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Pac

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Standard Liquid Filled 1 to 5 L Pillow

Packaging material: Foam pad14 3⁄4" x 10 3⁄4" x 1⁄2"

Packaging material: Polybag (2)12" x 16", 1.5 mil; or 18" x 24", 4 mil

Container: Corrugated box

Container: Corrugated boxPackaging material: sealing tape, label


214

Integrated Single-Use Mixing Systems for Volumesof 50 to 200 L

�Mixtainer BioProcess Container

Line 1 MPC Insert, PolycarbonateTubing:Length: 36” (91 cm)ID x OD: 3/8” x 5/8” (9.5 mm x 15.9 mm)Dip Tube Length: 10” (25 cm)

Line 2 Male Luer Lock, PolypropyleneTubing:Length: 36” (91 cm)ID x OD: ¼” x 3/8” (6.4 mm x 9.5 mm)

Line 3 Female Luer Lock, PolypropyleneTubing:Length: 36” (91 cm)ID x OD: ¼” x 3/8” (6.4 mm x 9.5 mm)

Line 4 MPC Body, PolycarbonateTubing:Length: 36” (91 cm)ID x OD: 3/8” x 5/8” (9.5 mm x 15.9 mm)

Line 5 1½” (38.1 mm) Triclamp PowderAddition Port

For powder/liquid mixing

Product # Size

SH30687.04 50 L

SH30687.05 100 L

SH30687.06 200 L

Top dispense, with clamps

�Conical Drum

Product # Size Dimensions (D x H)

SV50517.11 50 L 23½” x 23”(60 cm x 58 cm)

SV50517.12 100 L 23½” x 29¾”(60 cm x 76 cm)

SV50517.13 200 L 23½” x 44¾”(60 cm x 114 cm)

�Magnetic Stirring Plate

�DollyStainless Steel

Product # Dimensions (L xW x H)

SV50109.01 24¼” x 24¼” x 6”(61.6 cm x 61.6 cm x 15.2cm)

Product # Area Description

IKAMAG Motor

SV30097.01 EU 23½” x 23”(60 cm x 58 cm)

SV30097.02 US 23½” x 29¾”(60 cm x 76 cm)

Stainless Steel Support

SV30097.03 EU Stainless Steel Support

SV30097.04 US Stainless Steel Support


215

BPCs

Single-Use Bioreactor (S.U.B.) from 50 to 1000 L

Key Features

• Provides all the advantages of single-usebioprocessing without having to buy a com-plete new bioreactor system

• Offered as a system with a reusable outersupport container providing a variable speedmixer motor, electrical heating blanket orwater jacket, control unit for integration withexternal control system and a mobile skid

• The Single-Use Bioreactor (S.U.B.) is a fullyfunctional bioreactor vessel includingsingle-use sterile contact surfaces formixing, venting, sparging and temperaturesensing. Additional ports are included toallow aseptic connections for filling,emptying and sampling

• Unique patented mixing system which pro-vides conventional overhead mixing whilemaintaing sterility and integrity of theS.U.B. BPC

• Standard S.U.B. product configurationsavailable with custom designed ports toutilize conventional 12 mm monitoringprobes for pH and DO. The probes must beautoclaved separately and inserted into theBPC using the bioreactor probe assembly

• Currently available in maximum workingvolumes of 50, 100, 250, 500 and 1000 L.Minimum working volume is half of themaximum

• Intended as a retrofit product to replacethe stainless steel bioreactor vessel inexisting bioreactor systems rather than as acomplete turn-key bioreactor system

• Flexible, rapid and economic option toupdate or increase your bioreactorcapacity

• Setup time in hours rather than days

Applications

• The intended use for the S.U.B. is an animalcell culture bioreactor

• Suitable for both suspension and microcar-rier culture

• Comparability with conventional systemsproven by customer evaluations

• Not suitable for use as a microbial fermenter

Product Support

• The Thermo Scientific HyClone line is uniquein that it provides both bioreactors and cellculture products

• The S.U.B. is used in our cell culture R&Dprogram, which enables offering practicalsupport customers expect for the optimiza-tion and set-up of their system

• A full validation package is supplied witheach S.U.B.

Development Program

• The rapid acceptance of the S.U.B. hasestablished the Thermo Scientific HyCloneproducts as a leading line of bioreactors

• An active development program is inprogress to expand the product portfolio toinclude a full range of working volumes from50–2000 L, with a wider range of outersupport container options

• Research is continuing to find suppliers withcontrol systems and non-invasive probetechnologies to facilitate integration ofsystems into the S.U.B.

Supporting Information

• S.U.B. User’s Guide, UG003

• 50 L S.U.B. Data Sheet, 026





• S.U.B. Capabilities Brochure, SM0497.01

• S.U.B. Microcarriers Application Note,AN001

• S.U.B. SP2/0 Application Note, AN003

Associated Products

• Thermo Scientific HyQtainerPatent Pending

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Mixing System

Cap

Latch Pin

Motor

Drive Shaft

Seal/BearingAssembly

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Impeller

Drive ShaftHead

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216

Key Features

• The Thermo Scientific HyClone MixtainerSystem has a unique BioProcess Container(BPC) incorporating a magnetic stir barproviding a closed system with single-usecontact surfaces

• Optimized support hardware allows mixing,moving, storage, shipping and discharge ofthe BPC contents while maintaining sterilityof the contents (if desired)

• Standard products available for sterileliquid/liquid or powder/liquid mixing

• Available in 50, 100, and 200 L unit volumes

• Recommended for applications where theBPC contents are post filtered

Applications

• Hydration of powdered media and buffers

• Sterile mixing of cell culture media with seraand reagents

• Sterile mixing of buffer solutions or otherprocess fluids

• Pooling of sterile liquid fractions

Supporting Information

• CX5-14 Film Fact Sheet

• Mixtainer User’s Guide, UG009

• Mixtainer with Powder Port Data Sheet, 006

• BPC Handling Data Sheet, 047

Associated Products

• Thermo Scientific HyClone Powdertainer,

Patents: 7,278,780,B2 and 7,153,021

Thermo Scientific HyCloneMixtainer System Components

Mixtainer BioProcess Container—TheMixtainer BPC is supplied sterilizedby irradiation. It is constructed of ThermoScientific HyClone CX5-14 film andcontains a magnetic stir-bar located in aplastic dish welded in the bottom of theBPC. The dish contains a locking ring toensure the containment of the stir-barduring shipping, handling and operation.A powder port option is available on oneof the standard configurations. Asepticliquid transfers are facilitated byconnectors and tubing attached to portsat the top of the BPC

Conical Drum—TheMixtainer systemutilizes a polyethylene drum with aspecial enlarged opening at the bottomto accommodate the stir-bar dish

Magnetic Stirring Plate—A commer-cially available standard unit with thepower and variable speed capability toprovide a range of mixing rates

Dolly—The dolly is designed to interfacebetween the drum and the magneticstirring plate to create the vertical andconcentric alignment necessary foroperation. It also provides mobility

Integrated Single-Use Mixing Systems for Volumesof 50 to 200 L

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217

BPCs


S.U.B. BPC Components

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� Vent FilterSingle-use capsule filter for exhaust gasexchange

� Gas Overlay PortProtected by gas filter

� PortsFor addition of media and other liquids

� Seal/Bearing AssemblyLinks with mixer motor and allows impellerto turn while retaining integrity of theS.U.B. BPC

� ImpellerMolded polyethylene linked to seal/bearingassembly by C-Flex tubing forms the con-tact material for the shaft

� Ports with Kleenpak ConnectorsFor integration of standard ½” monitoringpH and DO probes

�Temperature RTD PortFor integration of temperature probe whileretaining integrity of the S.U.B. BPC

�Sampling PortFor needleless sampling or connection forsampling manifold

Drain PortUsed when draining the S.U.B.

Gas SpargePermeable disc integrated into the chamberprotected by gas filter on all sizes, as wellas by a check valve

Ordering InformationBioreactor Probe Assembly Non-sterileUsed to package the pH and DO probes forsterilization and to aseptically connect them tothe S.U.B. BPC

Product # Size

SH30720.01 50-1000 L

Heavy Duty Tubing ClampReusable tubing clamp used on probe portswhen connecting probe assemblies

Product # Description

SV20664.01 Single Pack

SV20664.02 4 Pack

SV20664.03 10 Pack

S.U.B. Temperature/Sample PortUsed for RTD calibration/validation

Product # Size

SV20750.01 50-1000 L

Product # Size

SV50177.01 50-1000 L

Autoclave Tray for Probe KitsStainless SteelSupport tray which allows the probes to besafely autoclaved

Product # Size

SH30845.01 Single Pack

SH30845.02 10 Pack

Sterile Sampling Manifold with luer locksAvailable to weld onto the sample line to takesample sets

500 and 1000 L BPC

50, 100 and 250 L BPC

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50, 100 and 250 L S.U.B. BPCSingle-Use Bioreactor BPC (with MPC Body)


SH30715.01 50 L SV50171

SH30744.01 100 L SV50197

SH30716.01 250 L SV50172


SH30725.01 50 L SV50171

SH30726.01 250 L SV50172

Line # Description Details

1 Overlay Gas Sparge 4” (10 cm) length with Acro 50 Vent Filter

2 Addition 96” (243.8 cm) length branching to 2 SmartSite Valve Ports

3 Inoculum Addition 72” (182.9 cm) length with plugged end

4 Media Addition Branch total 81” (205.7 cm) length with plugged end and Branch total 78” (198.1 cm) lengthwith MPC Body

5 Supplement/Feed Addition Branch total 81” (205.7 cm) length with plugged end and Branch total 78” (198.1 cm) lengthwith MPC Body

6, 7, 8, 9 ACD Connection for pH probe Pall Female Aseptic Connector

10 Thermo-Well/Small Volume Sample Thermo-Well Adapter for ¼” Diameter, 12” (30.5 cm) length with SmartSite Valve Port

11 Bottom Drain Harvest Branch total 93” (236.2 cm) length with plugged end and Branch total 90” (228.6 cm) lengthwith MPC Body

12 Exhaust Filter 0.22 Micron Exhaust Vent Filter

13 Direct Gas Sparge 54” (137.2 cm) length with Check Valve and Acro 50 Vent Filter

Single-Use Bioreactor BPC(with Triclamps)

Line # Description Details

1 Overlay Gas Sparge 4” (10 cm) length with Acro 50 Vent Filter

2 Addition 96” (243.8 cm) length branching to 2 SmartSite Valve Ports

3 Inoculum Addition 72” (182.9 cm) length with SIP Triclamp

4 Media Addition Branch total 81” (205.7 cm) length with plugged end and Branch total 78” (198.1 cm) lengthwith ¾” SIP Triclamp[

5 Supplement/Feed Addition Branch total 81” (205.7 cm) length with plugged end and Branch total 78” (198.1 cm) lengthwith ¾” SIP Triclamp[

6, 7, 8, 9 ACD Connection for pH probe Pall Female Aseptic Connector

10 Thermo-Well/Small Volume Sample Thermo-Well Adapter for ¼” Diameter, 12” (30.5 cm) length with SmartSite Valve Port

11 Bottom Drain Harvest Branch total 93” (236.2 cm) length with plugged end and Branch total 90” (228.6 cm) lengthwith 1½” SIP Triclamp[

12 Exhaust Filter 0.22 Micron Exhaust Vent Filter

13 Direct Gas Sparge 54” (137.2 cm) length with Check Valve and Acro 50 Vent Filter


219

BPCs


S.U.B. Hardware System Components

� Container304 stainless steel with cut-out sections tofacilitate S.U.B. BPC loading

�Mixer MotorIntegrates with seal/bearing assembly onS.U.B. BPC

� Electrical Heater JacketMaintains bioreactor temperature andcontrolled by RTD probe in S.U.B. BPC

�Water JacketControls and maintains bioreactortemperature

� PlatformLocation points for S.U.B. BPC with con-tainment for spills and casters for mobility

� Control UnitFor mixer and heater, also can integratewith customer’s existing system

� DoorAvailable on the 1000 L model only

� Load Cells (not shown)Standard on 1000 L model only

50, 100, 250 and 500 L S.U.B.Electric Resistive Heater

50, 100, 250 and 500 L S.U.B.Water Jacket

1000 L S.U.B.Electric Resistive Heater

1000 L S.U.B.Water Jacket

Product # Size Description

SV50171.01 50 L 120 VAC

SV50171.02 50 L 240 VAC

SV50197.01 100 L 120 VAC

SV50197.02 100 L 240 VAC

SV50172.01 250 L 120 VAC

SV50172.02 250 L 240 VAC

SV50200.02 500 L 240 VAC

SV50174.01 1000 L Modbus Plus Load Cell Interface

SV50174.02 1000 L Allen-Bradley Load Cell Interface

SV50174.03 1000 L Analog Output Load Cell Interface

SV50174.04 1000 L Profibus Load Cell Interface

SV50174.05 1000 L DeviceNet Load Cell Interface

Electric Resistive Heater

Product # Size Description

SV50171.03 50 L 120 VAC

SV50171.04 50 L 240 VAC

SV50197.03 100 L 120 VAC

SV50197.04 100 L 240 VAC

SV50172.03 250 L 120 VAC

SV50172.04 250 L 240 VAC

SV50200.04 500 L 240 VAC

SV50174.11 1000 L 240 VAC, Modbus Plus Load Cell Interface

SV50174.12 1000 L 240 VAC, Allen-Bradley Load Cell Interface

SV50174.13 1000 L 240 VAC, Analog Output Load Cell Interface

SV50174.14 1000 L 240 VAC, Profibus Load Cell Interface

SV50174.15 1000 L 240 VAC, DeviceNet Load Cell Interface

Water Jacket

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Standard Terms and Conditions of SaleUNLESS OTHERWISE EXPRESSLY AGREED INWRITING, ALL SALES ARE SUBJECT TO THEFOLLOWING TERMS AND CONDITIONS:

1. GENERAL. Hyclone Laboratories, Inc.(“Seller”), a wholly owned subsidiary ofThermo Fisher Scientific Inc. hereby offersfor sale to the person or entity ("Buyer")that receives a quote from, or places awritten or verbal order with, Seller for thesale of consumable products (e.g., serum,serum-free media, animal-derived compo-nent free medium, microcarriersand single-use BioProcess Containers)(“Consumable Product(s)”) and/or equip-ment (e.g, Single-Use Bioreactor andSingle-Use Mixer) (“Equipment”) ordered byBuyer (generally and collectively, the "Prod-uct(s)") on the express condition that Buyeragrees to accept and be bound by the termsand conditions set forth herein (the“Terms”). Any provisions containedin any document issued by Buyer areexpressly rejected and if the terms andconditions in these Terms differ from theterms of Buyer’s offer, this document willbe construed as a counter offer and will notbe effective as an acceptance of Buyer’sdocument. Buyer’s receipt of Productsprovided hereunder will constitute Buyer’sacceptance of these Terms. This is thecomplete and exclusive statement of thecontract between Seller and Buyer withrespect to Buyer's purchase of the Products.No waiver, consent, modification, amend-ment or change of the terms containedherein will be binding unless in writing andsigned by Seller and Buyer. Seller's failureto object to terms contained in any subse-quent communication from Buyer will notbe a waiver or modification of the terms setforth herein. All orders are subject to ac-ceptance in writing by an authorized repre-sentative of Seller.

2. CUSTOM GOODS. If Buyer desires topurchase customized or special goods("Custom Goods") from Seller (uniqueraw materials, special manufacturingprocesses or labeling or otherwise) and ifSeller, in its sole discretion, is willing toconsider the same, then Seller willprepare a written proposal for the priceindicated, either as part of a price

quotation, part of a bid submission orotherwise (a “Quote”) for such CustomGoods. Any change to specificationsrequires a new Quote. Seller will notaccept an Order for Custom Goods unlessSeller has issued a Quote for thoseCustom Goods. Orders for Custom Goodsmay not be cancelled after 48 hours ofOrder placement. Seller disclaims anyliability for: (1) the efficacy or compatibilityof components provided or specified byBuyer in the manufacture of Custom Goods;and (2) the performance of Custom Goodswithin ranges desired by Buyer (e.g., pHlevel for media), even if those ranges arecommunicated to Seller or are included inspecifications for the Custom Goods. Buyermust pay for initial lots of Custom Goodsso long as they comply with Seller'sspecifications, even if those Custom Goodsfall outside of Buyer's desired performanceranges for Buyer's own applications. In theevent of an overage or shortage in a batchof Custom Goods that Seller manufacturesto fill an Order, Seller may ship the entirebatch, which shall be deemed to satisfythe Order so long as the variance does notexceed +/- 10% of the quantity Ordered.Buyer shall indemnify Seller against anyclaims, damages and expenses (includingSeller's attorneys' fees in defending againstclaims and enforcing thisindemnity) resulting from infringement ofpatents or other intellectual property rightsof a third party arising from Seller'scompliance with Buyer's specialspecifications or instructions.

3. PRICE. All prices published by Seller orquoted by Seller's representatives maybe changed at any time without notice.All prices quoted by Seller or Seller’srepresentatives are valid for thirty (30)days, unless otherwise stated in writing.All prices for the Products will be asspecified by Seller or, if no price has beenspecified or quoted, will be Seller's pricein effect at the time of shipment. All pricesare subject to adjustment due to specifica-tions, quantities, raw materials, cost ofproduction, shipment arrangements orother terms or conditions which are notpart of Seller's original price quotation.

4. TAXES AND OTHER CHARGES. Prices forthe Products exclude all sales, value addedand other taxes and duties imposed withrespect to the sale, delivery, or use of anyProducts covered hereby, all of which taxesand duties must by paid by Buyer. If Buyerclaims any exemption, Buyer must providea valid, signed certificate or letter ofexemption for each respective jurisdiction.

5. TERMS OF PAYMENT. Seller may invoiceBuyer upon shipment for the price andall other charges payable by Buyer inaccordance with the terms on the facehereof. If no payment terms are stated onthe face hereof, payment will be net thirty(30) days from the date of invoice. If Buyerfails to pay any amounts when due, Buyerwill pay Seller interest thereon at aperiodic rate of one and one-half percent(1.5%) per month (or, if lower, the highestrate permitted by law), together with allcosts and expenses (including withoutlimitation reasonable attorneys' fees anddisbursements and court costs) incurredby Seller in collecting such overdueamounts or otherwise enforcing Seller'srights hereunder. Seller reserves theright to require from Buyer full or partialpayment in advance, or other security thatis satisfactory to Seller, at any time thatSeller believes in good faith that Buyer'sfinancial condition does not justify theterms of payment specified. All paymentswill be made in U.S. Dollars. Credit cardinformation sent via fax or email will notbe processed.

6. DELIVERY; CANCELLATION OR CHANGESBY BUYER. The Products will be shipped tothe destination specified by Buyer, F.O.B.Seller's shipping point for destinationswithin the U.S. and FCA place of shipment(Incoterms 2000) for destinations outsidethe U.S, with packaging and carriers asdesignated by Seller, unless otherwisespecified in an accepted Order. Seller willhave the right, at its election, to makepartial shipments of the Products and toinvoice each shipment separately. Sellerreserves the right to stop delivery ofProducts in transit and to withholdshipments in whole or in part if Buyer failsto make any payment to Seller when due or


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otherwise fails to perform its obligationshereunder. All shipping dates areapproximate only, and Seller will not beliable for any loss or damage resulting fromany delay in delivery or failure to deliver. Inthe event of a delay due to any causebeyond Seller's reasonable control, Sellerreserves the right to terminate the order orto reschedule the shipment within areasonable period of time, and Buyer willnot be entitled to refuse delivery orotherwise be relieved of any obligations asthe result of such delay. Products as towhich delivery is delayed due to any causewithin Buyer's control may be placed instorage for Buyer by Seller at Buyer's riskand expense. Orders in process may becanceled only with Seller's written consentand upon payment of Seller's cancellationcharges. Orders for Custom Goods andOrders in process may not be changedexcept with Seller's written consent andupon agreement by the parties as to anappropriate adjustment in the purchaseprice therefor. Credit will not be allowedfor Products returned without the priorwritten consent of Seller.

7. TITLE AND RISK OF LOSS.Notwithstanding the trade terms indicatedabove and subject to Seller's right to stopdelivery of Products in transit, title to andrisk of loss of the Products will pass toBuyer upon delivery of possession of theProducts by Seller to the carrier.

8. WARRANTY. Products are warranted toperform in substantial conformity withpublished Product specifications in effect atthe time of sale, as set forth in the Productdocumentation, specifications and/oraccompanying package inserts(“Documentation”) and to be free fromdefects in material and workmanship. Thewarranty provided herein is valid only whenused by properly trained individuals. Unlessotherwise stated in the Documentation,this warranty is limited to forty-five (45)days from date of shipment for ConsumableProducts and one (1) year for Equipmentwhen subjected to normal, proper andintended usage. This warranty does notextend to anyone other than Buyer. Noother warranties, express or implied, are

granted, including without limitation,implied warranties of merchantability,fitness for any particular purpose, or noninfringement. Buyer’s exclusive remedyfor non-conforming Consumable Productsduring the warranty period is limited toreplacement of or refund for the non-conforming Product(s), at Seller’s soleoption. Buyer’s exclusive remedy for non-conforming Equipment during the warrantyperiod is limited to repair, replacement ofor refund for the non-conformingEquipment, at Seller’s sole option. Sellershall have no obligation to repair, replaceor refund Products as the result of (i)accident, disaster or event of forcemajeure, (ii) misuse, fault or negligence ofor by Buyer, (iii) use of the Products in amanner for which they were not designed,or (iv) improper storage and handling of theProducts. For Equipment, replacement partsmay be new or refurbished, at the electionof Seller. All replaced parts shall becomethe property of Seller. Shipment to Buyerof repaired or replacement Products shallbe made in accordance with the Deliveryprovisions of the Seller’s Terms andConditions of Sale.

9. RETURNS. Promptly after receipt ofProducts, Buyer must inspect the Productsand advise Seller of any defects orshipment errors within ten (10) days ofreceipt. Except for defective Productscovered under the above warranties orSeller's shipment errors, Buyer may notreturn Products to Seller unless Buyermakes a return request within 30 days afterBuyer receives the Products and Seller, inits sole discretion, consents to the return inwriting by issuing a return authorizationnumber (obtained from Seller's customerservices department). Seller will notconsent to returns for shipments ofProducts: (1) made from the wrong Sellerlot number when Buyer does not specify, atthe time of placing the Order, the specificlot number on reserve for Buyer from whichthe Order must be fulfilled; (2) made withinadequate import documentation forshipments to destinations outside of theUnited States if Seller has complied withBuyer's prior import instructions; (3) withexpiration dates sooner than a particular

date if Buyer does not specify that date atthe time of placing the Order; (4) that havealready expired or that are within 60 daysof expiration; or (5) that are CustomProducts or that otherwise are custommanufactured. If a return is authorized,then Buyer shall return the Products toSeller within 10 days after authorizationindicating the return authorization number;shall ship the Products F.O.B. place ofdestination Seller's facility (for shipmentfrom places within the United States) andDDP place of destination Seller's facility(Incoterms 2000; for shipment from placesoutside the United States) and shall paySeller a processing charge of US $50.00 or25% of the sales price, whichever isgreater. Seller shall give Buyer a credit forreturned Products only if Seller receives theProducts, inspects them and deems theProducts to be re-saleable, in Seller's solediscretion.

10. INDEMNIFICATION. 10.1 By Seller. Selleragrees to indemnify, defend and saveBuyer, its officer, directors, and employeesfrom and against any and all damages,liabilities, actions, causes of action, suits,claims, demands, losses, costs andexpenses (including without limitationreasonable attorney’s fees) for injury to ordeath of persons or damage to property tothe extent caused by the negligence orwillful misconduct of Seller, its employees,agents or representatives or contractors.Buyer will provide Seller prompt writtennotice of any third party claim covered bySeller’s indemnification obligationshereunder. Seller will have the right toassume exclusive control of the defense ofsuch claim or, at the option of the Seller, tosettle the same. Buyer agrees to cooperatereasonably with Seller in connection withthe performance by Seller of its obligationsin this Section.

10.2 By Buyer. Buyer will indemnify, defendwith competent and experienced counseland hold harmless Seller, its parent,subsidiaries, affiliates and divisions, andtheir respective officers, directors,shareholders and employees, from andagainst any and all damages, liabilities,actions, causes of action, suits, claims,


222

demands, losses, costs and expenses(including without limitation reasonableattorneys' fees and disbursements andcourt costs) to the extent arising from or inconnection with (i) the negligence or willfulmisconduct of Buyer, its agents,employees, representatives or contractors;(ii) use of the Products for any non-researchpurpose or by a non-technically qualifiedindividual; (iii) use of a Product incombination with equipment or softwarenot supplied by Seller where the Productitself would not be infringing; (iv) Seller'scompliance with designs, specifications orinstructions supplied to Seller by Buyer; (v)use of a Product in an application orenvironment for which it was not designed;or (vi) modifications of a Product by anyoneother than Seller without Seller's priorwritten approval.

11. LIMITATION OF LIABILITY.NOTWITHSTANDING ANYTHING TO THECONTRARY CONTAINED HEREIN, THELIABILITY OF SELLER UNDER THESETERMS AND CONDITIONS (WHETHER BYREASON OF BREACH OF CONTRACT, TORT,INDEMNIFICATION, OR OTHERWISE, BUTEXCLUDING LIABILITY OF SELLER FORBREACH OFWARRANTY (THE SOLEREMEDY FORWHICHWILL BE ASPROVIDED UNDER SECTION 8 ABOVE))WILL NOT EXCEED AN AMOUNT EQUALTO THE TOTAL PURCHASE PRICE PAID BYBUYER TO SELLERWITH RESPECT TO THEPRODUCT(S) GIVING RISE TO SUCHLIABILITY. NOTWITHSTANDINGANYTHING TO THE CONTRARYCONTAINED HEREIN, IN NO EVENTWILLSELLER BE LIABLE FOR ANY INDIRECT,SPECIAL, CONSEQUENTIAL ORINCIDENTAL DAMAGES (INCLUDINGWITHOUT LIMITATION DAMAGES FORLOSS OF USE OF FACILITIES OREQUIPMENT, LOSS OF REVENUE, LOSS OFDATA, LOSS OF PROFITS OR LOSS OFGOODWILL), REGARDLESS OFWHETHERSELLER (a) HAS BEEN INFORMED OF THEPOSSIBILITY OF SUCH DAMAGES OR (b) ISNEGLIGENT.

12. EXPORT RESTRICTIONS. Buyeracknowledges that each Product and anyrelated technology, including technicalinformation supplied by Seller or containedin documents (collectively “Items”), issubject to export controls of the U.S.government. The export controls mayinclude, but are not limited to, those of theExport Administration Regulations of theU.S. Department of Commerce (the “EAR”),which may restrict or require licenses forthe export of Items from the United Statesand their re-export from other countries.Buyer will comply with the EAR and allother applicable laws, regulations, laws,treaties, and agreements relating to theexport, re-export, and import of any Item.Buyer will not, without first obtaining therequired license to do so from theappropriate U.S. government agency; (i)export or re-export any Item, or (ii) export,re-export, distribute or supply any Item toany restricted or embargoed country or to aperson or entity whose privilege toparticipate in exports has been denied orrestricted by the U.S. government. Buyerwill cooperate fully with Seller in anyofficial or unofficial audit or inspectionrelated to applicable export or importcontrol laws or regulations, and willindemnify and hold Seller harmless from, orin connection with, any violation of thisSection by Buyer or its employees,consultants, agents, or customers.

13. CONFIDENTIALITY. Buyer agrees that allpricing, discounts and technical informationthat Seller provides to Buyer are theconfidential and proprietary information ofSeller. Buyer agrees to (i) keep suchinformation confidential and not disclosesuch information to any third party, and (ii)use such information solely for Buyer’sinternal purposes and in connection withthe Products supplied hereunder. Nothingherein will restrict the use of informationavailable to the general public.

14. MISCELLANEOUS. 14.1 The Products areintended to be used only for furthermanufacturing or research use and are notintended for diagnostic or therapeutic useor administration to animals or humans.

Due to the nature of serum products,virtually every serum has some type ofcontaminant or virus, which may affect theuses intended by Buyer.

14.2 Buyer may not delegate any dutiesnor assign any rights or claims hereunderwithout Seller's prior written consent, andany such attempted delegation orassignment will be void.

14.3 The rights and obligations of theparties hereunder will be governed by andconstrued in accordance with the laws ofthe State of Delaware, without referenceto its choice of law provisions. In the eventof any legal proceeding between the Sellerand Buyer relating to these Terms, neitherparty may claim the right to a trial by jury,and both parties waive any right they mayhave under applicable law or otherwise toa right to a trial by jury. Any action arisingunder these Terms must be brought withinone (1) year from the date that the cause ofaction arose. The application to theseTerms of the U.N. Convention on Contractsfor the International Sale of Goods ishereby expressly excluded.

14.4 In the event that any one or moreprovisions contained herein will be held bya court of competent jurisdiction to beinvalid, illegal or unenforceable in anyrespect, the validity, legality andenforceability of the remaining provisionscontained herein will remain in full forceand effect, unless the revision materiallychanges the bargain.

14.5 Seller's failure to enforce, or Seller'swaiver of a breach of, any provisioncontained herein will not constitute awaiver of any other breach or of suchprovision.

14.6 Any notice or communication requiredor permitted hereunder will be in writingand will be deemed received whenpersonally delivered or three (3) businessdays after being sent by certified mail,postage prepaid, to a party at the addressspecified herein or at such other address aseither party may from time to timedesignate to the other.


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AcronymsABS Adult Bovine Serum

ACS American Chemical Society

ADCF Animal Derived Component Free

AQIS Australian Quarantine and Inspection Service

ASC Adult Stem Cell

BEVS Baculovirus Expression Vector System

BCS Bovine Calf Serum

BGS Bovine Growth Serum

BME Basal Medium Eagle

BPC BioProcess Container

BSE Bovine Spongiform Encephalopathy

BT Bluetongue

BVD Bovine Viral Diarrhea

C Celsius

CET Cellular Engineering Technologies

cfu colony forming unit

CFR Code of Federal Regulations

cGMP current Good Manufacturing Practices

CHO Chinese Hamster Ovary

CIT Continuous Improvement Team

CIP Clean-in-Place

cm centimeter

CO2 Carbon Dioxide

COA Certificate of Analysis

COS Certificate of Suitability

CPMP Committee for Proprietary Medicinal Products

CVMP Committee for Veterinary Medicinal Products

dl deciliter

DMEM Dulbecco's Modified Eagle's Medium

DMSO Dimethyl Sulfoxide

DNA Deoxyribonucleic Acid

DPBS Dulbecco's Phosphate Buffered Saline

DPM Dry Powdered Media

EBSS Earle's Balanced Salt Solution

EDTA Ethylene-Diamine-Tetra-Acetic Acid

EEC European Economic Community

EMEA European Agency for the Evaluation of Medical Products

EP European Pharmacopeia

ERP Enterprise Resource Planning

ESC Embryonic Stem Cell

EU Endotoxin Units

FBS Fetal Bovine Serum

FCC Food Chemical Codex

FDA Food and Drug Administration

FMD Food and Mouth Disease

FOB Freight on Board

g gram

HAE Human amniotic epithelial

HAESC Human amniotic epithelial stem cells

HAMSC Human adipose-derived mesenchymal stem cells

HBSS Hank's Balanced Salt Solution

HCBHSC Human Cord Blood Hematopoietic Stem Cell

HDPE High density Polyethylene

HEK Human Embryonic Kidney

HEPA High Efficiency Particulate Arrestance

HFS Human Foreskin Fibroblast

HI heat inactivation

hMSC Human Mesenchymal Stem Cell

HPMSC Human placental mesenchymal stem cells

HWJSC HumanWharton's Jelly stem cells

IBR Infectious Bovine Rhinotracheitis

IgG Immunoglobulin G

IMDM Iscove's Modified Dulbecco's Medium

IQ Installation Qualification

IR irradiation

ISO International Standards Organization

JIT Just-In-Time

JP Japanese Pharmacopeia

kGy kilogray

L liter

LAL limulus amebocyte lysate

M Molar

M199 Medium 199

MAb Monoclonal Antibody

MB Molecular Biology

MCB Master Cell Bank

MCBUSSC Multipotent Cord Blood Unrestricted Somatic Stem Cells

MDCK Madin Darby Canine Kidney

MEM Minimum Essential Medium

mESC Mouse Embryonic Stem Cells


224

mg milligram

mL milliliter

MSC Mesenchymal Stem Cell

mw molecular weight

NaCl sodium chloride

NEAA Non-essential amino acids

NF National Formulary

nm nanometer

NZ New Zealand

OQ Operational Qualification

PBS Phosphate Buffered Saline

PCD Production Control Document

PCR Polymerase Chain Reaction

PETE Polyethylene Terephthalate

PETG Polyethylene Terephthalate Glycol

PF Protein-free

PI3 Parainfluenza 3

ppb parts per billion

PPV Porcine parvovirus

PQ Performance Qualification

QA Quality Assurance

QC Quality Control

R&D Research and Development

R&PD Research and Product Development

RNA Ribonucleic Acid

rpm revolutions per minute

RPMI Roswell Park Memorial Institute

SAL Sterility Assurance Level

SAM Sequential Adaptation Medium

SCM Serum-containing medium

SFM Serum-free media

SIP Steam-in-Place

TIBC Total Iron Binding Capacity

TOC Total Organic Carbon

TPB Tryptose Phosphate Broth

UF ultrafiltrate

Rg micrograms

UK United Kingdom

RL microliter

Rm micrometer

USDA United States Department of Agriculture

USP United States Pharmacopeia

WFI Water for injection


225

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Product Index (alphabetical)AADCF-MAb (liquid) (SH30349) 20, 42ADCF-MAb (powder) (SH30635) 42ADCF-MAb without L-glutamine (liquid) (SH30547) 20, 42Adipogenic Differentiation Kit (SH30876.KT) 31Adipogenic Differentiation Medium (SH30886) 31Adult Bovine Serum, New Zealand Origin (SH30409) 90Adult Bovine Serum, New Zealand Origin, Heat Inactivated (SH30409.HI) 90Adult Bovine Serum, New Zealand Origin, Irradiated (SH30409.IR) 90Adult Bovine Serum, New Zealand Origin, Non-Sterile (SH30402) 90AdvanceSTEM DMEM4SC with 4.5 mg/L Glucose, without L-glutamine andSodium Pyruvate (SH30824)

27

AdvanceSTEM ES Qualified Dulbecco's Phosphate Buffered Saline (DPBS)without Calcium or Magnesium (SH30850)

27

AdvanceSTEM ES Qualified HEPES (1 M) (SH30851) 27AdvanceSTEM ES Qualified L-glutamine 200 mM (SH30852) 27AdvanceSTEM ES Qualified Non-Essential Amino Acids (100X (SH30853) 27AdvanceSTEM IMDM4SC without L-glutamine (SH30822) 27AdvanceSTEM Low Osmo DMEM without L-glutamine (SH30870) 27AdvanceSTEM Serum Replacement (SH30874) 27Alpha Calf Fraction, Iron-Supplemented, Heat Inactivated (SH30076.HI) 87Alpha Calf Fraction, Iron-Supplemented, Irradiated (SH30076.IR) 87Alpha Calf Fraction, without Iron (SH30212) 87Alpha Calf Fraction, without Iron, Heat Inactivated (SH30212.HI) 87Alpha Calf Fraction, without Iron, Irradiated (SH30212.IR) 87Alpha Calf Fraction, Iron-Supplemented (SH30076) 87Amniotic Epithelial Expansion Kit (SH30904) 31Amniotic Epithelial Expansion Medium (SH30900) 31Amphotericin B (Fungizone) Solution, 250 Rg/mL (SV30078) 59Antibiotic Antimycotic Solution (Pen/Strep/Fungizone), 100X, 10,000 U/mLPenicillin G, 10,000 Rg/mL Streptomycin, 25 Rg/mL Amphotericin B (SV30079)

59

Antifoam Irradiated (SH30896) 64Antifoam Irradiated, ADCF (SH30897) 64BBEVS PlaKit (SH30340) 48BME/EBSS with L-Glutamine (SH30157) 16BME/EBSS with L-glutamine (SH30159) 22Bovine Calf Serum, US Origin (SH30073) 83Bovine Calf Serum, US Origin (non-EMEA equivalent SH30073) (SH30544) 92Bovine Calf Serum, US Origin, Heat Inactivated (SH30073.HI) 83Bovine Calf Serum, US Origin, Irradiated (SH30073.IR) 83Bovine Growth Serum, US Origin (SH30541) 83Bovine Growth Serum, US Origin, Heat Inactivated (SH30541.HI) 83Bovine Growth Serum, US Origin, Irradiated (SH30541.IR) 83Bovine Serum Albumin (BSA), Cell Culture Grade, pH 7.0, Lyophilized Powder(SH30574)

53

BPCs, 2D, 2 Ports, End Dispense (SH30658, SH30662, SH30657) 93BPCs, 2D, 3 Ports, End Dispense (SH30713, SH30712, SH30714,SH30709, SH30667)

94

CCalcium Chloride Solution, CM Solution (SH30289) 53CCM1 (liquid) (SH30043) 42CCM1 (powder) (SH30058) 42CCM1 (powder) (SH30059) 42CCM3 (DPM) (SH30061) 48CCM3 (liquid) (SH30065) 48CCM5 (liquid) (SH30100) 38CDM4CHO (powder) (SH30556) 37

CDM4CHO with L-glutamine (liquid) (SH30557) 20, 37CDM4CHO without L-glutamine (liquid) (SH30558) 20, 37CDM4HEK293 without L-glutamine (liquid) (SH30858) 20, 45CDM4HEK293 without L-glutamine (powder) (SH30859) 45CDM4MAb (powder) (SH30800) 41CDM4MAb with L-glutamine (liquid) (SH30801) 20, 41CDM4MAb (liquid) (SH30802) 20, 41CDM4NS0 (powder) (SH30578) 40CDM4NS0 (liquid) (SH30579) 20, 40CDM4PERMAb without L-glutamine (liquid) (SH30871) 20, 45CDM4PERMAb without L-glutamine (powder) (SH30872) 45CDM4Retino (liquid) (SH30520) 46CDM4Retino (powder) (SH30519) 46Cell Boost 1 (R05.2) (SH30584) 52Cell Boost 2 (R15.4) (SH30596) 52Cell Boost 3 (JM3.5) (SH30825) 52Cell Boost 4 (PS307) (SH30857) 52Cell Boost 5 (CN-F) (SH30865) 52Cell Boost 6 (CN-T) (SH30866) 52Cell Boost Process Supplement Kit (SH30890) 52CET Human Adipose-Derived Mesenchymal Stem Cells (SV30102) 32CET Human Amniotic Epithelial Stem Cells (SV30104) 33CET Human Amniotic Mesenchymal Stem Cells (SV30103) 32CET Human Bone Marrow Mesenchymal Stem Cells (SV30110) 32CET Human Cord Blood CD133+ Stem Cells (SV30107) 33CET Human Cord Blood CD34+ Stem Cells (SV30106) 33CET Human Umbilical Cord Blood (SV30105) 33CET Human Wharton's Jelly Mesenchymal Stem Cells (SV30101) 32Characterized Australian FBS (non-EMEA equivalent SH30084) (SH30545) 92Characterized Australian Foetal Bovine Serum (SH30084) 74Characterized Australian Foetal Bovine Serum, Heat Inactivated (SH30084.HI) 74Characterized Australian Foetal Bovine Serum, Irradiated (SH30084.IR) 74Characterized FBS, US Origin (non-EMEA equivalent SH30071) (SH30532) 92Characterized Fetal Bovine Serum, Canadian Origin (SH30396) 73Characterized Fetal Bovine Serum, Canadian Origin, Heat Inactivated(SH30396.HI) 73Characterized Fetal Bovine Serum, Canadian Origin, Irradiated (SH30396IR) 73Characterized Fetal Bovine Serum, US Origin (SH30071) 73Characterized Fetal Bovine Serum, US Origin, Heat Inactivated (SH30071.HI) 73Characterized Fetal Bovine Serum, US Origin, Irradiated (SH30071.IR) 73Charcoal/Dextran Treated Fetal Bovine Serum, US Origin (SH30068) 79Charcoal/Dextran Treated Fetal Bovine Serum, US Origin, Heat Inactivated(SH30068.HI) 79Charcoal/Dextran Treated Fetal Bovine Serum, US Origin, Irradiated(SH30068.IR) 79Chondrogenic Differentiation Medium (SH30889) 31Cosmic Calf Serum, US Origin (SH30087) 83Cosmic Calf Serum, US Origin, Heat Inactivated (SH30087.HI) 83Cosmic Calf Serum, US Origin, Irradiated (SH30087.IR) 83Cosmic Calfq Serum, US Origin (non-EMEA equivalent SH30087) (SH30533) 92Cryopreservation Medium (SH30894) 31CultiSpher GL Microcarriers, Macroporous gelatin bead (SV30006) 62CultiSpher S Microcarriers, Macroporous gelatin bead (SV30009) 62CultiSpher G Microcarriers, Macroporous gelatin bead (SV30008) 62D

Defined Fetal Bovine Serum (FBS), US Origin (non-EMEA equivalent SH30070)(SH30531) 92


226

Product Index (alphabetical)Defined Fetal Bovine Serum, US Origin (SH30070) 73Defined Fetal Bovine Serum, US Origin, Heat Inactivated (SH30070.HI) 73Defined Fetal Bovine Serum, US Origin, Irradiated (SH30070.IR) 73D-Glucose (powder) (SH30371) 53Dialyzed Fetal Bovine Serum, US Origin (SH30079) 79Dialyzed Fetal Bovine Serum, US Origin, Heat Inactivated (SH30079.HI) 79Dialyzed Fetal Bovine Serum, US Origin, Irradiated (SH30079.IR) 79DMEM High Modified, with Sodium Pyruvate, without L-glutamine, with25 mM HEPES (SH30563)

17

DMEM with High Glucose, 4.0 mM L-glutamine, Sodium Pyruvate (SH30243) 17, 20DMEM with High Glucose, 4.0 mM L-glutamine, without Sodium Pyruvate(SH30022)

16, 20

DMEM with High Glucose, Modified, without Phenol Red, L-glutamine, withSodium Pyruvate (SH30604)

17

DMEM with High Glucose, Modified, without Phenol Red, Sodium Pyruvate,without L-glutamine (SH30585)

17

DMEM with High Glucose, L-glutamine, with HEPES, without SodiumPyruvate (SH30249)

17

DMEM with High Glucose, L-glutamine, without Phenol Red, SodiumPyruvate (SH30284)

17

DMEM with High Glucose, Sodium Pyruvate, without L-glutamine andPhosphate (SH30607)

17

DMEM with High Glucose, Sodium Pyruvate, without L-glutamine,Methionine, Cystine (SH30606)

17

DMEM High Modified with Sodium Pyruvate, without L-glutamine,with 25 mM HEPES (SH30563)

17

DMEM with High Glucose, without Calcium, without Magnesium (SH30262) 17DMEMwith High Glucose, without L-glutamine and Sodium Pyruvate (SH30081) 16, 20DMEMwith High Glucose, without L-glutamine, with Sodium Pyruvate (SH30285) 17, 20DMEMwith LowGlucose, 4.0mML-glutamine, 110mg/ SodiumPyruvate (SH30021) 17, 20DMEM/F12 1:1 with L-glutamine, HEPES (SH30004) 23DMEM/F12 1:1 with L-glutamine, without HEPES (SH30069) 23DMEM/F12 1:1, with 2.50 mM L-glutamine, 15 mM HEPES (SH30023) 17, 20DMEM/F12 1:1, with L-glutamine, HEPES, Sodium Pyruvate (SH30261) 17DMEM/F12 1:1, with L-glutamine, without HEPES (SH30271) 17, 20DMEM/F12 1:1, with L-glutamine, without HEPES, Phenol Red (SH30272) 17DMEM/F12 1:1, without L-glutamine, with HEPES (SH30126) 17, 20DMEM/HIGH with L-glutamine, Sodium Pyruvate (SH30045) 22DMEM/HIGH with L-glutamine, without Sodium Pyruvate (SH30003) 22DMEM/HIGH with L-glutamine, without Sodium Pyruvate, Phenol Red(SH30211) 22DMEM/HIGH without L-glutamine, Sodium Pyruvate (SH30053) 22DMEM/HIGH without L-glutamine, Sodium Pyruvate, Calcium,Magnesium(SH30346)

22

DMEM/LOW with L-glutamine, Sodium Pyruvate (SH30002) 23DMEM/LOW with L-glutamine, Sodium Pyruvate, without Phenol Red(SH30044)

23

DMEM-RS with L-glutamine (SH30565) 25DMEM-RS with L-glutamine, Complete Powder (SH30628) 25Donor Adult Bovine Serum, US Origin (SH30075) 90Donor Adult Bovine Serum, US Origin (non-EMEA equivalent SH30075) (SH30527) 92Donor Adult Bovine Serum, US Origin, Heat Inactivated (SH30075.HI) 90Donor Adult Bovine Serum, US Origin, Irradiated (SH30075.IR) 90Donor Bovine Serum, New Zealand Origin (SH30417) 90Donor Bovine Serum, New Zealand Origin, Heat Inactivated (SH30417.HI) 90Donor Bovine Serum, New Zealand Origin, Irradiated (SH30417.IR) 90Donor Equine Serum, US Origin (SH30074) 90Donor Equine Serum, US Origin, Heat Inactivated (SH30074.HI) 90Donor Equine Serum, US Origin, Irradiated (SH30074.IR) 90Dulbecco's Phosphate Buffered Saline (DPBS), 10X, with Calcium,Magnesium (SH30597)

56

Dulbecco's Phosphate Buffered Saline (DPBS), 10X, without Calcium,Magnesium, Phenol Red (SH30378)

56

Dulbecco's Phosphate Buffered Saline (DPBS), 1X, with Calcium, Magnesium,without Phenol Red (SH30264)

21, 56

Dulbecco's Phosphate Buffered Saline (DPBS), 1X, without Calcium,Magnesium, Phenol Red (SH30028)

21, 56

Dulbecco's Phosphate Buffered Saline (DPBS), without Calcium,Magnesium (powder) (SH30013)

56

E

Earle's Balanced Salt Solution (EBSS), 1X, with Calcium,Magnesium, PhenolRed (SH30029)

56

Earle's Balanced Salt Solution (EBSS), with Calcium, Magnesium, Phenol Red(powder) (SH30014)

56

ES Screened FBS, U.S. Origin (SH30070.02E) 27, 31, 79F

FetalClone I (SH30080) 87

FetalClone I, Heat Inactivated (SH30080.HI) 87FetalClone I, Irradiated (SH30080.IR) 87FetalClone II (SH30066) 87FetalClone II, Heat Inactivated (SH30066.HI) 87FetalClone II, Irradiated (SH30066.IR) 87FetalClone III (SH30109) 87FetalClone III (non-EMEA equivalent SH30109) (SH30530) 92FetalClone III, Heat Inactivated (SH30109.HI) 87FetalClone III, Irradiated (SH30109.IR) 87G

G418 Sulfate Solution, 50 mg/mL (SV30069) 59G418 Sulfate, >90%Purity (SV30068) 59Gentamicin Solution, 50 mg/mL (SV30080) 59Grace's Unsupplemented (liquid) (SH30610) 18, 48Growth Supplement (SH30878) 31GS-MAX (SH30586) 52H

Ham's F10, with L-glutamine (SH30009) 23Ham's F10, with L-glutamine (SH30025) 18Ham's F12, 1X, with 1.00 mM L-glutamine (SH30026) 18, 20Ham's F12, Kaighn's Modification (SH30526) 18Ham's F12, with L-glutamine (SH30010) 23Ham's F12, without L-glutamine (SH30056) 23Ham's F12-RS with L-glutamine (SH30623) 25Hank's Balanced Salt Solution (HBSS), 1X, with Calcium, Magnesium,without Phenol Red (SH30268)

21, 57

Hank's Balanced Salt Solution (HBSS), 1X, with Calcium,Magnesium,Phenol Red (SH30030)

56

Hank's Balanced Salt Solution (HBSS), 1X, without Calcium, Magnesium,Phenol Red (SH30588)

57

Hank's Balanced Salt Solution (HBSS), 1X, without Calcium, withoutMagnesium, with Phenol Red (SH30031)

21, 56

Hank's Balanced Salt Solution (HBSS), with Calcium, Magnesium, PhenolRed (powder) (SH30015)

56

Hank's Balanced Salt Solution (HBSS), without Calcium, Magnesium, PhenolRed (powder) (SH30107)

57

Hank's Balanced Salt Solution (HBSS), without Calcium, Magnesium, withPhenol Red (powder) (SH30016)

56

HEPES 1 M Solution (SH30237) 53, 57HEPES Buffer (powder) (SH30337) 57HEPES Solution, 50 mM, 0.15 M NaCl, 20 mM EDTA, pH 8.0 (SH30291) 53, 57Human Mesenchymal Stem Cell Screened Fetal Bovine Serum, US Origin(SH30070.03M)

31, 79

Hygromycin B Solution, 50 mg/mL (SV30070) 59Hypoxanthine/Thymidine (HT) Solution, 50X (SH30614) 53

HyQSphere Microcarrier Starter Kit Contains 5 Grams Each of Pro-F 102 -L,P 102-L, P Plus 102-L, FACT 102-L, CGEN 102-L, and HLX II-170Microcarriers (SV30092)

61

HyQSphere Microcarriers, CGEN 102-L Same as fact bead without surfacecharge. (SV30044)

61

HyQSphere Microcarriers, FACT 102-L Collagen-coated plastic with surfacecharge. General purpose bead. (SV30040)

61

HyQSphere Microcarriers, HLX II-170 Polystyrene microcarrier coated withcationic trimethyl ammonium with a nominal specific gravity of 1.11 andbead diameter of 160–180 Rm. This bead has a surface charge. (SV30090)

61

HyQSphere Microcarriers, P 102-L (SV30056) 61


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Product Index (alphabetical)HyQSphere Microcarriers, plus 102-L ADCF Plastic bead with surfacecharge. General purpose bead for use with serum-containing mediacultures or other non-fastidious cell lines. (SV30052)

61

HyQSphere Microcarriers, Pro-F 102-L ADCF Plastic bead coated withProNectine F, a recombinant protein. Superior performance bead facilitatesattachment of fastidious cell lines in ADCF conditions (SV30048)

61

HyQTase Cell Detachment Solution, in DPBS with EDTA(non-mammalian) (SV30030)

54

HyStem Kit (SV30138) 34HyStem-C Kit (SV30139) 34HyStem-HP Kit (SV30140) 34I

IMDM with L-glutamine, HEPES, without Alpha-Thioglycerol (SH30005) 23IMDM with L-glutamine, HEPES, without Alpha-Thioglycerol (SH30228) 18, 20IMDM without L-glutamine (SH30259) 18IMDM without L-glutamine, with HEPES (SH30380) 23Insect Cell Screened Fetal Bovine Serum, US Origin (SH30070.03I) 79IPL-41 (powder) (SH30282) 48Iron-Supplemented Bovine Calf Serum, US Origin (SH30072) 83Iron-Supplemented Bovine Calf Serum, US Origin (non-EMEA equivalentSH30072) (SH30534)

92

Iron-Supplemented Bovine Calf Serum, US Origin, Heat Inactivated(SH30072.HI)

83

Iron-Supplemented Bovine Calf Serum, US Origin, Irradiated (SH30072.IR) 83L

Lactalbumin Hydrolysate (LAH) growth enhancement supplement (SH30322) 53Leibovitz L-15 Medium, with 2.05 mM L-glutamine (SH30525) 18Leibovitz L-15 with L-glutamine (SH30048) 23L-glutamine (powder) (SH30336) 53L-glutamine, 200 mM Solution 29.2 mg/mL L-glutamine in 0.85% NaCl(SH30034)

53

Lipid-Reduced Fetal Bovine Serum, US Origin (SH30855) 79Lipid-Reduced Fetal Bovine Serum, US Origin, Heat Inactivated (SH30855.HI) 79Lipid-Reduced Fetal Bovine Serum, US Origin, Irradiated (SH30855.IR) 79LS1000 Lipid Supplement, ADCF (liquid) (SH30554) 42, 52, 53LS250 Lipid Supplement, ADCF (liquid) (SH30555) 42, 52, 53

M

M199 with Earle's Balanced Salt Solution (EBSS), L-glutamine (SH30253) 18, 20M199 with Hank's Balanced Salt Solution (HBSS), L-glutamine (SH30233) 18M199 with Hank's Balanced Salt Solution (HBSS), L-glutamine, L-AminoAcids (SH30330)

18

M199/EBSS Filter Friendly, with L-glutamine (SH30351) 23M199/EBSS with L-glutamine, L-Amino Acids (SH30297) 23M199/EBSS with L-glutamine, without Phenol Red (SH30254) 23McCoy's 5A Iwakata and Grace Modification, with L-glutamine (SH30049) 23McCoy's 5A with L-glutamine (SH30200) 18, 20McCoy's 5A with L-glutamine, HEPES (SH30602) 18McCoy's 5A with L-glutamine, without Phenol Red (SH30270) 18MEM (Richter'sModification) with L-glutamine and Phenol Red (SH30601) 19MEM (Richter'sModification), without Phenol Red (SH30600) 19MEM Alpha Modification with L-glutamine, Ribo- andDeoxyribonucleosides (SH30007)

24

MEM Alpha Modification with L-glutamine, without Ribo- andDeoxyribonucleosides (SH30219)

24

MEM Alpha Modification without L-glutamine, Ribo- andDeoxyribonucleosides (SH30205)

24

MEM Alpha Modification, with L-glutamine, Ribo- andDeoxyribonucleosides (SH30265)

19, 20

MEM Amino Acid Solution, 50X (SH30598) 53MEM for Suspension Cultures, without L-glutamine, Calcium Chloride, orMagnesium (SH30603)

19

MEM Glasgow Modification with L-glutamine (SH30269) 24MEM Vitamin Solution, 100X (SH30599) 53

MEMwith Earle's Balanced Salt Solution (EBSS), 2.0 mM L-glutamine (SH30024) 19, 20MEM with Earle's Balanced Salt Solution (EBSS), Suspension Modification,with L-glutamine (SH30235)

19

MEMwith Earle's Balanced Salt Solution (EBSS), without L-glutamine (SH30244) 19, 20MEM/Alpha Modification without L-glutamine, Ribo- andDeoxyribonucleosides (SH30568)

19,20

MEM/EBSS (Autoclavable) (SH30327) 24MEM/EBSS with L-glutamine (SH30008) 24MEM/EBSS with L-glutamine, Non-Essential Amino Acids (SH30050) 24MEM/EBSS with L-glutamine, without Phenol Red (SH30171) 24MEM/EBSS without L-glutamine (SH30054) 24MEM/HBSS with L-glutamine (SH30193) 24MEM-RS with L-glutamine (SH30564) 25MEM-RS with L-glutamine, Complete Powder (SH30629) 25Mesenchymal Stem Cell Basal Medium (SH30879) 31Mesenchymal Stem Cell Expansion Kit (SH30875.KT) 31N

Neural Differentiation Kit (SH30892.KT) 31Neural Differentiation Medium (SH30893) 31New Zealand Adult Bovine Serum (non-EMEA equivalent SH30409) (SH30528) 92

New Zealand Bovine Calf Serum Iron Supplemented (non-EMEA equivalentSH30626) (SH30592)

92

New Zealand Bovine Growth Serum (SH30591) 84New Zealand Bovine Growth Serum, Heat Inactivated (SH30591.HI) 84New Zealand Bovine Growth Serum, Irradiated (SH30591.IR) 84New Zealand Newborn Bovine Serum (SH30401) 84New Zealand Newborn Bovine Serum (non-EMEA equivalent SH30401) (SH30560) 92New Zealand Newborn Bovine Serum, Heat Inactivated (SH30401.HI) 84New Zealand Newborn Bovine Serum, Irradiated (SH30401.IR) 84New Zealand Cosmic Calf (non-EMEA equivalent SH30413) (SH30625) 92New Zealand Cosmic Calf Serum, Heat Inactivated (SH30413.HI) 84New Zealand Cosmic Calf Serum, Irradiated (SH30413.IR) 84New Zealand Cosmic Calf Serum (SH30413) 84New Zealand Donor Bovine Serum (non-EMEA equivalent SH30417) (SH30624) 92New Zealand FBS (non-EMEA equivalent SH30406) (SH30611) 92New Zealand Foetal Bovine Serum (SH30406) 73New Zealand Foetal Bovine Serum, Heat Inactivated (SH30406.HI) 73New Zealand Foetal Bovine Serum, Irradiated (SH30406.IR) 73New Zealand Iron-Supplemented Calf Serum (SH30626) 84New Zealand Iron-Supplemented Calf Serum, Heat Inactivated (SH30626.HI) 84New Zealand Iron-Supplemented Calf Serum, Irradiated (SH30626.IR) 84New Zealand Standard Foetal Bovine Serum (SH30559) 73New Zealand Standard Foetal Bovine Serum, Heat Inactivated (SH30559.HI) 73New Zealand Standard Foetal Bovine Serum, Irradiated (SH30559.IR) 73Newborn Bovine Calf Serum, US Origin (SH30118) 83Newborn Bovine Calf Serum, US Origin, Heat Inactivated (SH30118.HI) 83Newborn Bovine Calf Serum, US Origin, Irradiated (SH30118.IR) 83Non-Essential Amino Acid, (NEAA) Solution, 100X (SH30238) 53OOsteogenic Differentiation Kit (SH30877.KT) 31Osteogenic Differentiation Medium (SH30881) 31PPen/Strep/Glutamine Solution (SV30082) 59Penicillin-Streptomycin Solution 10,000 U/mL Penicillin, 10,000 µg/mLStreptomycin in 0.85% NaCl (SV30010)

59

PF CHO LS with L-glutamine (liquid) (SH30359) 20, 38PF-293 (liquid) (SH30356) 46PF-293 MPS (powder) (SH30355) 46PF-CHO (liquid) (SH30220) 38PF-CHO (powder) (SH30333) 38


228

Product Index (alphabetical)PF-MAb (liquid) (SH30138) 42Phosphate Buffered Saline (PBS) 10X, 0.067 M PO4, without Calcium,Magnesium, Phenol Red (SH30258)

57

Phosphate Buffered Saline (PBS), 1X, 0.0067 M PO4, without Calcium,Magnesium, Phenol Red (SH30256)

21, 57

Pluronic (powder) (SH30612) 53Pluronic 10% Solution (SH30594) 53Porcine Serum, New Zealand Origin (SH30908) 90Porcine Serum, New Zealand Origin, Heat Inactivated (SH30908) (SH30908.HI) 90Porcine Serum, New Zealand Origin, Irradiated (SH30908IR) 90Primogenix PRX-129/X1Mouse Embryonic Stem Cells (mESCs) (SV30098) 28Primogenix PRX-B6NMouse Embryonic Stem Cells (mESCs) (SV30109) 28Puromycin 2 HCl, USP (SV30075) 59R

Research Grade Fetal Bovine Serum, European Origin, Heat Inactivated(SV30143HI)

74

Research Grade Fetal Bovine Serum, European Origin, Irradiated (SV30143) 74Research Grade Fetal Bovine Serum, Heat Inactivated South AmericanOrigin, European Customers (SV30160.HI)

74

Research Grade Fetal Bovine Serum, Irradiated, South American Origin,European Customers (SV30160.IR)

74

Research Grade Fetal Bovine Serum, South American Origin, ChinaCustomers (SV30087)

74

Research Grade Fetal Bovine Serum, South American Origin, EuropeanCustomers (SV30160)

74

RPMI 1640 Medium, with 25 mM HEPES, L-glutamine (SH30255) 19, 20RPMI 1640, 1X, with 2.05 mM L-glutamine (Same as SH30027) (SH30091) 19RPMI 1640, 1X, with 2.05 mM L-glutamine (Same as SH30091) (SH30027) 19, 20RPMI 1640, Bakers X-Tra Soluble with L-glutamine (SH30012) 24RPMI 1640, with L-glutamine (SH30011) 24RPMI 1640, with L-glutamine, without Phenol Red (SH30197) 24RPMI 1640, without L-glutamine (SH30057) 24RPMI 1640, without L-glutamine (SH30096) 19, 20RPMI 1640, without L-glutamine or Phenol Red (SH30605) 19RPMI 1640, without L-glutamine, with HEPES (SH30203) 19S

SFM4CHO (liquid) (SH30549) 21, 37SFM4CHO (powder) (SH30518) 38SFM4CHO without L-glutamine (liquid) (SH30548) 20, 37SFM4CHO-Utility (powder) (SH30517) 38SFM4CHO-Utility with L-glutamine (liquid) (SH30516) 20, 38SFM4CHO-A with L-glutamine (liquid) (SH30821) 20, 38SFM4CHO-A without L-glutamine (liquid) (SH30820) 20SFM4HEK293 (powder) (SH30522) 46SFM4HEK293 with L-glutamine (liquid) (SH30521) 21, 46SFM4Insect with L-glutamine (liquid) (SH30913) 48SFM4Insect with L-glutamine (powder) (SH30912) 48SFM4MAb (powder) (SH30535) 41SFM4MAb with L-glutamine (liquid) (SH30513) 21, 41SFM4MAb without L-glutamine (liquid) (SH30391) 41SFM4MAb-Utility (powder) (SH30550) 41SFM4MAb-Utility with L-glutamine (liquid) (SH30382) 21, 41SFM4MegaVir (powder) (SH30587) 49SFM4MegaVir without L-glutamine (liquid) (SH30552) 21, 49SFM4HEK293 with L-glutamine (liquid) (SH30521) 21, 46SFM4Transfx-293 without L-glutamine (liquid) (SH30860) 20, 45SFM4Transfx-293 without L-glutamine (powder) (SH30861) 45SFX-CHO (liquid) (SH30187) 38SFX-Insect (liquid) (SH30278) 21, 48SFX-Insect (powder) (SH30350) 48SFX-MAb (liquid) (SH30206) 42SG-200, (Stable dipeptide of L-Alanyl L-glutamine) 200 mM, in 0.85% NaClSolution (SH30590)

53

Sodium Bicarbonate (powder) (SH30173) 57Sodium Bicarbonate Solution, with 75.0 g/L Sodium Bicarbonate (SH30033) 57

Sodium Pyruvate 100 mM Solution (SH30239) 53Soy Hydrolysate UF (25%) (liquid) (SH30357) 53Standard Australian Foetal Bovine Serum (SV30370) 74Standard Australian Foetal Bovine Serum, Heat Inactivated (SH30370.HI) 74Standard Australian Foetal Bovine Serum, Irradiated (SH30370.IR) 74Standard Fetal Bovine Serum, Canadian Origin (SH30397) 73Standard Fetal Bovine Serum, Canadian Origin, Heat Inactivated (H30397.HI) 73Standard Fetal Bovine Serum, Canadian Origin, Irradiated (SH30397IR) 73Standard Fetal Bovine Serum, US Origin (SH30088) 73Standard Fetal Bovine Serum, US Origin, Heat Inactivated (SH30088.HI) 73Standard Fetal Bovine Serum, US Origin, Irradiated (SH30088.IR) 73Super Low IgG Fetal Bovine Serum, US Origin (SH30898) 79Super Low IgG Fetal Bovine Serum, US Origin, Heat Inactivated (SH30898.HI) 79Super Low IgG Fetal Bovine Serum, US Origin, Irradiated (SH30898.IR) 79Super Safe New Zealand Fetal Bovine Serum, Irradiated (SH30406.IR) 79TTetracycline Screened Fetal Bovine Serum, US Origin (SH30070.03T) 79TNM-FH (liquid) (SH30280) 18, 48Trypan Blue Solution, 0.4%in Phosphate Buffered Saline (SV30084) 54Trypsin 0.25% 1X Solution, with 2.5 g Porcine Trypsin (1:250/L) in HBSS,without Calcium and Magnesium, with 0.1%EDTA (SV30031)

54

Trypsin 2.5% 10X Solution, with 25.0 g Porcine Trypsin (1:250/L) in HBSS,without Calcium and Magnesium, without EDTA or Phenol Red (SV30037)

54

Trypsin, 0.05% 1X, with 0.5 g porcine Trypsin (1:250/L gamma irradiated) inHBSS with 0.2 g/L EDTA, without Calcium, Magnesium (liquid) (SH30236)

54

Trypsin, 0.25% 1X, with 2.5 g porcine Trypsin (1:250/L gamma irradiated) inHBSS with 0.2 g/L EDTA, without Calcium, Magnesium (liquid) (SH30042)

54

Tryptose Phosphate Broth (TPB) (powder) (SH30394) 53UUSDA-Tested Fetal Bovine Serum (SV30014) 74USDA-Tested Fetal Bovine Serum, Heat Inactivated (SV30014.HI) 74USDA-Tested Fetal Bovine Serum, Irradiated (SV30014.IR) 74

WWater, Cell Culture Grade, Endotoxin-free, (SH30529) 21, 57Water, Molecular Biology Grade, DNase, RNase, Protease free (SH30538) 21, 57Water, WFI Quality, (SH30221) 21, 57Wear Testing Fluid for Artificial Joints 20, US Origin, Heat Inactivated (SH30891) 90Wear Testing Fluid for Artificial Joints 30, US Origin (SH30856) 90William's E Medium with L-glutamine (SH30094) 24


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Product Index (by part number)

SH30002 DMEM/LOW with L-glutamine, Sodium Pyruvate 23SH30003 DMEM/HIGH with L-glutamine, without Sodium Pyruvate 22SH30004 DMEM/F12 1:1 with L-glutamine, HEPES 23SH30005 IMDM with L-glutamine, HEPES, without Alpha-Thioglycerol 23SH30007 MEM Alpha Modification with L-glutamine, Ribo- and

Deoxyribonucleosides24

SH30008 MEM/EBSS with L-glutamine 24SH30009 Ham's F10, with L-glutamine 23SH30010 Ham's F12, with L-glutamine 23SH30011 RPMI 1640, with L-glutamine 24SH30012 RPMI 1640, Bakers X-Tra Soluble with L-glutamine 24SH30013 Dulbecco's Phosphate Buffered Saline (DPBS), without Calcium,

Magnesium (powder)56

SH30014 Earle's Balanced Salt Solution (EBSS), with Calcium, Magnesium,Phenol Red (powder)

56

SH30015 Hank's Balanced Salt Solution (HBSS), with Calcium, Magnesium,Phenol Red (powder)

56

SH30016 Hank's Balanced Salt Solution (HBSS), without Calcium, Magnesium,with Phenol Red (powder)

56

SH30021 DMEM with Low Glucose, 4.0 mM L-glutamine, 110 mg/ SodiumPyruvate

17, 20

SH30022 DMEM with High Glucose, 4.0 mM L-glutamine, without SodiumPyruvate

16, 20

SH30023 DMEM/F12 1:1, with 2.50 mM L-glutamine, 15 mM HEPES 17, 20SH30024 MEM with Earle's Balanced Salt Solution (EBSS), 2.0 mM L-glutamine 19, 20SH30025 Ham's F10, with L-glutamine 18SH30026 Ham's F12, 1X, with 1.00 mM L-glutamine 18, 20SH30027 RPMI 1640, 1X, with 2.05 mM L-glutamine (Same as SH30091) 19, 20SH30028 Dulbecco's Phosphate Buffered Saline (DPBS), 1X, without Calcium,

Magnesium, Phenol Red21, 56

SH30029 Earle's Balanced Salt Solution (EBSS), 1X, with Calcium, Magnesium,Phenol Red

56

SH30030 Hank's Balanced Salt Solution (HBSS), 1X, with Calcium, Magnesium,Phenol Red

56

SH30031 Hank's Balanced Salt Solution (HBSS), 1X, without Calcium,Magnesium, with Phenol Red

21, 56

SH30033 Sodium Bicarbonate Solution, with 75.0 g/L Sodium Bicarbonate 57SH30034 L-glutamine, 200 mM Solution 29.2 mg/mL L-glutamine in 0.85% NaCl 53SH30042 Trypsin, 0.25% 1X, with 2.5 g porcine Trypsin (1:250/L gamma irradi-

ated) in HBSS with 0.2 g/L EDTA, without Calcium, Magnesium (liquid)54

SH30043 CCM1 (liquid) 42SH30044 DMEM/LOW with L-glutamine, Sodium Pyruvate, without Phenol Red 23SH30045 DMEM/HIGH with L-glutamine, Sodium Pyruvate 22SH30048 Leibovitz L-15 with L-glutamine 23SH30049 McCoy's 5A Iwakata and Grace Modification, with L-glutamine 23SH30050 MEM/EBSS with L-glutamine, Non-Essential Amino Acids 24SH30053 DMEM/HIGH without L-glutamine, Sodium Pyruvate 22SH30054 MEM/EBSS without L-glutamine 24SH30056 Ham's F12, without L-glutamine 23, 166SH30057 RPMI 1640, without L-glutamine 24SH30058 CCM1 (powder) 42SH30059 CCM1 (powder) 42SH30061 CCM3 (DPM) 48SH30065 CCM3 (liquid) 48SH30066 FetalClone® II 87SH30066.HI FetalClone II, Heat Inactivated 87SH30066.IR FetalClone II, Irradiated 87SH30068 Charcoal/Dextran Treated Fetal Bovine Serum, US Origin 79SH30068.HI Charcoal/Dextran Treated Fetal Bovine Serum, US Origin, Heat

Inactivated79

SH30068.IRCharcoal/Dextran Treated Fetal Bovine Serum, US Origin, Irradiated 79SH30069 DMEM/F12 1:1 with L-glutamine, without HEPES 23SH30070 Defined Fetal Bovine Serum, US Origin 27, 73SH30070.02EES Screened FBS, US Origin 27, 79SH30070.03I Insect Cell Screened Fetal Bovine Serum, US Origin 79

SH30070.03M Human Mesenchymal Stem Cell Screened Fetal Bovine Serum,US Origin

31, 79

SH30070.03T Tetracycline Screened Fetal Bovine Serum, US Origin 79SH30070.HI Defined Fetal Bovine Serum, US Origin, Heat Inactivated 73SH30070.IR Defined Fetal Bovine Serum, US Origin, Irradiated 73SH30071 Characterized Fetal Bovine Serum, US Origin 73SH30071.HI Characterized Fetal Bovine Serum, US Origin, Heat Inactivated 73SH30071.IR Characterized Fetal Bovine Serum, US Origin, Irradiated 73SH30072 Iron-Supplemented Bovine Calf Serum, US Origin 83SH30072.HI Iron-Supplemented Bovine Calf Serum, US Origin, Heat Inactivated 83SH30072.IR Iron-Supplemented Bovine Calf Serum, US Origin, Irradiated 83SH30073 Bovine Calf Serum, US Origin 83SH30073.HI Bovine Calf Serum, US Origin, Heat Inactivated 83SH30073.IR Bovine Calf Serum, US Origin, Irradiated 83SH30074 Donor Equine Serum, US Origin 90SH30074.HI Donor Equine Serum, US Origin, Heat Inactivated 90SH30074.IR Donor Equine Serum, US Origin, Irradiated 90SH30075 Donor Adult Bovine Serum, US Origin 90SH30075.HI Donor Adult Bovine Serum, US Origin, Heat Inactivated 90SH30075.IR Donor Adult Bovine Serum, US Origin, Irradiated 90SH30076 Alpha Calf Fraction, Iron-Supplemented 87SH30076.HI Alpha Calf Fraction, Iron-Supplemented, Heat Inactivated 87SH30076.IR Alpha Calf Fraction, Iron-Supplemented, Irradiated 87SH30079 Dialyzed Fetal Bovine Serum, US Origin 79SH30079.HI Dialyzed Fetal Bovine Serum, US Origin, Heat Inactivated 79SH30079.IR Dialyzed Fetal Bovine Serum, US Origin, Irradiated 79SH30080 FetalClone I 87SH30080.HI FetalClone I, Heat Inactivated 87SH30080.IR FetalClone I, Irradiated 87SH30081 DMEM with High Glucose, without L-glutamine and Sodium Pyruvate 16, 20SH30084 Characterized Australian Foetal Bovine Serum 74SH30084.HI Characterized Australian Foetal Bovine Serum, Heat Inactivated 74SH30084.IR Characterized Australian Foetal Bovine Serum, Irradiated 74SH30087 Cosmic Calf Serum, US Origin 83SH30087.HI Cosmic Calf Serum, US Origin, Heat Inactivated 83SH30087.IR Cosmic Calf Serum, US Origin, Irradiated 83SH30088 Standard Fetal Bovine Serum, US Origin 73SH30088.HI Standard Fetal Bovine Serum, US Origin, Heat Inactivated 73SH30088.IR Standard Fetal Bovine Serum, US Origin, Irradiated 73SH30091 RPMI 1640, 1X, with 2.05 mM L-glutamine (Same as SH30027) 19SH30094 William's E Medium with L-glutamine 24SH30096 RPMI 1640, without L-glutamine 19, 20SH30100 CCM5 (liquid) 38SH30107 Hank's Balanced Salt Solution (HBSS), without Calcium,

Magnesium, Phenol Red (powder)57

SH30109 FetalClone III 87SH30109.HI FetalClone III, Heat Inactivated 87SH30109.IR FetalClone III, Irradiated 87SH30118 Newborn Bovine Calf Serum, US Origin 83SH30118.HI Newborn Bovine Calf Serum, US Origin, Heat Inactivated 83SH30118.IR Newborn Bovine Calf Serum, US Origin, Irradiated 83SH30126 DMEM/F12 1:1, without L-glutamine, with HEPES 17, 20SH30138 PF-MAb (liquid) 42SH30898 Super Low IgG Fetal Bovine Serum, US Origin 79SH30898.HI Super Low IgG Fetal Bovine Serum, US Origin, Heat Inactivated 79SH30898.IR Super Low IgG Fetal Bovine Serum, US Origin, Irradiated 79SH30157 BME/EBSS with L-glutamine 16SH30159 BME/EBSS with L-glutamine 22SH30171 MEM/EBSS with L-glutamine, without Phenol Red 24SH30173 Sodium Bicarbonate (powder) 57SH30187 SFX-CHO (liquid) 38SH30193 MEM/HBSS with L-glutamine 24SH30197 RPMI 1640, with L-glutamine, without Phenol Red 24SH30200 McCoy's 5A with L-glutamine 18, 20


230


SH30203 RPMI 1640, without L-glutamine, with HEPES 19SH30205 MEM Alpha Modification without L-glutamine, Ribo- and

Deoxyribonucleosides24

SH30206 APCF-MAb (liquid) 42SH30211 DMEM/HIGH with L-glutamine, without Sodium Pyruvate,

Phenol Red22

SH30212 Alpha Calf™ Fraction, without Iron 87SH30212.HI Alpha Calf Fraction, without Iron, Heat Inactivated 87SH30212.IR Alpha Calf Fraction, without Iron, Irradiated 87SH30219 MEM Alpha Modification with L-glutamine, without Ribo-

and Deoxyribonucleosides24

SH30220 PF-CHO (liquid) 38SH30221 Water, WFI Quality 21, 57SH30228 IMDM with L-glutamine, HEPES, without Alpha-Thioglycerol 18, 20SH30233 M199 with Hank's Balanced Salt Solution (HBSS), L-glutamine 18SH30235 MEM with Earle's Balanced Salt Solution (EBSS),

Suspension Modification, with L-glutamine19

SH30236 Trypsin, 0.05% 1X, with 0.5 g porcine Trypsin (1:250/L gammairradiated) in HBSS with 0.2 g/L EDTA, without Calcium,Magnesium (liquid)

54

SH30237 HEPES 1 M Solution 53, 57SH30238 Non-Essential Amino Acid, (NEAA) Solution, 100X 53SH30239 Sodium Pyruvate 100 mM Solution 53SH30243 DMEM with High Glucose, 4.0 mM L-glutamine, Sodium

Pyruvate17, 20

SH30244 MEM with Earle's Balanced Salt Solution (EBSS), withoutL-glutamine

19, 20

SH30249 DMEM with High Glucose, L-glutamine, HEPES, withoutSodium Pyruvate

17

SH30253 M199 with Earle's Balanced Salt Solution (EBSS), L-glutamine 18, 20SH30254 M199/EBSS with L-glutamine, without Phenol Red 23SH30255 RPMI 1640 Medium, with 25 mM HEPES, L-glutamine 19, 20SH30256 Phosphate Buffered Saline (PBS), 1X, 0.0067 M PO4, without

Calcium, Magnesium, Phenol Red21, 57

SH30258 Phosphate Buffered Saline (PBS) 10X, 0.067 M PO4, withoutCalcium, Magnesium, Phenol Red

57

SH30259 IMDM without L-glutamine 18SH30261 DMEM/F12 1:1, with L-glutamine, HEPES, Sodium Pyruvate 17SH30262 DMEM with High Glucose, without Calcium, without

Magnesium17

SH30264 Dulbecco's Phosphate Buffered Saline (DPBS), 1X, withCalcium, Magnesium, without Phenol Red

21, 56

SH30265 MEM Alpha Modification, with L-glutamine, Ribo- andDeoxyribonucleosides

19, 20

SH30268 Hank's Balanced Salt Solution (HBSS), 1X, with Calcium,Magnesium, without Phenol Red

21, 57

SH30269 MEM Glasgow Modification with L-glutamine 24SH30270 McCoy's 5A with L-glutamine, without Phenol Red 18SH30271 DMEM/F12 1:1, with L-glutamine, without HEPES 17, 20SH30272 DMEM/F12 1:1, with L-glutamine, without HEPES, Phenol Red 17SH30278 SFX-Insect (liquid) 21, 48SH30280 TNM-FH (liquid) 18, 48SH30282 IPL-41 (powder) 48SH30284 DMEM with High Glucose, with L-glutamine, without Phenol

Red, Sodium Pyruvate17

SH30285 DMEM with High Glucose, without L-glutamine, with SodiumPyruvate

17, 20

SH30289 Calcium Chloride Solution, 1 M Solution 53SH30291 HEPES Solution, 50 mM, 0.15 M NaCl, 20 mM EDTA, pH 8.0 53, 57SH30297 M199/EBSS with L-glutamine, L-Amino Acids 23SH30322 Lactalbumin Hydrolysate (LAH) growth enhancement

supplement53

SH30327 MEM/EBSS (Autoclavable) 24SH30330 M199 with Hank's Balanced Salt Solution (HBSS), L-glutamine,

L-Amino Acids18

SH30556 CDM4CHO (powder) 37SH30333 PF-CHO (powder) 38SH30336 L-glutamine (powder) 53

SH30337 HEPES Buffer (powder) 57SH30340 BEVS PlaKit 48SH30346 DMEM/HIGH without L-glutamine, Sodium Pyruvate, Calcium,

Magnesium22

SH30349 ADCF-MAb (liquid) 20, 42SH30350 SFX-Insect (powder) 48SH30351 M199/EBSS Filter Friendly, with L-glutamine 23SH30352 PF-Vero 49SH30352 SFM4Transfx-293 without L-glutamine (liquid) 20, 45SH30355 PF-293 MPS (powder) 46SH30356 PF-293 (liquid) 46SH30357 Soy Hydrolysate UF (25%) (liquid) 53SH30359 PF CHO LS with L-glutamine (liquid) 20, 38SH30370.HI Standard Australian Foetal Bovine Serum, Heat Inactivated 74SH30370.IR Standard Australian Foetal Bovine Serum, Irradiated 74SH30371 D-Glucose (powder) 53SH30378 Dulbecco's Phosphate Buffered Saline (DPBS), 10X, without

Calcium, Magnesium, Phenol Red56

SH30380 IMDM without L-glutamine, with HEPES 23SH30382 SFM4MAb-Utility with L-glutamine (liquid) 21, 41SH30391 SFM4MAb without L-glutamine (liquid) 41SH30394 Tryptose Phosphate Broth (TPB) (powder) 53SH30396 Characterized Fetal Bovine Serum, Canadian Origin 73SH30396.HI Characterized Fetal Bovine Serum, Canadian Origin, Heat Inactivated 73SH30396.IR Characterized Fetal Bovine Serum, Canadian Origin Irradiated 73SH30397 Standard Fetal Bovine Serum, Canadian Origin 73SH30397.HI Standard Fetal Bovine Serum, Canadian Origin, Heat Inactivated 73SH30397.IR Standard Fetal Bovine Serum, Canadian Origin, Irradiated 73SH30401 New Zealand Newborn Bovine Serum 84SH30401.HI New Zealand Newborn Bovine Serum, Heat Inactivated 84SH30401.IR New Zealand Newborn Bovine Serum, Irradiated 84SH30402 Adult Bovine Serum, New Zealand Origin, Non-Sterile 90SH30406 New Zealand Foetal Bovine Serum 73SH30406.HI New Zealand Foetal Bovine Serum, Heat Inactivated 73SH30406.IR New Zealand Foetal Bovine Serum, Irradiated 73SH30406.IR Super Safe New Zealand Fetal Bovine Serum, Irradiated 79SH30409 Adult Bovine Serum, New Zealand Origin 90SH30409.HI Adult Bovine Serum, New Zealand Origin, Heat Inactivated 90SH30409.IR Adult Bovine Serum, New Zealand Origin, Irradiated 90SH30413 New Zealand Cosmic Calf Serum 84SH30413.HI New Zealand Cosmic Calf Serum, Heat Inactivated 84SH30413.IR New Zealand Cosmic Calf Serum, Irradiated 84SH30417 Donor Bovine Serum, New Zealand Origin 90SH30417.HI Donor Bovine Serum, New Zealand Origin, Heat Inactivated 90SH30417.IR Donor Bovine Serum, New Zealand Origin, Irradiated 90SH30513 SFM4MAb with L-glutamine (liquid) 21, 41SH30516 SFM4CHO-Utility with L-glutamine (liquid) 20, 38SH30517 SFM4CHO-Utility (powder) 38SH30518 SFM4CHO (powder) 38SH30519 CDM4Retino (powder) 46SH30520 CDM4Retino (liquid) 46SH30521 SFM4HEK293 with L-glutamine (liquid) 21, 46SH30522 SFM4HEK293 (powder) 46SH30525 Leibovitz L-15 Medium, with 2.05 mM L-glutamine 18SH30526 Ham's F12, Kaighn's Modification 18SH30527 Donor Adult Bovine Serum, US Origin (non-EMEA equivalent

SH30075)92

SH30528 New Zealand Adult Bovine Serum (non-EMEA equivalentSH30409)

92

SH30529 Water, Cell Culture Grade, Endotoxin-free 21, 57SH30530 FetalClone III (non-EMEA equivalent SH30109) 92SH30531 Defined Fetal Bovine Serum (FBS), US Origin (non-EMEA

equivalent SH30070)92

SH30532 Characterized FBS, US Origin (non-EMEA equivalent SH30071) 92SH30533 Cosmic Calf Serum, US Origin (non-EMEA equivalent SH30087) 92


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SH30534 Iron-Supplemented Bovine Calf Serum, US Origin (non-EMEAequivalent SH30072)

92

SH30535 SFM4MAb (powder) 41SH30538 Water, Molecular Biology Grade, DNase, RNase, Protease free 21, 57SH30541 Bovine Growth Serum, US Origin 83SH30541.HI Bovine Growth Serum, US Origin, Heat Inactivated 83SH30541.IR Bovine Growth Serum, US Origin, Irradiated 83SH30544 Bovine Calf Serum, US Origin (non-EMEA equivalent

SH30073)92

SH30545 Characterized Australian FBS (non-EMEA equivalent SH30084) 92SH30547 ADCF-MAb without L-glutamine (liquid) 20, 42SH30548 SFM4CHO without L-glutamine (liquid) 20, 37SH30549 SFM4CHO (liquid) 21, 37SH30550 SFM4MAb-Utility (powder) 41SH30552 SFM4MegaVir without L-glutamine (liquid) 21, 49SH30554 LS1000 Lipid Supplement, ADCF (liquid) 42, 52, 53SH30555 LS250 Lipid Supplement, ADCF (liquid) 42, 52, 53SH30556 CDM4CHO (powder) 37SH30557 CDM4CHO with L-glutamine (liquid) 20, 37SH30558 CDM4CHO without L-glutamine (liquid) 20, 37SH30559 New Zealand Standard Foetal Bovine Serum 73SH30559.HI New Zealand Standard Foetal Bovine Serum, Heat Inactivated 73SH30559.IR New Zealand Standard Foetal Bovine Serum, Irradiated 73SH30560 New Zealand Calf Serum (non-EMEA equivalent SH30401) 92SH30563 DMEM High Modified, with Sodium Pyruvate, without

L-glutamine, with 25 mM HEPES17

SH30564 MEM-RS with L-glutamine 25SH30565 DMEM-RS with L-glutamine 25SH30568 MEM/Alpha Modification without L-glutamine, Ribo- and

Deoxyribonucleosides19, 20

SH30574 Bovine Serum Albumin (BSA), Cell Culture Grade, pH 7.0,Lyophilized Powder

53

SH30578 CDM4NS0 (powder) 40SH30579 CDM4NS0 without L-glutamine (liquid) 20, 40SH30584 Cell Boost 1 (R05.2) 52SH30585 DMEM with High Glucose, Modified, without Phenol Red,

without Sodium Pyruvate, without L-glutamine17

SH30586 GS-MAX 52SH30587 SFM4MegaVir (powder) 49SH30588 Hank's Balanced Salt Solution (HBSS), 1X, without Calcium,

Magnesium, Phenol Red57

SH30590 SG-200, (Stable dipeptide of L-Alanyl L-glutamine) 200 mM,in 0.85% NaCl Solution

53

SH30591 New Zealand Bovine Growth Serum 84SH30591.HI New Zealand Bovine Growth Serum, Heat Inactivated 84SH30591.IR New Zealand Bovine Growth Serum, Irradiated 84SH30592 New Zealand Bovine Calf Serum Iron Supplemented

(non-EMEA equivalent SH30626)92

SH30594 Pluronic 10% Solution 53SH30596 Cell Boost 2 (R15.4) 52SH30597 Dulbecco's Phosphate Buffered Saline (DPBS), 10X, with

Calcium,Magnesium56

SH30598 MEM Amino Acid Solution, 50X 53SH30599 MEM Vitamin Solution, 100X 53SH30600 MEM (Richter's Modification), without Phenol Red 19SH30601 MEM (Richter's Modification) with L-glutamine and Phenol Red 19SH30602 McCoy's 5A with L-glutamine, HEPES 18SH30603 MEM for Suspension Cultures, without L-glutamine, Calcium

Chloride, or Magnesium19

SH30604 DMEM with High Glucose, Modified, without Phenol Red,L-glutamine, with Sodium Pyruvate

17

SH30605 RPMI 1640, without L-glutamine or Phenol Red 19SH30606 DMEM with High Glucose, Sodium Pyruvate, without

L-glutamine, Methionine, Cystine17

SH30607 DMEM with High Glucose, Sodium Pyruvate, withoutL-glutamine and Phosphate

17

SH30610 Grace's Unsupplemented (liquid) 18, 48SH30611 New Zealand FBS (non-EMEA equivalent SH30406) 92SH30612 Pluronic (powder) 53

SH30614 Hypoxanthine/Thymidine (HT) Solution, 50X 53SH30623 Ham's F12-RS with L-glutamine 25SH30624 New Zealand Donor Bovine Serum (non-EMEA equivalent

SH30417)92

SH30625 New Zealand Cosmic Calf (non-EMEA equivalent SH30413) 92SH30626 New Zealand Iron-Supplemented Calf Serum 84SH30626.HI New Zealand Iron-Supplemented Calf Serum, Heat Inactivated 84SH30626.IR New Zealand Iron-Supplemented Calf Serum, Irradiated 84SH30628 DMEM-RS with L-glutamine, Complete Powder 25SH30629 MEM-RS with L-glutamine, Complete Powder 25SH30635 ADCF-MAb (powder) 42SH30800 CDM4MAb (powder) 41SH30801 CDM4MAb with L-glutamine (liquid) 20, 41SH30802 CDM4MAb without L-glutamine (liquid) 20, 41SH30820 SFM4CHO-A without L-glutamine 20SH30821 SFM4CHO-A with L-glutamine 20, 38SH30822 AdvanceSTEM IMDM4SC without L-glutamine 27SH30824 AdvanceSTEM DMEM4SC with 4.5 mg/L Glucose, without

L-glutamine and Sodium Pyruvate27

SH30825 Cell Boost 3 (JM3.5) 52SH30850 AdvanceSTEM ES Qualified Dulbecco's Phosphate Buffered

Saline (DPBS) without Calcium or Magnesium27

SH30851 AdvanceSTEM ES Qualified HEPES (1 M) 27SH30852 AdvanceSTEM ES Qualified L-glutamine 200 mM 27SH30853 AdvanceSTEM ES Qualified Non-Essential Amino Acids (100X 27SH30855 Lipid-Reduced Fetal Bovine Serum, U.S. Origin 79SH30855.HI Lipid-Reduced Fetal Bovine Serum, U.S. Origin, Heat Inactivated 79SH30855.IR Lipid-Reduced Fetal Bovine Serum, U.S. Origin, Irradiated 79SH30856 Wear Testing Fluid for Artificial Joints 30, U.S. Origin 90SH30857 Cell Boost 4 (PS307) 52SH30858 CDM4HEK293 without L-glutamine (liquid) 20, 45SH30859 CDM4HEK293 without L-glutamine (powder) 45SH30860 SFM4Transfx-293 without L-glutamine (liquid) 20, 45SH30861 SFM4Transfx-293 without L-glutamine (powder) 45SH30865 Cell Boost 5 (CN-F) 52SH30866 Cell Boost 6 (CN-T) 52SH30870 AdvanceSTEM Low Osmo DMEM without L-glutamine 27SH30871 CDM4PERMAb without L-glutamine (liquid) 20, 45SH30872 CDM4PERMAb without L-glutamine (powder) 45SH30874 AdvanceSTEM Serum Replacement 27SH30875.KT Mesenchymal Stem Cell Expansion Kit 31SH30876.KT Adipogenic Differentiation Kit 31SH30877.KT Osteogenic Differentiation Kit 31SH30878 Growth Supplement 31SH30879 Mesenchymal Stem Cell Basal Medium 31SH30881 Osteogenic Differentiation Medium 31SH30886 Adipogenic Differentiation Medium 31SH30889 Chondrogenic Differentiation Medium 31SH30890 Cell Boost Process Supplement Kit 52SH30891 Wear Testing Fluid for Artificial Joints 20, U.S. Origin, Heat

Inactivated90

SH30892.KT Neural Differentiation Kit 31SH30893 Neural Differentiation Medium 31SH30894 Cryopreservation Medium 31SH30896 Antifoam Irradiated 64SH30897 Antifoam Irradiated (ADCF) 64SH30898 Super Low IgG Fetal Bovine Serum, U.S. Origin 79SH30898, HI Super Low IgG Fetal Bovine Serum, U.S. Origin, Heat Inactivated 79SH30898, IR Super Low IgG Fetal Bovine Serum, U.S. Origin, Irradiated 79SH30908 Porcine Serum, New Zealand Origin 90SH30908.HI Porcine Serum, New Zealand Origin, Heat Inactivated 90SH30908.IR Porcine Serum, New Zealand Origin, Irradiated 90SH30910 USDA-Tested Fetal Bovine Serum 74SH30910.HI USDA-Tested Fetal Bovine Serum, Heat Inactivated 74SH30912 SFM4 Insect with L-glutamine, Powder 48SH30913 SFM4 Insect with L-glutamine, Liquid 48


232


SV30080 Gentamicin Solution, 50 mg/mL 59SV30082 Pen/Strep/Glutamine Solution 59SV30084 Trypan Blue Solution, 0.4% in Phosphate Buffered Saline 54SV30087 Research Grade Fetal Bovine Serum, South American Origin,

China Customers74

SV30090 HyQSphere Microcarriers, HLX II-170 Polystyrene microcarriercoated with cationic trimethyl ammonium with a nominalspecific gravity of 1.11 and bead diameter of 160–180 µm.This bead has a surface charge.

61

SV30092 HyQSphere Microcarrier Starter Kit Contains 5 Grams Each ofPro-F 102-L, P 102-L, P Plus 102-L, FACT 102-L, CGEN 102-L,and HLX II-170 Microcarriers

61

SV30098 Primogenix 129/X1 Mouse Embryonic Stem Cells (mESCs) 28SV30101 CET HumanWharton's Jelly Mesenchymal Stem Cells ≥ 100,000 32SV30102 CET Human Adipose-Derived Mesenchymal Stem Cells ≥ 100,000 32SV30103 CET Human Amniotic Mesenchymal Stem Cells ≥ 100,000 32SV30104 CET Human Amniotic Epithelial Stem Cells ≥ 100,000 33SV30105 CET Human Umbilical Cord Blood ≥ 100,000 33SV30106 CET Human Cord Blood CD34+ Stem Cells ≥ 100,000 33SV30107 CET Human Cord Blood CD133+ Stem Cells ≥ 100,000 33SV30109 Primogenix B6NMouse Embryonic Stem Cells (mESCs) 28SV30110 CET Human Bone Marrow Mesenchymal Stem Cells ≥ 100,000 32SV30138 HyStem Kit 34SV30139 HyStem C-Kit 34SV30140 HyStem HP-Kit 34SV30143 Research Grade FEtal Bovine Serum, European Union Origin 74SV30160 Research Grade Fetal Bovine Serum, South American Origin,

European Customers74

SV30160.HI Research Grade Fetal Bovine Serum, Heat Inactivated SouthAmerican Origin, European Customers

74

SV30160.IR Research Grade Fetal Bovine Serum, Irradiated, SouthAmerican Origin, European Customers

74

SV30370 Standard Australian Foetal Bovine Serum 74

SV30006 CultiSpher GL Microcarriers, Macroporous gelatin bead 62SV30008 CultiSpher G Microcarriers, Macroporous gelatin bead 62SV30009 CultiSpher S Microcarriers, Macroporous gelatin bead 62SV30010 Penicillin-Streptomycin Solution 10,000 µg/mL Penicillin,

10,000 µg/mL Streptomycin in 0.85% NaCl59

SV30014 USDA-Tested Fetal Bovine Serum 74SV30014.HI USDA-Tested Fetal Bovine Serum, Heat Inactivated 74SV30014.IR USDA-Tested Fetal Bovine Serum, Irradiated 74SH30908 Porcine Serum, New Zealand Origin 90SH30908.HI Porcine Serum, New Zealand Origin, Heat Inactivated 90SV30030 HyQTase Cell Detachment Solution, in DPBS with EDTA

(non-mammalian)54

SV30031 Trypsin 0.25% 1X Solution, with 2.5 g Porcine Trypsin (1:250/L)in HBSS, without Calcium and Magnesium, with 0.1% EDTA

54

SV30037 Trypsin 2.5% 10X Solution, with 25.0 g Porcine Trypsin(1:250/L) in HBSS, without Calcium Magnesium, EDTA orPhenol Red

54

SV30040 HyQSphere Microcarriers, FACT 102-L Collagen-coated plasticwith surface charge. General purpose bead.

61

SV30044 HyQSphere Microcarriers, CGEN 102-L Same as fact beadwithout surface charge.

61

SV30048 HyQSphere Microcarriers, Pro-F 102-L ADCF Plastic beadcoated with ProNectine F, a recombinant protein. Superiorperformance bead facilitates attachment of fastidious celllines in ADCF conditions

61

SV30052 HyQSphere Microcarriers, plus 102-L ADCF Plastic beadwith surface charge. General purpose bead for use with serum-containing media cultures or other non-fastidious cell lines.

61

SV30056 HyQSphere Microcarriers, P 102-L 61SV30068 G418 Sulfate, >90% Purity 59SV30069 G418 Sulfate Solution, 50 mg/mL 59SV30070 Hygromycin B Solution, 50 mg/mL 59SV30075 Puromycin 2 HCl, USP 59SV30078 Amphotericin B (Fungizone) Solution, 250 Rg/mL 59SV30079 Antibiotic Antimycotic Solution (Pen/Strep/Fungizone), 100X,

10,000 U/mL Penicillin G, 10,000 Rg/mL Streptomycin,25 Rg/mL Amphotericin B

59

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Classical Media

Reagents

Serum

Stem Cell Products

Serum-Free Media

Process Supplements

Specialty Waters

Custom Products


Thermo Scientific HyClone Cell Culture Products and Capabilities

2010/2011

THerm

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TifiC HyClo

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CTS an

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iliTieS 2010/2011

SM0508.01

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