CELL CULTURING

Girija Maganti

M.Pharm (pharmacology)

What is Cell Culture?

Cell culture refers to the removal of cells from an animal or plant and their subsequent growth in a favorable artificial environment. 

•Mouse, mammals,•Embryo•Embryonated Eggs because stage of differentiation)

organ

explant

Grow in media-monolayer-suspension cells

Cell culture

Finely cut

Finely cut tissue or explant

Enzymic digestion

STAGES OF CULTURE Isolated tissue

(disaggregation)

Primary cell culture(limited lifespan after certain proliferations undergo senescence)

Finite cell cultures

continous cell lines(immortalized cell line acquires ability to proliferate indefinitely by transformation)

Growth Curve

ISOLATION OF TISSUES

• Sterilize the site with 70% alcohol.• Remove tissue aseptically.• Transfer to the laboratory in transport medium• If delay in transporting to lab, keep at 4°C for

up to 72hour.

Enzymatic disaggregation•Warm trypsin, 37˚C for 30 mins, cell damaged if too long

exposure.•Cold pre exposure, soak at 4°C overnight and 37°C for less 30

mins. Advantage: higher yield of viable cells, preserve more cell types

•Other enzyme-collagenase benefit for connective tissues and muscle

(fibrous tissue)- pronase, dipase, DNase, hyaluronidase

Mechanical disaggregation (prevent proteolytic damage)

•Scrapping or spillage•Sieving•Syringes•Trituration by pipette

DISAGGREGATION

CULTURE CONDITIONS Culture conditions vary widely for each cell

type. a substrate or medium that supplies the

essential nutrients (amino acids, carbohydrates, vitamins, minerals)

growth factors hormones gases (O2, CO2)

a  regulated physico-chemical environment (pH, osmotic pressure, temperature)

MEDIA AND SERUM Common basal medium

include Eagle minimal effective medium,Dulbecco’s Modified eagle medium,RPMI 1640,Ham f10.

The above medium includes aminoacids,glucose,salts,vitamins and nutrients.

L-glutamate,serum,antibiotics,

fungicides are added for complete medium.

Serum may or may not be used Most frequently used serum-fetal calf

serum Less expensive sera such as horse or calf

sera can be used. Serum supports growth of cell. Antibiotics and fungicides prevent microbial

contamination. Some cell types like primary cell requrie

additional supliments(collagen ,fibronectin,epidermal growth factor)to attach culture vessel and proliferate.

Sterility of the medium is tested by incubating small aliquot at 37°c for 48 hours,ifany microial growth the medium should be discarded.

CULTURE VESSELS

MODES OF PROPAGATION Depending on their origin, animal

cells grow either as adherent monolayers or in suspension.

Adherent cells are anchorage dependent and propagate as monolayer attached to the cell culture vessel.

Eg:cells derived from tissues Suspension cells:proliferate without

being attached to a substratum.Eg:haemopoietic cell,cells derived

from tumours

PRIMARY CULTURE Capable of only limited number of cell

divisions cells that are placed in culture directly

from the tissue of origin. these are called primary cultures until

the first subculture

Epithelial cell

TYPES OF PRIMARY CELL CULTURE

Mouse embryosChick embryosHuman biopsy materialsTransplantable animal tumourChick embryo organ rudiments ( brain,

heart, lungs, liver, kidney, spinal cord, skin,)

SUBCULTURING Subculturing or "splitting cells," is

required to periodically provide fresh nutrients and growing space for continuously growing cell lines.

The frequency of subculture and the split ratio, or density of cells plated depend on the characteristics of each cell line being carried.

Sub culturing -Adherent Cells Suspension culture.

CONTINOUS CELL LINES • After the first subculture, primary

culture may be called secondary cultures, and thereafter, if continued passage is possible, a continous cell line are formed.

• An established or immortalised cell line has acquired the ability to proliferate indefinitely either through random mutation or deliberate modification, such as artificial expression of the telomerase gene.

EXAMPLES OF ESTABLISHED CELL LINES

May be derived from Normal or Tumor cells.Cell line Organism Origin Tissue

HeLa Human Cervical cancer

293-T Human Kidney (embryonic)

A-549 Human Lung carcinoma

ALC Murine Bone marrow

CHO Hamster Ovary

HB54 Hybridoma Hybridoma

FM3 Human Metastatic lymph node

Mouse kidney cells

Secondary Hamster kidney cellsPrimary cell cultures split several times:

INCUBATION CONDITIONS

Cell cultures should incubated in incubators with tightly regulated temperatures(eg:water jacketed incubator)and co2 con.

Most cell lines grows at 37°c and 5%co2 with saturating humidity.

STERILIZATION OF MEDIUM

The various media constituents and other reagents used in cell cultures must be carefully sterilized either by autoclaving or by filtration. Heat stable constituents tike water, salts, supplements like peptone or tryptase etc. are autoclaved at 121°C for 20 min.

But heat labile constituents like serum, trypsin, proteins, growth factors etc. must be sterilized by filtration through a 0.2 mm porosity membrane filter. Each filtrate should be tested for sterility to avoid failure due to contamination.

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CRYOPRESERVATION Freeze preservation of animal

cells is now routine in all cell line banks.

A cryoprotective agent like DMSO or glycerol (at-130°cis generally added to minimize injury to cells during freezing and thawing.

Frozen ampoules are generally stored in liquid nitrogen refrigerators which are rather convenient and quite safe.

CELL THAWING preserved

culture collection will arrive frozen and in order to use the cells they must be thawed and put into culture

Contamination

Contamination with other cell lines(cross contamination) Yeast Fungi Viruses Bacteria mycoplasma

CHARACTERISTIC FEATURE O0F MICROIAL CONTAMINATION

BACTERIA YEAST FUNGI

CHANGE IN PH PH DROP PH CHANGE WITH HEAVY INFECTIONS

PH CHANGES SOME TIMES

CLOUDY MEDIUM

SHIMMERING IN SPACES BETWEEN CELLS;RODS OR COCCI MAY BE OBSERVED

ROUND OR OVOID PARTICLES THAT BUDD OFF SMALLER PARTICLES

THIN FILAMENTOUS MYCELIA;SOMETIMES CLUMPS OF SPORES

Cross contamination

APPLICATIONSModel Systems : Cell cultures provide a

good model system for studying 1) basic cell biology and biochemistry, 2) the interactions between disease-causing agents and cells, 3) the effects of drugs on cells, 4) the process and triggers for aging, and 5) nutritional studies

Drug screening and development Toxicity testing Cancer research Virology Largescale production of

monoclonal antibodies and vaccines Genetic counselling Gene therapy

Thank you