ANIMAL CELL CULTURE
Salient Features of Animal cell culture
a) Animal cells can grow in simple glass or plastic containers in nutritive media but they grow only to limited generations.
b) Animal cells exhibit contact inhibition.
c) There is a difference in the in vitro and in vivo growth pattern of cells.
d) The maintenance of growth of cells under laboratory conditions in suitable culture medium is known as PRIMARY CELL CULTURE.
e) Cells are dissociated form tissues by mechanical means and by enzymatic digestion using proteolytic enzymes.
f) Cells can grow as adherent cells (anchorage dependent) or as suspension cultures (anchorage independent).
g) The primary culture is subcultured in fresh media to establish SECONDARY CULTURES.
h) The various types of cell lines are categorized into two types as Finite cell line and Continuous cell line.
i) Finite cell lines are those cell lines which have a limited life span and grow through a limited number of cell generations.
j) Cell lines transformed under in vitro conditions give rise to continuous cell lines.
k) The physical environment includes the optimum pH, temperature, osmolality and gaseous environment, supporting surface and protecting the cells from chemical, physical, and mechanical stresses.
l) Nutrient media is the mixture of inorganic salts and other nutrients capable of sustaining cell survival in vitro.
m) Serum is essential for animal cell culture and contains growth factors which promote cell proliferation. It is obtained as exuded liquid from blood undergoing coagulation and filtered using Millipore filters.
n) Cryo preservation is storing of cells at very low temperature (-180oC to -196oC) using liquid nitrogen.
o) DMSO is a cryopreservative molecule which prevents damage to cells.
p) In order to maintain the aseptic conditions in a cell culture, a LAF hood is used.
q) Based on the nature of cells and organism the tissue culture hoods are grouped into three types: Class I, Class II, and Class III.
r) CO2 incubators are used and designed to mimic the environmental conditions of the living cells.
s) An inverted microscope is used for visualizing cell cultures in situ.
t) For most animal cell cultures low speed centrifuges are needed.
Animal cells can grow in simple glass and plastic containers in a nutritive medium. As pointed earlier there are certain features typical of animal cell cultures.
A) Mortality- Animal cells, depending on the tissue they have been isolated from can grow to only limited generations in spite of growing them in the best nutritive media.
B) Contact Inhibition- The animal cells in culture divide and fill the surface of the container they are growing in and then stop growing. This phenomenon is similar to what happens in the normal body where the body grows to a certain size after which it stops. This is due to Contact Inhibition. In cultures when the cells come in close contact with each other, they stop growing.
C) In vivo and in vitro environment difference- The cells in culture behave differently from the cells in vivo environment. There is an absence of cell-cell interaction, cell matrix interaction, lack of three dimensional architecture and alteration in hormonal and nutritional environment. Due to these differences, the cells in culture adhere to the glass or plastic container in a different manner, the shape of the cells change and the way cells proliferate or grow also changes.
In fact these parameters help us to distinguish the cancer cells in culture from the normal cells because the cancer cells in culture, change shape (more rounded), loose contact inhibition, pile on each other due to overgrowth and uncontrolled growth.
REQUIREMENTS FOR ANIMAL CELL CULTURE
Among the essential requirements for animal cell culture are special incubators to maintain the levels of oxygen, carbon dioxide, temperature, humidity as present in the animal’s body. The synthetic media with vitamins, amino acids and fetal calf serum. Following parameters are essential for successful animal cell culture:
a) Temperature- In most of the mammalian cell cultures, the temperature is maintained at 370C in the incubators as the body temperature of Homo sapiens is 370C.
b) Culture media- The culture media is prepared in such a way that it provides-
1) The optimum conditions of factors like pH, osmotic pressure, etc.
2) It should contain chemical constituents which the cells or tissues are incapable of synthesizing. Generally the media is the mixture of inorganic salts and other nutrients capable of sustaining cells in culture such as amino acids, fatty acids, sugars, ions, trace elements, vitamins, cofactors, and ions. Glucose is added as energy source-it’s concentration varying depending on the requirement. Phenol Red is added as a pH indicator of the medium.
3) Natural Media - The Natural Media used to promote cell growth fall in three categories.
i) Coagulant, such as plasma clots.
ii) Biological fluids such as serum. Serum is one of the very important components of animal cell culture which is the source of various amino acids, hormones, lipids, vitamins, polyamines, and salts containing ions such as calcium, ferrous, ferric, potassium etc. It also contains the growth factors which promotes cell proliferation, cell attachment and adhesion factors.
iii) Tissue extracts for example Embryo extracts- Other biological fluids used as natural media include amniotic fluids, ascetic and pleural fluids, aqueous humour (from eye), serum ultra filtrate insect haemolymph etc.
4) pH- Most media maintain the pH between 7 and 7.4. The optimum pH is essential to maintain the proper ion balance, optimal functioning of cellular enzymes and binding of hormones and growth factors to cell surface receptors in the cell cultures. The regulation of pH is done using a variety of buffering systems. Most media use a bicarbonate-CO2 system as its major component.
5) Osmolality- A change in osmolality can affect cell growth and function. Salt, Glucose and Amino acids in the growth media determine the osmolality of the medium. All commercial media are formulated in such a way that their final osmolality is around 300 mOsm.
