Techniques of Animal Cell Culture
Unlike plant and microbial cells, animal cells are also cultured in vitro on specific culture media. Before studying the culturing methods, it is essential to know the characteristics of growth of animal cells in culture.
1. Some Characteristics of Animal Cell Growth in Culture
The animal cells can grow only to a limited generations even in the best nutritive media. This growth also depends on the sources of tissue isolated. The special features of different cell cultures are briefly discussed.
(i) Neuronal cells constitute the nervous system. In culture the neuronal cells cannot divide and grow.
(ii) The cells that form connective tissue (skin) is called fibroblast. Fibroblast can divide and grow in culture to some generations. After completing several generations they die. It means that all normal animal cells are mortal.
(iii) In culture the animal cells divide and grow. Consequently they fill the surface of the container in which they are growing. Thereafter, they stop further growth. This phenomenon is termed as contact inhibition i.e. inhibition of further cell growth after reaching the wall of container.
(iv) The environment of cell growth in culture differs from that of in vitro, for example (i) absence of cell-cell interaction and cell matrix interaction, (ii) lack of three-dimensional architectural appearance, and (iii) changed hormone and nutritional environment. The way of adherence to glass or plastic container in which they grow, cell proliferation and shape of cell results in alterations.
(v) In culture the cancer cells apparently differ from the normal cells. Due to uncontrolled growth and more rounded shape, they loose contact inhibition (Fig. 9.4). Therefore, they are piled on each other. These features are exploited by cancer specialists i.e. the cerologists to identify can-cer cells from the normal cells.
2. Primary Cell Cultures
The first step in establishing cells in culture is to dissociate organs (e.g. kidney or liver) or tissues into a single cell suspension. It is done by mechanical or enzymatic methods.
The cells are transferred into special glass or plastic containers containing culture medium. Under these conditions, maintenance of growth of such cells is called primary cell culture.
The cells are enumerated together by a proteinaceous material. Therefore, crude preparation of proteo-lytic enzymes (trypsin and collagenase) are commonly used to break the cementing mater and separate cells of a given tissue. The characteristics of the animal cells govern the characteristi of the cells in culture.
The growing cells are of two different types: (i) anchorage-dependent (adherei cells and (ii) anchorage-independent cells (suspension culture). Commonly the adherent cells can obtained from such organs that are fixed at a place (e.g. kidney, liver, etc.). The cells too are r mobile but remain fixed in connective tissues.
In contrast the suspension cells grow continuously in liquid medium and do not attach to the surface of the container. The source of cells is the governing factor for suspension non-adherent cells. All suspension cultures are raised by culturing the blood cells. You know that all blood cells are vascular in nature and get dissolved in plasma.
3. Secondary Cell Cultures and Cell Lines :
The primary cell culture cannot remain viable for a long time because the cell utilise all nutrients of the medium. Therefore, sub- culturing needs to be done on another fresh medium.
Hence, the primary culture is removed; adherent cells are dissociated enzymatic ally or by repeated pipe ting. Then the cells are diluted with fresh medium and passed into fresh culture flask. These sub-cultures are called secondary cell culture or cell lines.
Sub-culturing is done on fresh medium at certain intervals. It provides sufficient nutrient and space to growing cell lines. Characteristic features of such cell type govern that how quickly sub-culturing shall be done. However, the cells may exhaust the medium and die if they are not split frequently.
During sub-culturing the in vitro conditions produce mostly undefined selection pressure. Consequently, a certain cell type (e.g. fibroblast) is selected. After some generations the normal diploid cells die. Growth of fibroblast cells in culture.
Sometimes some cells of secondary cell cultures in vitro can be transformed spontaneously or chemically. Such transformed cells are immortal (i.e. they will not die) and hence give rise to continuous cell lines i.e. cancerous cell lines.
These cells proliferate indefinitely with a doubling time of about 10 to 25 hours. Such cultures consist of a mixture of cell types where a particular cell type may dominate over the others. For detail see preceding section.
4. Type of Cell Lines :
There are different types of cell lines produced from, different tissues or organs. Broadly they are grouped into two types: finite cell lines and continuous cell lines.
(a) Finite Cell Lines:
The cell lines which grow through a limited number of cell generations and have a limited life are called finite cell lines. The cells grow slowly and form monolayer. Their doubling time ranges from 24 to 96 hours. The characteristics of cell lines are anchorage dependence, contact inhibition and density limitation.
(b) Continuous Cell Lines:
Continuous cell lines are obtained either from transformed cell lines in vitro or cancerous cells. They divide rapidly. Their generation time is 12 to 14 hours. They have no or reduced density limitation. The transformed cells in vitro bear the following differences:
i. enhanced growth and proliferation due to rapid growth rate,
ii. existence of altered ploidy i.e. aneuploidy or heteroploidy due to altered chromosome number,
iii. different cell shape and organisation of microfilaments,
iv. ability to translocate,
v. different energy metabolism,
vi. no contact inhibition and no anchorage dependence, and
vii.different growth factor requirements and responses to regulate molecules.
5. Environmental Factors Required Culturing the Animal cells :
Nutritional requirements of the animal cells are specific. The cells are not suitable to survive independently.
Therefore, mechanical, physical and chemical (nutritional and hormonal) requirements of the cells need to be fulfilled for temperature for growth of mammalian proteins and nucleic acids in medium. Consequently, cell cultures including human is 37°C. Separation and purification of products become difficult.
Therefore, these cells of warm blooded in suspension culture cell death releases DN A which animals are grown in vitro at 37°C. In stimulates cell aggregation and hence more cell death.
DNA a compact loop bioreactor this acts as mediator of cell-cell adhesion. The cell cluster temperature can be maintained by can be disrupted in stirred bioreactor.
6. Cryopreservation of Animal cells :
Cryopreservation is done by using liquid nitrogen at a very low temperature (470°C to – 197°C) (Fig. 9.9). Due to cryopreservation ice crystals are formed which damage cells. Cell damage can be prevented by using glycerol or dimethyl sulfoxide (DMSO) with high concentration of serum. DMSO is soluble in lipids.
It enters inside the cell by diffusion via bilipid layer of cell membrane. The crystals are not formed in the presence of DMSO inside the cells. DMSO also causes protein permeability. Hence high concentration of serum maintains the proper concentration of proteins inside the cell. It also maintains the structural integrity of the animal cells.
When thawing needs to be done, a suitable temperature should be maintained in a waterbath. The vials containing frozen cells should be quickly thawed by emerging them inside the waterbath. This decreases the ice crystals and cell damage. The stored cells can directly be transferred onto a fresh culture medium and incubated properly.
7. Equipment Required for Animal Cell Culture :
The equipment required for animal cell culture are given below:
(a) Laminar Air Flow (LAF):
LAF hood acts as aseptic working table for inoculation of animal cells. Culture manipulation in aseptic conditions protects from contamination by any microorganisms such as bacteria and fungi. The contaminated bacterial/fungal cells grow more rapidly than the cultured animal cells.
Therefore, the growth of animal cells fails to occur in the presence of contaminants. The working area of LAF hood is first made sterile by using 70% ethanol. Manipulation of any cell is done by keeping the LAF in ON position.
The LAF hood performs two functions: (i) it provides a sterile environment for cell manipulation (i.e. protects tissue culture from operator), and (ii) protects the operator from the potential infection risk from the culture. There are different types of LAF hoods.
(b) CO2 Incubators:
The CO2 incubators provide the suitable environmental conditions to the growing animal cells. A silicon gasket is used on the inner door which makes the chamber of incubator airtight. It separates the internal environment situ. Because, the cells are found on bottom of the tissue culture vessel (e.g. Petri plates).
The culture medium remains above the growing cells in plates. If such plates are put over the stage of an ordinary microscope, the growing cells at bottom cannot be observed. Therefore, the inverted microscope is used for such purpose.
In inverted microscope the optical system is at the bottom and light source at the top. When a plate is observed in the inverted microscope, the cells of the culture growing at the bottom of the plate can easily be observed. If anyone wants to count cells using a counting chamber, a standard microscope with movable slide holder is required for this purpose.