Culture techniq and type of animal cell culture

Culture techniques and Types of animal cell culture

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Primary culture and cell lineA primary culture refers to the starting

culture of cells, tissues or organs, taken directly from an organism.

It is the initial culture before the first subculture.

Cell line is used for the propagation of culture after the first subculture

Primary cultures are usually prepared from large tissue masses.

Therefore it may contain a variety of differentiated cells eg. Fibroblasts, lympocytes, macrophages, epithelial cells

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Characteristics of primary culture1. Embryonic tissues rather than adult tissues are

preferred for primary cultures. As these cells can be easily disaggregated and yield more viable cells, besides rapidly proliferating in vitro

2. The quantity of the cells used in primary culture should be higher since survival rate is lower

3. The tissues should be processed with minimum damage to cells for use in primary culture.

4. Proper selection of media is suggested5. It is necessary to remove the enzymes used for

disaggregation of cells by centrifugation

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Techniques for primary culture

1. Mechanical disaggregation2. Enzymatic disaggregation3. Primary explant technique

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Mechanical disaggregationFor soft tissues such as spleen, brain,

embryonic liver, soft tumor this technique is used.

It involve careful chopping or slicing of tissue into pieces and collection of spill out cells. The cells are collected by any one of the following methods-

Pressing the tissue pieces through a series of sieves with gradual reduction in the mesh size

Forcing the tissue fragments through a syringe and needle

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Enzymatic disaggregationIt is the mostly used technique when high

recovery of cells is required from a tissue. Enzymatic disaggregation can be carried out

by using trypsin, collagenase or some other enzymes.

The term trypsinization is commonly used for disaggregation by trypsin. Two techniques are there

1. Warm trypsinization 2. Cold trypsinization

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Collagenase disaggregation has been successfully used for human brain, lung and several other epithelial tissues, besides various human tumors and other animal tissues.

Addition of another enzyme hyaluronidase , trypsin promotes disaggregation

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Primary explant techniqueThe tissue in basal salt solution is finely

chopped and washed by settlings. The basal salt solution is then removed.

The tissue pieces are spread evenly over the growth surface. After addition of appropriate medium, incubation is carried out for 3-5 days.

Then the medium is changed at weekly intervals until a substantial out growth of cells is observed.

Now the explants are removed and transferred to a fresh culture vessel

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Cell linesThe term cell line refers to the propagation of

culture after the first subculture.In other words, once the primary culture is

subcultured it becomes a cell line. A given cell line contains several cell lineages of either similar or distinct phenotypes.

It is possible to select a particular cell lineage by cloning or physical cell separation or some other selection method.

Such a cell line derived by selection or cloning is referred to as cell strain.

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Difference between finite and continuous cell linePROPERTY FINITE CELL LINE CONTINUOUS

CELL LINEGROWTH RATE SLOW FASTMode of growth monolayer Suspension or

monolayeryield low Hightransformation normal Immortal,

tumorigenicAnchorage dependence

yes No

Contact inhibition yes no

Cloning efficiency Low HighSerum requirement High HighMarkers Tissue specific Chromosomal,

antigenic or enzymatic

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Organ cultureIn vitro culture and growth of organs or parts

thereof in which their various tissue components eg. Parenchyma and stroma are preserved both in term of their structure and function, so that culture organ resembles closely with the organ in vivo then called organ culture.

New growth is in the form of differentiated structures eg. Glandular structures in case of glands, bronchioles in case of lungs

Cultured organ should retain their physiological features

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Techniques of organ cultureFollowing are some techniques of organ

culture:1. Plasma clot or watch glass method2. Raft method3. Combination of 1 and 24. Agar gel5. Grid method6. Cyclic exposure to medium and gas phase

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Plasma clot or watch glass methodExplant is placed and cultured on suitably prepared

plasma clot kept in watch glass.Plasma clot is prepared by mixing 3 drops of chicken

plasma and 1 drop of chick embryo extract (50 %) One or two such watch glasses are kept in Petri dishes

lined with cotton wool or moist filter paper to minimize evaporation of clot. May or may not be closed with lid and sealed with paraffin wax.

Petri dish is then incubated at 37.5 0C. Fresh clots have been provided every 2 to 3 days for avian tissues and every 3 to 4 days for mammalian tissues.

Maximow single slide technique: it is the modification of the above mentioned method

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Advantages:1. In expensive2. Permits light microscope hence suitable to

study hair growth, fetal mouse skin differentiation

Disadvantages:1. Clot liquefies in vicinity of explants so explants

can become immersed in the medium2. Short duration of culture (Less than 4 weeks)3. Biochemical analysis is not possible due to

complexity of medium

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ApplicationsTo study morphogenesis in embryonic organ

rudimentsTo study action of hormones, vitamins,

carcinogens on adult mammalian tissues.

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Raft methodIn this method explants are placed on the raft

of glass/ lens/paper/ rayon acetate, which is then floated on serum in watch glass.

Four or more explants are placed on single raft. Floatability of lens paper/rayon acetate is enhanced by it treatment with silicone

COMBINATION METHOD: In this method explants are first placed on

the raft of glass or suitable material then place on plasma clot. This prevents sinking of explant into liquefied plasma

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Agar gel methodMedium is gelled with 1 % agar.Gels are then kept in embryological watch

glass and explant is then transferred on surface of agar, sealed with paraffin wax.

Sub cultured into fresh agar gels every 5 to 7 days. Explants can be examined with stereoscopic microscope .

The technique is used to study many developmental aspects of normal organs as well as tumors

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Grid methodGrid utilizes 25 mm X25 mm pieces of suitable wire

mesh or perforated stainless steel sheet, whose edges are about to form 4 legs of about 4 mm height.

Skeletal tissues are kept directly on grid while soften tissues are firstly kept on raft and then on grid.

Then grid is kept in culture chamber filled with fluid medium up to the grid.

Chamber is flushed with O2/CO2 to maintain required O2 concentration.

It is used to study growth and differentiation of adult and embryonic tissues.

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Cyclic exposure to medium and gas phaseUseful for long term (4-5 months) culture of

human adult tissues. 2 to 8 explants per dish are attached at bottom of plastic culture dish and covered with fluid medium.

Dishes are enclosed in chamber containing suitable gas mixture, mounted on rocker platform for cyclic exposures of medium and gas.

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Advantages:Explant remain comparable, so suitable to study

physiological studies May replace whole animals in experimentation, as

results from them are easy to interpret.APPLICATIONS:To study pattern of growth, differentiation,

development of organ rudimentsTo study action of drugs and carcinogens on organsDevelopment of tissues called tissue engineering

for implantation in patients eg. Artificial cartilage, artificial skin.

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