animal cell culture i

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Animal Cell Culture

W.M.M.S BandaraB.Sc (Hons), MSc (Biotechnology)

MS (USA), M.I.Biol



What is animal Cell Culture?

Removal of cells from an animaland their subsequent growth in a

favorable artificial environment.

Introduction to animal cell culture



•

by enzymatic or mechanical means beforecultivation

• or they may be derived from a cell line or cellstrain that has already been established

Removal of cells from the tissue



History of animal cell culture

• Cell culture was first successfully undertaken

by Ross Harrison in 1907

• Roux in 1885 for the first time maintained

embryonic chick cells in a cell culture

• 1940s: The use of the antibiotics penicillin and

streptomycin in culture medium decreased

the problem of contamination in cell culture.



Historical events in the development of cell culture

• 1878: Claude Bernard proposed that physiologicalsystems of an organism can be maintained in a livingsystem after the death of an organism.

• 1885: Roux maintained embryonic chick cells in a salineculture.

• 1897: Loeb demonstrated the survival of cells isolatedfrom blood and connective tissue in serum and plasma.

• 1903: Jolly observed cell division of salamanderleucocytes in vitro.

•1911: Lewis and Lewis made the first liquid mediaconsisted of sea water, serum, embryo extract, salts andpeptones. They observed limited monolayer growth.

• 1913: Carrel introduced strict aseptic techniques so thatcells could be cultured for long periods.



• 1916: Rous and Jones introduced proteolytic enzymetrypsin for the subculture of adherent cells.

• 1927: Carrel and Rivera produced the first viral vaccine – Vaccinia.

•

1940s: The use of the antibiotics penicillin andstreptomycin in culture medium decreased the problemof contamination in cell culture.

• 1948: Earle isolated mouse fibroblasts which formedclones from single cells. Fischer developed a chemicallydefined medium, CMRL 1066.

• 1952: Gey established a continuous cell line from ahuman cervical carcinoma known as HeLa (Helen Lane)cells. Dulbecco developed plaque assay for animal

viruses using confluent monolayers of cultured cells.


Contd..



Contd..

•1965: Harris and Watkins were able to fuse human andmouse cells by the use of a virus.

• 1975: Kohler and Milstein produced the first hybridomacapable of secreting a monoclonal antibody.

•

1982: Human insulin became the first recombinantprotein to be licensed as a therapeutic agent.

• 1985: Human growth hormone produced fromrecombinant bacteria was accepted for therapeutic use.

• 1990s: Many recombinant products in clinical trial

• 1980 to date: Tissue culture becomes less of anexperimental research field, and more of a widelyaccepted research tool




Safety is very important !

Cell culture laboratory has a number of specific

hazards associated with handling and manipulatinghuman or animal cells and tissues, as well as toxic,corrosive or mutagenic solvents and reagents.

Strict adherence to standard microbiologicalpractices and techniques is essential.

Cell culture laboratory



Cell culture laboratory



Safe Laboratory

• Always wear appropriate personal protective equipment.

Change gloves when contaminated, and dispose of usedgloves.

•

Wash your hands after working with potentiallyhazardous materials.

• Do not eat, drink, smoke, handle contact lenses, apply

cosmetics.

• Be careful when handling of sharps (i.e., needles,

scalpels, pipettes).



•Decontaminate all work surfaces , glasswarebefore and after your experiments, andimmediately after any spill with an appropriatedisinfectant.

• Decontaminate all potentially infectious materialsbefore disposal.

• Report any incidents that may result in exposureto infectious materials to appropriate personnel(e.g., laboratory supervisor, safety officer).

Safe Laboratory



Equipments required for Cell Culture

-Depends mainly on the type of research conducted.



Basic Equipment

• Cell culture hood (i.e., laminar-flow hood or biosafety cabinet)

• Incubator (humid CO2 incubator recommended)

• Water bath

• Centrifuge

• Refrigerator and freezer (–20°C)

• Cell counter (e.g., CountessR Automated Cell Counter or

hemacytometer)

• Inverted microscope

• Liquid nitrogen (N2) freezer or cryostorage container• Sterilizer (i.e., autoclave)




Expanded Equipment

• Aspiration pump (peristaltic or vacuum) • pH meter

• Confocal microscope

• Flow cytometer

Additional Supplies

• Cell culture vessels (e.g., flasks, Petri dishes, roller bottles, multi-

well plates)

• Pipettes and pipettors• Syringes and needles

• Waste containers

• Media, sera, and reagents

• Cells




f




• Cell culture hood (laminar-

flow hood or biosafety

cabinet) –

Provides aseptic work area



• Horizontal flow -provides protection to the culture (if the air flowing towards the

user) or to the user (if the air is drawn in through the front)

• Vertical flow –provides significant protection to the user and the cell culture.

Basic Layout of a culture hood



• Class I

an open-front negative pressure cabinet The exhaust air from

the cabinet is filtered by a high-efficiency particulate air(HEPA) filter. The Class I biosafety cabinet will providepersonnel and environmental protection, but not productprotection.

•Class IIan open-front, ventilated cabinet. This cabinet provides aHEPA-filtered, recirculated mass airflow within the workspace. The exhaust air from the cabinet is also filtered byHEPA filters. Thus, the Class II biosafety cabinet will provide

personnel, environment and product protection

• Class III

a totally enclosed ventilated cabinet of gas-tight construction.Operations within the Class III cabinet are conducted through

attached rubber gloves.

Bio safety Cabinets



• Class I

Bio safety Cabinets

f b



• Class II

Bio safety Cabinets

i f bi



• Class III

Bio safety Cabinets



I b



• Incubator -provides the appropriate environment

for cell growth.

• 5% CO2

• 370C

• Appropriate humidity

Incubator

E i t i d f C ll C lt



Other equipments

• Water bath

• Centrifuge

• Refrigerator and freezer

( –20°C)

• Cell counter (Automated

Cell Counter or

hemacytometer

• Liquid nitrogen (N2)

cryostorage container

• Sterilizer (i.e., autoclave)




E i t i d f C ll C lt



Expanded Equipment

• Flowcytometer – To

identify, characterize

and/or sort desired

cell types.




Confocal Microscope


Additi l S li



Additional Supplies

• Cell culture vessels (e.g., flasks, Petri dishes, roller

bottles, multi-well plates)• Pipettes and pipettors

• Syringes and needles

•Waste containers

• Media, sera, and reagents

• Cells



C ti ll



Counting cells

Square # cells

1 5

2 6

3 5

4 8

Total No of Cells: 24

Total No of cells per square = 6

Total volume = 10 4

Dilution factor = D

Cell concentration = 6 X 10 4 X D / mL

C ti Vi bl ll



• Trypan Blue assay

• Mix cells with Trypan blue, fill the haemocytometer andobserve under the microscope.

• Dead cells stained blue with trypan blue can be countedseparately for a viability count

• Percentage viability =Live cell count X 100

Total cell count

Counting Viable cells



Cell culture vessels

• Flat-bottom and coated, cell

culture vessels are needed for cell

growth.

• Three types of culture vessels are

commonly used : flasks, dishes,and multiwell plates.

• All three types are available in

different sizes with different

surface areas.

• The choice of the vessel depends

on the nature of the procedures

and personal preference.



Culture Flasks

• PYREX glass flasks and Petri dishes

• Disposable plastic culture vessels are

commonly used

• Flasks, dishes and 96 well plates

made up of polystyrene, a longcarbon chain polymer with benzene

rings attached to every other carbon

are available commercially

• Polystyrene was chosen because it

has excellent optical clarity, easy to

mold and can be sterilized by

irradiation.



Tissue Culture Treated Flasks

• Drawback of polystyrene: it is a very

hydrophobic - cells have difficulty attaching.

• Hydrophobic polystyrene surface are modified

to a more hydrophilic surface.

• Corona discharge , extracellular matrix,

collagen, laminin and fibronectin, and heparin

sulfate, hyaluronidate and chondroitin sulfate,poly-D-lysine (PDL), as coatings

d d l l d

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Vented and Plug sealed caps

Flasks with vented caps

are preferred, since non-

vented caps need to beloose when the flasks are

in the incubator to allow

for exchange of gases





-Commercially available disposableculture vessels are sterile

-No need to sterilize

-Transfer of media must be done using a

sterile pipettes

Applications of Cell Culture



Applications of Cell Culture

Study the effects of

drugs and toxiccompounds on the cells

Excellent model systems

for studying the normal

physiology and

biochemistry of cells (e.g.,

metabolic studies, aging)

Drug screening and

development

Large scale manufacturing

of biological compounds

(e.g., vaccines, therapeutic

proteins)



Advantages and disadvantages of cell culture



• The major advantage of using cell culture is the

consistency and reproducibility of results that can be

obtained from using a batch of clonal cells.

• Represent the best experimental models for in vivo

situations.• Can have the same karyotype as the parent tissue

normal or abnormal.

• Can maintain cells in “undedifferentiated” states.






• Control of the environment

• Characterization and homogeneity of the samples

• In vitro modeling of in vivo conditions

• Economy, scale and mechanization of culture

• Avoid animal experiments

• Mimic of in vivo cell behavior (e.g cancer cells)



Cell Culture associated problems



Two main categories

1. Chemical contaminants such as impurities in

media, sera, and water, endotoxins, etc.

2. Biological contaminants such as bacteria, molds,

yeasts, viruses, mycoplasma, as well as cross

contamination by other cell lines.

It is essential to have understanding of the sources of

contaminants and follow good aseptic technique.

Cell Culture associated problems

Cell culture contaminations - Bacteria



Bacteria

- unicellular microorganisms- few micrometers in diameters

- variety of shapes (spheres, rods

and spirals)

- fast growth rates.

Cell culture contaminations - Bacteria

Bacterial contamination is easily detected by visual within a few days of

infection; infected cultures usually appear cloudy (turbid)

Sudden drops in the pH of the culture medium occurs.

Under a low power microscope, the bacteria appear as tiny granules

between the cells, and under a high-power microscope can identify the

shapes of individual bacteria.

Cell culture contaminations - Yeast



• Yeasts - unicellular eukaryotic microorganisms (Fungi)

• cultures become turbid, especially if the contamination isin an advanced stage.

• little change in the pH of the culture at the initiail stages.

•

Under microscopy, yeast appear as individual ovoid orspherical particles, that may bud off smaller particles.





Cell culture contaminations Yeast

Cell culture contaminations - Molds



• Molds -eukaryotic microorganisms (Fungi)

-grow as multicellular filaments called hyphae

-the pH of the culture remains stable in the initial stages

of contamination

•

Under microscopy, the mycelia usually appear as thin,wisp-like filaments, and sometimes as denser clumps of

spores.

Cell culture contaminations Molds

•Spores of many

mold species can

survive extremely

harsh

environments

Cell culture contaminations -Mycoplasma



• Mycoplasma -are simple bacteria that lack a cell wall, and they

are considered the smallest self-replicating organism.• Mycoplasma are very difficult to detect until they achieve

extremely high densities and cause the cell culture to deteriorate;

until then, there are often no visible signs of infection.

•

Detection: by testing the cultures periodically using fluorescentstaining, ELISA, PCR, immunostaining, autoradiography, or

microbiological assays.

Cell culture contaminations Mycoplasma

Cell culture contaminations - Viruses



• Viruses -microscopic infectious agents that take over the

host cells machinery to reproduce. T

• heir extremely small size makes them very difficult to detect

in culture, and to remove them from reagents used in cell

culture laboratories. can be a serious health hazard to the

laboratory• Viral infection of cell cultures can be detected by electron

microscopy, immunostaining with a panel of antibodies, ELISA

assays, or PCR with appropriate viral primers.

Cell culture contaminations Viruses

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