corning life sciences - · pdf filecorning life sciences – different brands & market...
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CORNING Life Sciences
Cell Culture TreatmentsCell Culture Treatments
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Corning – A brief History
- A brief history
- Corning’s Brands
- Corning’s Workflows- Cell Culture Workflow
- Cell Culture surfaces- Suspension cells
- Non treated
© 2013 Corning Incorporated 2
- ULA = Ultra Low Attachment
- Adherent Cells- TC = standard treated culture
- Synthetic non specific
- Biological coating
- Syntethic defined
- Syntehtic specific
- Other products with specific cell culture surfaces- Inserts cell culture
- Assays microplates
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Founded in 1851 : world leader in specialty glass and ceramics.
Corning created and make keystone components that enable high-technologysystems for consumer electronics, mobile emissions control, telecommunicationsand life sciences : bubble (first one it Edison ; touchable screens ; …
Headquarters : Corning, New York
Employees:
Approximately 25,000 worldwide
Corning – A brief History
© 2013 Corning Incorporated 3
Corning acquired Costar Corporation to become Corning Costar in 1993.
Corning acquired Axygen end of 2009
Corning acquired Gosselin end of 2010
Corning acquired Falcon DL end of 2012
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Corning Life Sciences – Different Brands & Market segments
• Whatever is your application, you can now get a more comprehensive line of labresearch tools with Corning’s quality.
FALCON
© 2013 Corning Incorporated 4
cellgro® is available in Europe as Mediatech™.
Cell Culture, Drug Discovery &Liquid Handling
Molecular BiologyGenomics
Microbiology
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Corning Life Science - Workflows
© 2013 Corning Incorporated 5
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Corning Life Science - Workflows
Cell Culture surfaces :
- Suspension cells
- Adherent cells
Adherent Cells :
- Standard - TC
- Synthetic improved
© 2013 Corning Incorporated 6
- Synthetic improved(CellBIND ; Primaria)
- Biological(ECM – Biocoat)
- Synthetic defined(Purecoat)
- Synthetic specific(OsteoAssays / Synthemax)
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StationaryMoving
Adherent culture Suspension culture
• Spinner
StationaryMoving
• Flasks• Rollerbottles
• Flasks
Corning Life Science – Cell Culture surfaces
© 2013 Corning Incorporated 7
• Spinnerflasks
• Shakerflasks
• Rollerbottles
• Bags
• Flasks• Storagebottles
• Dishes• Microplates• Transwellinserts
• Bags
bottles
• Micro-carriersin spinnerflasks
• Flasks onrockers
• Microplates
• Dishes
• Transwellinserts
• HYPERFlask
• CellSTACK
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Moving
Adherent culture Suspension culture
Corning Life Science – Cell Culture surfaces
© 2013 Corning Incorporated 8
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• Suspension Cell Culture :
− NT (Not Treated)
• For standard suspension cell culture
− ULA (Ultra Low Attachment)
Corning Life Science – Cell Culture surfacesSuspension Cells
© 2013 Corning Incorporated 9
• For advanced suspension cell applications (stem cells..)
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• Adherent Cell Culture :
- Standard – TC
- Synthetic improved (CellBIND ; Primaria)
Corning Life Science – Cell Culture surfacesAdherent Cells
Specificity to CellsAnimal Free
YES
YES
© 2013 Corning Incorporated 10
- Biological (ECM – Biocoat)
- Synthetic defined (Purecoat)
- Synthetic specific (OsteoAssays / Synthemax)
NO
YES
YES
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Cell Culture surfaces - Adherent CellsStandard Tissue Culture Surfaces
Standard Tissue Culture (TC) Treatement :
• To be used with adherent cells, the Polystyrene needs to be treated toadd charges and to get it hydrophylic.
• Polystyrene is fully hydrophobic and no natural.
• TC-surface treatment is achieved by (similar proceedure) :
− By gas plasma : placing polystyrene vessels into a vacuum chamber that
© 2013 Corning Incorporated 11
− By gas plasma : placing polystyrene vessels into a vacuum chamber thatreceives a mixture of gases with electric power.
− By corona discharge.
• The treatment will create a modification of the polystyrene altering it‘ssurface chemistry Negative charges
• Feature No specific
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Cell Culture surfaces - Adherent CellsStandard Tissue Culture Surfaces
- uncharged- hydrophobic- No cell attachment
e.g. "non-treated"rounder
40 ml droplet
Conveyor belt
High energy oxygen ions
Corona Discharge Plate
Conveyor belt
High energy oxygen ions
Corona Discharge PlateCultureVessels
© 2013 Corning Incorporated 12
- charged groups- hydrophilic- cell attachment
e.g. "TC"flatter
40 ml droplet
HH H H
HH H HHH
H
H
C C C C CC C CC
HH H H
HH H HHH
H H
H
H
C C C C CC C CC
HH H H
HH H HHH
H
H
C C C C CC C CC
HH H H
HH H HHH
H H
H
H
C C C C CC C CCHydrophobic
H H
H
O HH H
O
OHH
OC C C C C C C CC
H
H
O OHHH
H H
O
OH
OH
HO
H
OC C C C C C C CCHydrophilic
H H
H
O HH H
O
OHH
OC C C C C C C CC
H
H
O OHHH
H H
O
OH
OH
HO
H
OC C C C C C C CCHydrophilic
H H
H
O HH H
O
OHH
OC C C C C C C CC
H
H
O OHHH
H H
O
OH
OH
HO
H
OC C C C C C C CCHydrophilic
SurfacetreatmentSurfacetreatment
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Cell Culture surfaces - Adherent CellsSynthetic Non Specific – CellBIND & Primaria
Syntethic Non specific (Improved attachment) – CellBIND or Primaria :
• To be used with adherent cells, the Polystyrene needs to be treated toadd charges and to get it hydrophylic.
• Polystyrene is fully hydrophobic and no natural.
• TC-surface treatment is achieved by :
− a specific gaz mixture.
© 2013 Corning Incorporated 13
− a specific gaz mixture.
− A difference source of energy (microwaves).
• The treatment will create a modification of the polystyrene altering it‘ssurface chemistry more balanced – vs + positives
• Feature No specific ; more dedicated to difficult cells
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Cell Culture surfaces - Adherent CellsSynthetic Non Specific – CellBIND & Primaria
CellBIND® TCTBD
Primaria™Not Treated
Treatment N2O O2 NH4 /proprietary
% Oxygen/
Nitrogen35% Oxygen 22% Oxygen
14.1% Oxygen
11.1% Nitrogen1.8% Oxygen
Contact
© 2013 Corning Incorporated 14
TCT
CellBIND
Contact
angle23 - 26° 56 - 63° 75° 90 - 95°
Charge Negative Negative Positive
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Cell Culture surfaces - Adherent CellsECM – Biological Coating
Biological coating with ECM molecules :
• Cells are not living in 2D into a plastic box !
• They are normally „living“ in the context of other cells, surrounded and incontact with an extracellular matrix (ECM)
• The TC-treated surfaces can be coated with ECM components :
© 2013 Corning Incorporated 15
− Biological molecules from the ECM :
• Can be bought pre-coated – ready-to-use products.
• Can be bought as a chemical – to plate into the lab.
− Reconstituted ECM = Corning® Matrigel® Matrix :
• Can be bought pre-coated – ready-to-use products.
• Can be bought as a chemical – to plate into the lab.
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Cell Culture surfaces - Adherent CellsECM – Biological Coating
READY-TO-USE
Biological molecules from the ECM :
Collagen I
Collagen IV
Fibronectin
Gelatin
Laminin
© 2013 Corning Incorporated 16
Laminin
Poly-Lysine
Poly-D-Lysine/Laminin
Poly-L-Ornithine/Laminin
Laminin/Fibronectin
Reconstituted ECM = Corning® Matrigel® Matrix :
Matrigel
Matrigel Growth Factors Reduced (GFR)
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Cell Culture surfaces - Adherent CellsECM – Biological Coating
TO COAT INTO THE LAB :
Biological molecules from the ECM :
Collagen I, II, III, IV, V, VI
Fibronectin
Laminin
Osteopontin
Poly-Lysine
© 2013 Corning Incorporated 17
Poly-Lysine
Vitronectin
Reconstituted ECM = Corning® Matrigel® Matrix :
Matrigel
Matrigel hESC (human Embryonic Stem Cells) qualified
Matrigel HC to do tumor injection
Matrigel Phenol Red free
Matrigel GFR (Growth Factor Reduced)
Dispase & Cell Recovery Solution to recover cells from Matrigel
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Cell Culture surfaces - Adherent CellsECM – Biological Coating
APPLICATIONS :• Endothelial cell migration, invasion
• Angiogenesis tube formation
• Tumor invasion
• 3-D-cell culture
• Maintenance of human embryonic stem cells
• Neurite outgrowth
• Hepatocyte culture
© 2013 Corning Incorporated 18
SEM‘s of hepatocytes cultured for two days on collagen I (1),collagen I gels (2), or BD Matrigel Matrix (3). Note the cluster ofspherical cells for hepatocytes cultured on Corning Matrigel Matrix,typical of differentiated cells.
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TC Plates
After Wash,Before Wash After Wash Calcein AM Staining
APPLICATIONS :• HEK293
Cell Culture surfaces - Adherent CellsECM – Biological Coating
© 2013 Corning Incorporated 19
BD BioCoatPDL Plates
After Wash,Before Wash After Wash Calcein AM Staining
HEK-293 cells were cultured under serum-free conditions on TC-treated or BDBioCoat PDL 384-well black/clear plates. Samples were washed using aSkatron Washer (Molecular Devices).
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ADVANTAGES :• More in vivo like properties
• Better way of life :
− attachment, proliferation, growth
− including difficult cells as : primary cells, neural cells, hepatocytes,endothelial cells, …
• Differentiation and functional studies possible
Cell Culture surfaces - Adherent CellsECM – Biological Coating
© 2013 Corning Incorporated 20
• Differentiation and functional studies possible
• 3-dimensional culture of cells possible, when cells are embedded in a thick gel,and not only applied to a thin coating
• Testing packs to try different molecules (mix of different surfaces)
LIMITATION :• Not Animal components free
• Limit shelf life
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• An advanced cell culture surfaces, that uses a novel process tocoat the culture vessel in a very defined and reproducible way
• 2 options :− amine (+) residues
− carboxyl (-) residues
BD PureCoatTM
Cell Culture surfaces - Adherent CellsSynthetic defined - Purecoat
© 2013 Corning Incorporated 21
• Advantages :− Chemically defined
− Animal-free
− High consistency
− Low CV‘s in cell-based assays
− Stable at room temperature (3 years)
+ + + + + +
or- - - - - - -
BD PureCoatTM
Thin-film coatingCarboxyl or Amine
momomers
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Cell Culture surfaces - Adherent CellsSynthetic defined - Purecoat
© 2013 Corning Incorporated 22
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2 options :− Fibronectine
− Collagen
• ECM Mimetic Surface technology
Cell Culture surfaces - Adherent CellsSynthetic defined – Purecoat ECM Mimetic
© 2013 Corning Incorporated 23
• Main applications :− Human Mesenchymal Stem Cells
− Human Endothelial Progenitor Cells
− Human Keratinocytes
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PuraMatrix Petpide Hydrogel :- Synthetic matrix to create a defined 3D microenvironments
- origin: synthetic, animal free
- major components: - 16 aa peptide
- average pore size 50-200 nm
- no growth factors
Cell Culture surfaces - Adherent CellsSynthetic defined – PuraMatrix
© 2013 Corning Incorporated 24
- Applications :
- Puramatrix scaffold allows creation of highly defined environment throughaddition of factors & compounds
- Does not activate RGD-dependent integrin signaling
- Highly reproducible experiments
- Compatible with most cells types in vitro & in vivo (stem cell proliferation, tumorcell migration and invasion, angiogenesis assays, tissue regeneration)
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Synthemax Surface :
1 Application Stem cell
What is the Corning® Synthemax™ Surface ?
Cell Culture surfaces - Adherent CellsSynthetic Specific – Synthemax
© 2013 Corning Incorporated 25
polystyrenepolystyrene polystyrene
1. Uncoated surface 2. Acrylate coated 3. Peptide covalentlybound
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Feeders ECMs Synthemax
Storage
• Need to be usedwithin a week
• Need to be stored at -70C• When working they
• No special storageconditions; can bestored at room temp
• Advantages : Time Saving & Shelf Life
Cell Culture surfaces - Adherent CellsSynthetic Specific – Synthemax
© 2013 Corning Incorporated 26
need to be kept cold
Shelf Life
• Last only a week onceprepared
• Plates need to be usedas soon as possible
• 2 year shelf life
Easy-to-Use
• Harvest mouseembryos• Grow up theFibroblasts• Time intensive
• Laborious time-consuming prep
• No prep, just cutopen the package anduse
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Customer Needs Research Therapeutic Screening
Traceability
10-6 Sterility
Consistent Performance
Shelf Life
No Special Storage Conditions
Ease of Use
Cell Culture surfaces - Adherent CellsSynthetic Specific – Synthemax
© 2013 Corning Incorporated 27
Ease of Use
Purity Post-Harvesting
Lot Size
Multiple Formats
GMP Production
Expand & Differentiate on same Surface
Inventory Availability
Xeno-Free Substrate
Ability to visualize cells
Automation Friendly Format
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Synthemax
Consistency
• Manufactured under GMP conditions• Performs as good as Matrigel• Consistent performance
Cell Culture surfaces - Adherent CellsSynthetic Specific – Synthemax
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Consistency • Consistent performance• 2 year shelf life
Defined, Synthetic,Xeno-Free
• Defined• Synthetic• No animal components
Easy-to-Use
• No prep, just cut open the package and use
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Osteo Assay Surface :
1 Application Osteo/bone assays
Specifications :
Inorganic synthetic coating that mimics
in-vivo bone for in vitro bone cell assays
Cell Culture surfaces - Adherent CellsSynthetic Specific – Osteo Assay
© 2013 Corning Incorporated 29
in-vivo bone for in vitro bone cell assays
Surface applications :
- Consistent assay results for bone cell
- Differentiation and function
- Co-culture of bone cells / metastatic cancer cells
- Alternative to preparing dentine, bone slicesSEM image
magnified not truerepresentation
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Key Benefits Critical Attributes Differentiating Attributes
Time Reduce time and cost of surfacepreparation
Precoated, sterile, ready to use
Direct Readout Detect both cell differentiation andfunctional analysis of bone cells on thesame surface
Transparent to conventional imagingtechniques
Cell Culture surfaces - Adherent CellsSynthetic Specific – Osteo Assay
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same surfaceSupernatant can be sampled for cellfunction
Amenable toautomation
Standard SBS format Can be used with standard imagingtechnologies
Reliable cellresponse
Consistent assays Standard process for manufacturingUniform surface
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• You can find the different cell culture surfaces on multiple vessels :
• Flasks
• Microplates from 6 to 1536 wells
• Dishes
• For specific applications :
Cell Culture surfaces – Procuts available
© 2013 Corning Incorporated 31
• Assays microplates :
• Drug discovery
• Molecules screening
• Cells Culture inserts (Transwell®) :
• Coculture
• Migration
• Invasion
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• Cell Culture Selection Guide Corning
• Cell Culture Selection Guide Corning – Falcon
• Success Stories CellBIND
• Bibliography OsteoAssays
• Bibliography ULA (Ultra Low Attachment)
TOOLS TO FIND THE RIGHT SURFACE
© 2013 Corning Incorporated 32
• Different Protocols or Application notes
• European Scientific Support (2 scientists)
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© 2013 Corning Incorporated 33
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• Glass
− High optical quality and scratch-resistant glass
− Bottom thickness 200 µm
− Intra well bottom flatness <50µm
• Copolimer
− Cyclic Olefin Copolymer(COC)
− Bottom thickness: 127 µm
− Intra-well bottom flatness 5µm
− Inter-well bottom flatness 30
Glass Bottom Microplates
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− Inter-well bottom flatness 30µm
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Flowell
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Flowell
© 2013 Corning Incorporated 36
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Axygen® 5 mL Snaplock Microtubes
5mL
15mm • No necesita adaptador
© 2013 Corning Incorporated 37
• No necesita adaptador• Rotores 15 mm (15ml)
• Rotores 17 mm (15ml)
• 14.000 rpm
• 120ºC (15’ 15psi)
• Libre de ARNasas, ADNasas
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Corning Life Science - AXYGEN Biosciences
Thanks a lot for your time and attention.
© 2013 Corning Incorporated 38
QUESTIONS ?