Establishment of cell cultures
However, it is not necessary that the cells which are plated on medium will start immediate growth. On the basis of growth responses culture cells are divided into the three types: precursor or master cells or stem cells, undifferentiated but committed cells, and the mature differentiated cells. The precursor cells have the ability to proliferate but they do not differentiate until the proper conditions for induction are applied in the medium. This is done to facilitate some or all cells to mature to differentiated cells.
In addition, the types of cells growing in culture are determined by their respective sources from where these have been derived. For example, a high number of stem cells are found in cell lines derived from embryos because they are capable of frequent cell division as compared to adult cell culture. Also the culture of such adults comprises of stem cells which are capable of undergoing continuous removal in vivo such as intestinal epithelium, haemopoietic cells, epidermis, etc. In contrast, the cultures of such tissues consist of committed precursor cells that renew only in stress conditions such as muscles, glia and fibroblasts. The committed precurssor cells have limited life (Anon, 1988).
(i) Evolution of cell lines. This mixture of cells is in single cell suspension which may be used as a primary culture or starter culture. The primary culture (in the form of single-cell suspension) is subcultured by transferring into culture dishes/flasks containing special growth nutrients at optimal growth conditions. Consequently, some cells attach to the surface and proliferate to yield single cell line, inspite of being damaged in suspension. Therefore, while subculturing the suspension should be diluted with fresh medium at certain ratio and transferred into a flask (Anon, 1988).
The primary culture becomes a cell line only after its first subculture. The subculture is needed when the nutrients present in medium for cell growth diminish. These may be subcultured several times on the fresh medium and propagated accordingly. By doing so a continuous growth of cells is maintained. This results in multiple copies of a single type of cell with a negligible amount of non-proliferating cells. During the course of repeated subculture and selection the cell line gets evolved and properly established consisting of rapidly proliferating cells. Thus the unaltered form of cell line (only for a limited number of generations) is called continuous cell line which propagates in logarithmic ways (Fig. 6.5). Any change in continuous cell line may discontinue the increase in cell number. This may be brought about by chemicals, spontaneous mutation or viruses (e.g. Epstein Barr virus). The phenomenon of alteration in continuous cell line is called 'in vitro transformation'.
Table 6.6. Properties of finite and continuous cell lines and cell strains.
Cell lines | Source | Morphology | Age | Tissue | Characteristics |
1. Finite Cell Line | |||||
IMR90 | Human lung | Fibroblasts | Embryonic | Normal | Infected by human virus |
MRC5 | Human lung | Fibroblasts | Embryonic | Normal | Infected by human virus |
MRC9 | Human lung | Fibroblasts | Embryonic | Normal | Infected by human virus |
WI38 | Human lung | Fibroblasts | Embryonic | Normal | Infected by human virus |
2. Continuous Cell Line | |||||
EB | Human | Lymphocytic | Juvenile | NP | EB virus, +ve |
HeLa | Human | Epithelial | Adult | NP | G6PD Type A |
LS | Mouse | Fibroblastic | Adult | NP | Grow in L929 suspension |
P388D | Mouse | Lymphocytic | Adult | NP | Grow in suspension |
S180 | Mouse | Fibroblastic | Adult | NP | Cancer chemotherapy |
3T3A | Mouse | Fibroblastic | Embryonic | Normal | Readily transformed |
(ii) Factors affecting subculture in vitro.
There are several factors that influence differentiation and proliferation of cells when subcultured in vitro as below:
- The mammalian cells need attachment to a suitable surface. The maximum cell numbers are limited by the surface area available. Therefore, mammalian cells should be grown on microcarriers such as beads of anion exchange resin, etc.
- Serum is also added in medium. It allows better cell growth in agitated and/or aerated cultures. Serum is a highly complex mixture of several kinds of molecules that provides both growth-promoting and growth-inhibitory factors to the cells. Some i constituents which are very useful are hormones, albumin binding proteins, transport protein, growth factors, attachment factors and micronutrients. Low serum or serum-free cultures are more susceptible to fluid-mechanical damage. It protects the cell from physical damage caused by agitation in bioreactor because bubbles cause damage but in the absence of gas, cells are also damaged, therefore, slow agitation rate of medium in bioreactor is required.
- Various types of additives are also used to protect freely suspended animal cells in culture from agitation or aeration damage. These additives are: cellulose and starch derivatives, protein mixtures pluronic polyals, polyvinyl-pyrrolidones, dextrans polyvinyl glycol (PEG), polyvinyl alcohols (PVA), methylcelluloses (MCs), etc. Effect of these additives on physiological and product expression of cells, cell aggegation should be arrested under both static and bioreactor growth conditions before their use.
- MCs have been found as reliable protectant, therefore, MCs and other cellulose derivatives have been influenced as media additives in the culture of different types of cells. Tilly et al. (1982) got success in growing certain mammalian cells to a level to about 5 x 106 cell /ml of cell density by employing conventional conditions of medium, serum and oxygen, and suitable bead carriers.
- The optimum pH of medium should be maintained between 7.0 and 7.6. This is controlled with a dual system provided with acid and base. In animal culture the COD/bicarbonate buffering system is used in cullture medium.
- The animal cells are very sensitive to temperature, therefore, by using a thermostate temperature of bioreactor medium should be maintained at 37°C.