Cell culture is the process by which cells are grown under controlled conditions.

Most important area of research:

Cell and tissue culture based production of vaccine, monoclonal antibodies, pharmaceutical drugs, Cancer research, genetic manipulation etc.

Animal cell, tissue and organ can be cultured like plant cell, tissue and organ on artificial media in control conditions.

Alexis carrel (1912) used tissue and embryo extract as culture media.

Ross Harrison (1907) Made first attempt to culture animal cell.

Enders, Weller and Robbins (19940/19950) grew poliomylities virus in culture.

Interferon were named by Issac in 1966.

In 1980 recombinant DNA technology used to produce interferon Alpha, beta, gamma

In 1996 Wilmut and coworkers successfully produce a transgenic sheep named Dolly via neuclear transferring technique.

In 2002, Clonaid claimed to produce a cloned human baby named Eve.

Requirements for animal cell and tissue culture

Animal cell can grow only to a limited generation in even in the best nutritive media.

Growth also depends on the sources of the tissue isolated.

Special feature of different tissue: In culture Neuronal cell required tough

condition. Connective tissue (skin) cell fibroblast can

grow up to some generation (die)

Contact inhibition (inhibition of further growth after reaching to the wall of the container)

The environment in culture is different.

Cancerous cells apparently differ from the normal cells.

Basic requirement Air conditioning of room Incubator Microscope room etc

 Anchorage-dependent cells required support for their growth in vitro.

Therefore a Large number of substrates which may be adhesive (e.g. Plastic, Glass, Palladium, Metallic surface etc

Or non adhesive agar, agarose etc

 Media: (growth medium or culture medium is a liquid or gel designed to support the growth of microorganisms or cells

Generally: Natural and synthetic media

It is the natural source of nutrient sufficient for growth and proliferation of animals cell and tissue.

There are three types Coagulans or plasma clots: available in the

market in the form of liquid plasma kept in silicon ampoules or lyophilized plasma. (heparin)

Biological fluid: serum from human adult blood, placental, cord blood, horse blood calf blood or aminiotic fluid, pleural fluid insect haemolymph serum etc.

Tissue extract: such as embryo, liver spleen, leukocytes, tumor bone marrow etc

Prepared artificially by adding several nutrients (org and inorg), vitamins, salts, serum proteins, carbohydrates, cofactors, O2 and CO2 gas phases etc.

Different type of synthetic media may be prepared for a variety of cells and tissue to cultured. It can be prepared for different functions.

Basically of two type ; Serum containing media and serum free media Serum free could be selective . Serum should not be used

Advantages: Contains a complete set of essential

growth factors, hormones etc.

It bind and neutralises the toxin.

It contains protease inhibitors.

It increase buffering capacity.

Provides trace elements and other nutrients.

Disadvantages:

Not chemically define.

May be the source of contamination

Increase difficulties and cost downstream processing.

Sterilizing of glassware, equipment and culture media:

Equipment required for Animal cell culture:

Act as aseptic working table for inoculation of animal cells etc.

Treatment with 70% ethanol.

Perform two functions Provides a sterile environment for cell

manipulation.

Protect operator from the potential risk from the culture.

Provides suitable environmental condition to growing cells.

Silicon gasket makes the chamber of incubator air tight.

HEPA (high efficiency particulate air) maintained the internal environment sterile.

Relative humidity remains high therefore, correct osmolarity maintained and medium is protected from desiccation.

Co2 designed in such a way that:

Maintained sterile condition inside the chamber,

Maintain an atmosphere of the high relative humidity

Provides an atmosphere with fixed level of CO2

Maintained a constant temperature at which they are fixed.

Centrifuge: Commonly cells centrifuged at 20c.

Inverted microscope: Optical system is at the bottom and

light source at the top.

Use to observe cell cultures in situ.

Culture Room:

Well control temperature, humidity, illumination, air circulation etc.

Observation (DATA collection) Cultures are maintained at regular

intervals in the culture room for the growth and development of cultured tissues.

Observation is also made in laminar air flow.

Should not be contaminated.

Tissue washed with BSS (balanced Salt Solution) to avoid contamination. (1000 unites of penicillin and 0.5mg of streptomycin or neomycin per ml)

Tissue to be cultured properly sterilized with 70% ethanol and removed surgically under aseptic conditions.

Tissue must be disaggregated either mechanically or using enzymes or chemical to obtained cell suspension.

Press via 100 um mesh (repeat)

Pass via 50 um mesh (repeat)

Pass via 20 um mesh

Remove debris and count the cells in haemocytometer

Dilute the medium up to 104 if required.

Forcing via Syringe or repeated pipetting can also be used.

Embryonic tissue a high number of undifferentiated cells with least extracellular matrix is found.

(more readily disaggregated than that of adult and new born)

In timorous (Cancerous Cells) the chances of cell death and cell recovery are more than the normal.

Collagenase Trypsin

use for embryonic and malignant tissue. It degrade the collagen of intracellular

matrix.

Biopsy

Medium containing antibiotic

Dissected into pieces in basal salt solution containing antibiotics.

washed with sterile H2O

keeping in medium containing collagenase for five days.

Pipetting

Centrifugation to remove the mediumWashing with enrich medium

Primary culture

Use of trypsin for disaggregation of tissue is called Trypsinization.

In crude form is commonly used for embryonic tissue

b.c many kinds of tissue cell can tolerate it and different types of tissue are significantly affected.

Serum or trypsin inhibitors can neutralise its residual enzyme activity only in serum free medium.

Tissue chopped and washed with dH2O

Keep in BSS. Place on ice soaked in cold trypsin 4-6 hours.

This allow penetration of enzymes in tissue.

Remove trypsin and tissue is incubated at 36.5C for 20-30 minutes.

10 ml of serum added and pipetting to disaggregate

Primary culture

Chopped pieces 100 ml of trypsin 36.5 c and stir for 3-4 hrs

The disassociated cells are collected at every 30 minutes.

Add fresh trypsin to allow minimum exposure of cells to warm enzyme.

Remove the trypsin n and centrifuge add medium

Primary culture.