Animal Cell culture is the process by which animal cells (tissue) are grown under

controlled conditions, generally outside of their natural environment. Artificial

environments consist of a suitable vessel with substrate or medium that supplies the
essential nutrients (amino acids, carbohydrates, vitamins, minerals), growth
factors, hormones, and gases (CO2, O2), and regulates the physio-chemical
environment (pH buffer, osmotic pressure, temperature). Most cells require a surface or
an artificial substrate (adherent or monolayer culture) whereas others can be grown free
floating in culture medium (suspension).

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.

For-example
(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.
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. 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.
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.

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.

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- Usually 370C in the incubators as the body temperature of animal.

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.

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. 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.

Synthetic media

Syntheic media are prepared artificially by adding several organic and inorganic
nutrients 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.

4) pH- Most media maintain the pH between 7 and 7.4. A pH below 6.8 inhibits cell
growth. 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.
Steps involved in animal cell culture:

1. Separation of animal cells or tissue explants from a sacrificed animal by enzymatic or

mechanical method.
2. Preparation of suitable media and sterilization.
3. Establishing primary cell line i.e transfer of animal cells into the media
4. Further sub-culturing into fresh media
5. Evaluating the cells that have been established by microscopy or chemical method.
6. Preservation of the cell lines
7. Scale up of the animal cell culture for its application or research etc.

Applications of animal cell Culture:

a) Production of antiviral vaccines, which requires the standardization of cell lines for the
multiplication and assay of viruses.
b) Cancer research, which requires the study of uncontrolled cell division in cultures.
c) Cell fusion techniques.
d) Genetic manipulation, which is easy to carry out in cells or organ cultures.
e) Production of monoclonal antibodies requires cell lines in culture.
f) Production of pharmaceutical drugs using cell lines.
g) Chromosome analysis of cells derived from womb.
h) Study of the effects of toxins and pollutants using cell lines.
i) Use of artificial skin.
j) Study the function of the nerve cells.

Somatic Cell Fusion

One of the applications of animal cell culture is the production of hybrid cells by the fusion of different
cell types. These hybrid cells are used for a the following purposes:
(i) study of the control of gene expression and differentiation,
(ii) study of the problem of ‘ malignancy’,
(iii) viral application,
(iv) gene mapping,
(v) production of hybridomas for antibody production.

Many commercial proteins have been produced by animal cell culture and there medical application is
being evaluated.

Tissue Plasminogen activator (t-PA) was the first drug that was produced by the mammalian cell
culture by using rDNA technology.
Blood Factor VIII

The patients suffering from Haemophilia A lack factor VIII, which plays an important role in the clotting
of blood. Current therapy for this disease is the transfusion of blood factor VIII into patients. Using
rDNA technology, Factor VIII has been produced from mammalian cell culture e.g. Hamster kidney cell.

Erythropoietin (EPO)

Erythropoietin (EPO) is a hormone-like substance released by the kidney under hypoxic or anoxic
conditions caused by anaemia. r-HUEPO- recombinant human erythro- protein has been effectively
used to treat anemia associated with AIDS, renal failure etc.

The production of Monoclonal Antibodies using hybridoma technology: Antibodies are produced
by B cells by hybridoma technology. Monoclonal antibodies have various therapeutic, diagnostic,
analytical and research applications.

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