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.In culture the cancer cells apparently differ from the normal cells. Due to uncontrolled growth and more rounded shape, they loose contact inhibition and pile over each other.
c) There is a difference in the in vitro and in vivo growth pattern of cells.
(i) there is an absence of cell-cell interaction and cell matrix interaction,
(ii) there is a lack of three-dimensional architectural appearance, and
(iii) changed hormonal and nutritional environment. They way of adherence to glass or plastic container in which they grow, cell proliferation and shape of cell results in alterations.
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. Finite cell lines are those cell lines which have a limited life span and grow through a limited number of cell generations. The cells normally divide 20 to 100 times (i.e. is 20-100 population doublings) before extinction. Cell lines transformed under in vitro conditions give rise to continuous cell lines. The continuous cell lines are transformed, immortal and tumorigenic.
i) The physical environment includes the optimum pH, temperature, osmolality and gaseous environment, supporting surface and protecting the cells from chemical, physical, and mechanical stresses.
j) Nutrient media is the mixture of inorganic salts and other nutrients capable of sustaining cell survival in vitro
k) 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.
l) Cryo preservation is storing of cells at very low temperature (-1800C to -196 0C) using liquid nitrogen. DMSO is a cryopreservative molecule which prevents damage to cells.
m) In order to maintain the aseptic conditions in a cell culture, a LAF hood is used. Based on the nature of cells and organism the tissue culture hoods are grouped into three types: Class I, Class II, and Class III.
n) CO2 incubators are used and designed to mimic the environmental conditions of the living cells.
o) An inverted microscope is used for visualizing cell cultures in situ
p) For most animal cell cultures low speed centrifuges are needed.
q) Neuronal cells constitute the nervous system. In culture the neuronal cells cannot divide and grow.
r) The cells that form connective tissue (skin) is called fibroblast. The fibroblast can divide and grow in culture to some generations after which they die. All normal animal cells are mortal.
s) Organ culture- The culture of native tissue that retains most of the in vivo histological features is regarded as organ culture.
t) Histotypic culture- The culturing of the cells for their reaggregation to form a tissue-like structure represents histotypic culture.
u) Organotypic culture- This culture technique involves the recombination of different cell types to form a more defined tissue or an organ.
There are certain terms that are associated with the cell lines.
These are as follows:
(i) Split ratio- The divisor of the dilution ratio of a cell culture at subculture.
(ii) Passage number- It is the number of times that the culture has been cultured,
(iii) Generation number- It refers to the number of doublings that a cell population has undergone.
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.There are two types of media used for culture of animal cells and tissues- the natural media and the synthesized media.
3) Natural Media - The natural media are the natural sources of nutrient sufficient for growth and proliferation of animal cells and tissues. The Natural Media used to promote cell growth fall in three categories.
i) Coagulant, such as plasma clots. It is now commercially available in the form of liquid plasma kept in silicon ampoules or lyophilized plasma. Plasma can also be prepared in the laboratory taking out blood from male fowl and adding heparin to prevent blood coagulation.
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. Serum is obtained from human adult blood, placental, cord blood, horse blood, calf blood. The other forms of biological fluids used are coconut water, amniotic fluid, pleural fluid, insect haemolymph serum, culture filtrate, aqueous humour, from eyes etc.
iii) Tissue extracts for example Embryo extracts- Extracts from tissues such as embryo, liver, spleen, leukocytes, tumour, bone marrow etc are also used for culture of animal cells.
Syntheic media are prepared artificially by adding several organic and inorganic nutrients, vitamins, salts, serum proteins, carbohydrates, cofactors etc. Different types of synthetic media can be prepared for a variety of cells and tissues to be cultured. Synthetic media are of two types- Serum containing media (media containing serum) and serum- free media (media with out serum). Examples of some media are: minimal essential medium (MEM), RPMI 1640 medium, CMRL 1066, F12 etc.
Advantages of serum in culture medium are:
i) serum binds and neutralizes toxins,
(ii) serum contains a complete set of essential growth factors, hormones, attachment and spreading factors, binding and transport proteins,
(iii) it contains the protease inhibitors,
(iv) it increases the buffering capacity,
(v) it provides trace elements.
Disadvantages of serum in culture medium are:
(i) it is not chemically defined and therefore it’s composition varies a lot,
(ii) it is sometimes source of contamination by viruses, mycoplasma, prions etc,
(iii) it increases the difficulties and cost of down stream processing,
(iv) it is the most expensive component of the culture medium.
4) pH- Most media maintain the pH between 7 and 7.4. A pH below 6.8 inhibits cell growth. 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.