Establishment of cell cultures

There are many type of animal cells that can grow in vitro such as tumour cells, pigmented melanoma cells, neuroblastoma cells, steroid producing adrenal cells, growth hormone prolactin secreting cells, teratoma cells capable of differentiating in artificial conditions pigmented or cartilage cells, etc. On die basis of purpose of experiment, a continuous (immortal) cell line can be developed from cultured tissues. Healthy animal tissues capable of dividing are cultured on artificial nutrient media that proliferate and differentiate into heterogeneous mixture of different types of cells (Fig. 6.4).

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. These are totipotent (also called multipotent or piuripotent) cells which have the ability to differentiate into different types of cells. The entire blood and immune systems are produced by the totipotent stem cells. In contrast, the piuripotent cells differ from totipotent cells in being less general and still capable of differentiating into many kinds of cells. Now the cell cultures are at a stage of equilibrium of three type of cells viz., multipotent stem cells, undifferentiated but committed precurssor cells and mature differentiated cells. This equilibrium may be shifted by manipulating the growth conditions. For example, in the presence of low serum, suitable hormones, cell matrix interaction and high cell density, cell differentiation is promoted, whereas in the presence of high serum, suitable growth factors and low cell density, cell proliferation is promoted (Anon, 1988).

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'.

A cell line consists of several similar or dissimilar cell lineages. A defined cell lineage having specific properties is called 'cell strain¹. The cell strain is identified when cells of that culture are in bulk. On the basis of life span of culture, the cell line or cell strain may be continuous of finite. The other types of cell line may be immortal i.e. which will not die. The life of finite cell line is limited upto 20-80 generations, thereafter, they die. Properties of some continuous and finite cell lines are given in Table 6.6. It should be noted that the cell lines are represented in abbreviated form either derived from the sources of cells, name of institute or association with viruses (for example EB, Epstein Barr; WI, Wistar Institute). They are numbered if more than one cell line are derived from the same cell line (e.g. EB1, EB2). Furthermore, a cell line is given the number of population doubling e.g. EB1/1, etc.

Table 6.6. Properties of finite and continuous cell lines and cell strains. NP, Neoplastic tissue.
(ii) Factors affecting subculture in vitro.
There are several factors that influence differentiation and proliferation of cells when subcultured in vitro as below:

1. 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.
2. 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.
3. 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.
4. 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.
5. 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.
6. The animal cells are very sensitive to temperature, therefore, by using a thermostate temperature of bioreactor medium should be maintained at 37°C.