All steps in the animal cell culture technique must be carried out in highly aseptic conditions. It is, therefore, required to sterilize all the usable, media and solutions by following a standard protocol. Several methods of sterilization are available, as discussed below. None of the methods can be used for all items used in the technique.
Swabbing is a common practice of maintaining an aseptic condition. The work bench is swabbed with a 70% ethanol soaked sterilized cotton. The reagent bottles containing reagents and growth media are swabbed with 70% ethanol before and after the operation.
Culture and reagent bottles are capped with deep screw caps. The reagent bottles are capped immediately after using the reagent.
Flaming is another common practice of enforcing an aseptic condition. The necks of all bottles and the screw caps are flamed before and after opening and closing. All stainless steel dissecting instruments are flamed properly before use.
Glass ware and dissecting instruments are to be placed in a hot air oven at 160° C for l hr following sterilization by wet heat in an autoclave.
Glassware, stainless steel instruments, autoclavable plastic wares, distilled water, phosphate buffered saline and growth medium are sterilized in an autoclave at 121° C and 15 lb / square inch for 20 min. The steam generated sterilizes the contents under pressure. A pressure cooker may substitute for an autoclave.
The cell culture room is fitted with a UV lamp. The door of the room is closed and the UV lamp is switched on at least half an hour before the operation. The laminar air flow cabinet is also fitted with a UV lamp. The lamp is switched on half an hour before the operation of the work. The work bench is sterilized by the UV rays.
Heat labile constituents of the growth medium (polypeptides, hormones, inteleukins and fetal calf serum) are sterilized by this method, since these are unstable at the heat generated in the autoclave and hot air oven. These liquids are filtered through a micro-filter (0.2 M-m), which removes everything except 50 % Mycoplasmas and bacterial endotoxins, if any.
A tissue, the starting material
A tissue is the starting material for establishing an animal culture. A tissue is made up of numerous cells cemented together by an inter-cellular matrix, chiefly consisting of proteins.
The dissociation of the tissue is done by two methods, such as mechanical and enzymatic, one following the other.
Dissociation of the tissue into cells
The isolated tissue is chopped into small pieces in BSS and the chopped tissue is macerated by a disposable van. The suspension of cell aggregates in the BSS is passed through a mesh.
The residue is mixed with fresh BSS and the suspension is centrifuged. The supernatant is decanted out and the pellet is re-suspended in BSS and either refrigerated for future use or treated with a proteolytic enzyme, trypsin. Trypsin digests the inter-cellular matrix and frees the individual cells.
The trypsin- treated cells are again suspended in fresh BSS and centrifuged. The supernatant is discarded and the pellet is resuspended in the BSS. The pellet contains isolated cells, ready for inoculation.
Following this, the cell suspension having free cells is diluted to a cell density of 106 cells/ml and placed in a flat bottomed cured vessel made of glass or high grade plastic containing Dulbeco’s Modified Eagle’s media (DMEM). The cell density is computed by a haemocytometer or a Coulter counter.
The inoculated culture vessel is incubated at 37° C. The inner surface of the culture vessel has negatively charged (SO”3) groups. Normal animal cells (skin, kidney and liver) secrete collagen and other matrix components, which bind to the culture surface.
These cells are known as anchorage-dependent cells. Blood cells, bone marrow cells and spleen cells adhere poorly to the surface and grow in suspension. A culture of these cells is known as a suspension culture. These cells are cultured in a specially designed spinner bottle
Culture vessels for animal cell culture, (a) T-flask for anchorage-dependent cells and (b) spinner bottle for suspension culture.
A culture established directly from the differentiated tissue is known as a primary culture. After some time, the bottom of the vessel will be covered by a continuous layer of cells, often one cell thick. This layer of cells is known as a monolayer.
Since, primary cultures are prepared directly from tissues; it consists of a variety of differentiated cells, such as fibroblasts, macrophages, epithelial cells and lymphocytes. The cells of the primary culture can be detached from the culture vessel by tyrosine 01 EDTA (ethylene diamine tetra acetic acid) treatment and fed to fresh culture medium in a high density. Cells from a primary culture may be transferred serially to fresh media a number of times.
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. Some cells of the primary culture become transformed, change morphology and divide and re-divide relatively at a faster rate. Progeny derived from these cells have an infinite life, unlike those of the cell strain. These cells constitute a cell line. Such cells primarily divide and form a monolayer, but when the density increases, they grow in clumps.
The cells are irregularly oriented with respect to each other. These cell lines are referred to as transformed and neoplastic. However, in human, no cell line is shown to have arisen from normal primary cell cultures. Several cell lines have been established from tumor cells and these are maintained in the laboratories for research and investigation.
The fact of immortality of these cells may be due to a better adaptation to the culture environment. This again may be related to the acquisition of extra chromosomes, which is known as aneuploidy.
A long-term culture of cells or cell lines may result in the accumulation of harmful mutations and contamination with pathogenic organisms. To avoid all these problems, a laboratory must maintain a stable and continuous supply of the cells.
This is achieved by preserving the cells in a frozen state in liquid nitrogen in the presence of cryo-preservative agents and fetal calf serum.
The cells are harvested from the culture medium and centrifuged gently in BSS. The pellet is re-suspended in a medium containing 10% of the cryo-preservative, dimethyl sulfoxide (DMSO), 10% glycerol and 90% heat inactivated fetal calf serum. Prior to use, the vials containing the frozen cells are thawed gently in a water bath at 37° C.
The content is transferred to a centrifuge tube and diluted 1: 10 with the medium, added drop- wise over a period of 5 min. It is then centrifuged for 5 min at 500g. The supernatant is discarded and the pellet is cultured with fresh medium.
Primary cell cultures are grown either in plastic Petri plates or plastic T-flasks. 15-20 ml of the medium is taken in the T-flask and 106cells are seeded into it and incubated at 37° C. After 2-3 days, the bottom of the flask will be covered by a monolayer of cells.
These cells can be detached by trypsin or EDTA treatment. The isolated cells are fed to a fresh medium containing X-irradiated cells. These irradiated cells have lost the power of division. However, these are still metabolically active and produce important factors that will be used by the cultured cells.
The layer of these irradiated cells is known as the feeder layer. The transformed cells can be grown more efficiently in suspension culture in spinner bottle.
Large-scale culture of mammalian cells is carried out in suspension culture. Standard bioreactors or fermenters have been designed for this purpose. These fermenters are designed to run aseptically for a long period of time. The growth of anchorage- dependent cells is difficult in a fermenter, since a fermenter does not provide the required more surface area to volume ratio.
The roller bottle method is an alternate method for the anchorage-dependent cells. Roller bottles (500 ml-50 1), made of glass are filled with the required amount of the growth media. Inoculant cells are put into it in the required number. The bottles are plugged and put on their sides on a slowly revolving roller. As the roller revolves, the content of each roller bottle is mixed and uniformly distributed in the liquid growth medium. At the same time, the content is continuously washed by the nutrients and exposed to oxygen.
Applications of animal cell culture
Primarily the technique of animal cell culture was applied to study the infection cycle of animal viruses and their impact on animal cells. However, the applications are far beyond this. This technique is used to produce a wide range of biological products of commercial value.
These include immuno-regulators (cytokines), antibodies, polypeptide growth factors, enzymes and hormones. This is already used in the manufacture of viral vaccines, tissue plasminogen activator, interferon-a, monoclonal antibodies and tumor specific antigens.
Isolated cells in culture would be the targets for the delivery of transgenes and generation of transgenic animals. Another potential application is their use in evaluating the impact of new drugs. This would take much less time as compared to animal model systems.