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The foundation of animal cell and tissue culture was laid by Jolly (1903) when he showed that animal cells could not only survive but could divide in culture medium.
The actual beginning of animal cell culture and tissue culture was made by Harrison (1907) and later by Carrel (1912) who used frog’s tissue in tissue culture.
They successfully showed that animal cells can be grown indefinitely in culture medium just like microorganisms. Later tissues from warm blooded animals like chick and mammals were used as material for tissue culture purpose.
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Prior to the year 1950, parts of animal tissues were used as material for tissue culture, but later on dispersed cells were utilized for the culture purpose. With the advancement of techniques, cells and tissues from mammals are mainly used for tissue culture purpose.
Laboratory Facilities for Tissue Culture:
The laboratory facilities for animal tissue culture consist of (i) Sterile area (ii) tissue culture equipment’s.
(i) Sterile area:
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For processing the animal tissues for culture purpose a sterile or aseptic area is needed. This working place must be free from any kind of contamination. Two types of sterile work areas are generally recommended.
They are:
(a) Laminar flow cabinet
(b) Bio-safety cabinet.
Laminar flow cabinet (fig. 7.8):
It is a specially designed chamber inside which animal tissue for culture purpose is being handled in an aseptic condition. It is completely open in front to allow the researcher to work comfortably and handle the equipment’s present inside the laminar flow cabinet. A motor blows air into the laminar flow cabinet through a coarse filter, where large dust particles are separated.
This air then passes through a 0.3 μm HF.PA (High Efficiency Particulate Air). This keeps all contaminants away from the work surface. Such arrangement does not give protection to researcher against pathogenic organisms. Hence, laminar flow cabinet cannot be used in any cell or tissue culture which may contain a human pathogen (disease causing organism).
Bio-safety cabinet:
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Bio-safety cabinet provides a sterile environment for tissue culture in addition to making provision for the safety of researcher against human pathogens.
(ii) Tissue culture equipments:
The equipments required for the animal tissue culture are the followings:
(i) Autoclave,
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(ii) Centrifuge,
(iii) Incubator (capable of regulating the percentage of CO2),
(iv) Water bath,
(v) Refrigerator,
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(vi) Freezer (for–20°C),
(vii) pH meter,
(viii) Chemical balance,
(ix) Stirrer,
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(x) Bunsen burner/spirit lamp,
(xi) Culture vessels with screw cap
(xii) Pasteur pipettes,
(xiii) Inverted microscope,
(xiv) Liquid Nitrogen freezer,
(xv) Liquid Nitrogen storage flask,
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(xvi) Bench centrifuge
(xvii) Soaking bath,
(xviii) Deep washing sink,
(xix) Pipette cylinder (s),
(xx) Pipette washer,
(xxi) Water purifier.
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Culture Media for Cells and Tissues:
Culture medium is the most important factor for culturing cells and tissues. It provides optimum conditions of factors like pH, osmotic pressure etc. It also provides all nutritional requirements needed by growing cells in the culture.
The media used in animal cell and tissue culture are of two types:
(i) Natural and
(ii) Artificial medium.
The natural media include fluids of biological origin, such as plasma clots and serum. Plasma clot is prepared by treating the blood of an animal with an anticoagulant such as heparin. Serum is the clear fluid part of the blood, formed after blood coagulation when fibrin separates from the plasma. It is considered as an ideal growth medium for animal cells as if is formed of hundreds of proteins and hormones.
The artificial culture media primarily consist of balanced salt solution (BSS) which provide essential inorganic ions, correct osmolarity, required pH (7.0-7.3), energy (= glucose) and a pH indicator (such as phenol red). However, BSS lacks essential amino acids and vitamins. Earle’s balanced salt solution and Hanks’ balanced salt solution come under this category. These media cannot support growth of cells and tissues but can keep them alive for a period of 12 hours.
For longer survival of the culture, serum may be used or the balanced salt solution may be supplemented with amino acids, oxygen, vitamins and serum protein. One such medium was developed and modified by Eagle (1955), which is described as minimum essential medium (MFM). Other more complex, synthetic media include (i) CMRL 1066 (ii) RPMI 1640, (iii) F12 etc. The use of serum in culture media has several disadvantages, which led to the development of many serum free media.
Culture Procedures:
The procedure for tissue culture and cell culture involve preparation and sterilization of glassware, equipment, reagents and media. As the tissue culture is being carried out in highly aseptic condition, all the usable, media and solutions are required to be sterilized thoroughly. Some of the methods of sterilization include swabbing, capping, flaming, dry at, wet heat, radiation, filter sterilization etc.
The work bench, reagent bottles and growth media are swabbed (= cleaned) with 70% ethanol before and after operation. Culture and reagent bottles are capped with deep screw caps. The reagents bottles are capped immediately alter using the reagent. The necks of all bottles and the screw caps are flamed before and after opening and closing. This is a common practice to prevent infection and maintaining aseptic condition.
Glassware, stainless steel instruments, plastic containers, distilled water, phosphate buffered saline and growth medium are sterilized in autoclave at 121 °C and under pressure 15 lb/square inch for 20 minutes. This process of sterilization by means of an autoclave is called as wet heat. It is followed by dry heat. During dry heating, glass ware and dissecting instruments are placed within a hot air oven at 160°C for one hour. The culture room or laminar How cabinet is fitted with a UV lamp.
The lamp is switched on half an hour before the operation of the work for the purpose of sterilization. Some heat labile constituents of the growth medium (such as polypeptides, hormones etc.) are sterilized not by heating method but by another method called filter sterilization. In this method liquid substances are passed through a micro filter (0.2 μm) which removes everything except mycoplasma (50%) and bacterial endotoxins.
Preparation of Starting Material:
The starting material is an isolated tissue or a body part. The isolated tissue or the body part consists of numerous cells cemented together by proteinous substances. To start the cell culture, the starting tissue is to be dissociated into cells by two methods: (i) mechanical method, (ii) enzymatic method.
In mechanical method the isolated tissue is cut into small pieces in BSS (Balanced Salt Solution) and these are then cultured in suitable vessels. In enzymatic method, the enzyme trypsin is generally used to dissociate cells present in the tissue by digesting the proteinous cementing material.
Following dissociation of starting tissue into cells by the mechanical or enzymatic method, the dissociated cells are placed in flat bottomed culture vessel (either made of glass or high grade plastic) containing culture medium (Fig. 7.9). The culture vessel should have optimum number of cells and it should be incubated at 37°C. The inner surface of the culture vessel should have negatively charged (SO-3) group.
The transfer of dissociated cells into the culture medium is called as inoculation. A culture established directly from differentiated tissue (isolated tissue or body part) is known as primary culture. After sometime the bottom of the culture vessel will be covered by a continuous layer of cells, often one cell thick. This layer of cells is known as monolayer.
The cells from the primary culture can be detached from the culture vessel by trypsin or EDTA (Ethylene Diamine Tetraacetic Acid) treatment and transferred to fresh media. The culture so obtained is called cell lines. The process of transferring cultured cells into fresh culture vessels is termed as sub culturing.
The cells divide at a constant rate over successive transfers. Such cells comprise a cell strain. Cell strains do not have an infinite life. They divide 50-100 times before dying. For long term culture the cell lines are preserved in frozen state in liquid nitrogen in presence of cryo preservative agents and foetal calf serum.
Small-scale cultures are generally carried out either in plastic petriplates or plastic T-flasks. Large-scale cultures involving mammalian cells are carried out in bioreactors or fermenters.
Applications of Tissue Culture:
1. Animal cell culture was primarily aimed to study infection of animal viruses.
2. Later on it was used to produce a wide range of biological products of commercial importance such as antibodies, enzymes, hormones, immuno-regulators.
3. Recently tissue culture technique has been used in the manufacture of viral vaccines, tissue plasminogen activator, interferon-a, monoclonal antibodies and tumor specific antigens.
4. Production of Foot and Mouth disease vaccines (FMD vaccines) is the most important example of the use of large scale cell culture. There are several other vaccines including polio vaccine, bovine leukaemia virus (BLV) vaccines, rabies vaccines etc. which are produced on commercial basis using cell cultures
5. Impact of new drugs can be evaluated using cell and tissue culture techniques.
