Introduction:

The term tissue culture refers to the culture of whole organs, tissue
fragments as well as dispersed cells on a suitable nutrient medium. Tissue culture
can be divided into the following two broad groups (i) cultures that facilitate cell-to-
cell interactions and signalling among cells and allow their study, and (ii) those in
which cell-to-cell interactions and signalling are missing. The first group consists of
three distinct types of culture systems, viz., (i) organ cultures, (ii) histotypic cultures
and (iii) organotypic cultures. The second group, on the other hand, comprises cell
cultures either as monolayers or as suspension cultures. In organ cultures, whole
embryonic organs or small tissue fragments are cultured in vitro in such a manner
that they retain their tissue architecture, i.e., the characteristic distribution of various
cell types in the given organ. In contrast, cell cultures are obtained either by
enzymatic or mechanical dispersal of tissues into individual cells or by spontaneous
migration of cells from an explant. In histotypic cultures, individual cell lineages are
first isolated from an organ, puried and multiplied; they are then grown separately to
high density in a three-dimensional matrix to study interactions and signalling among
homologous cells. In an organotypic culture, cells of different lineages are mixed
together in specific ratios and spatial relationships in order to recreate a component
of the concerned organ.
Freshly isolated cell cultures are called primary cultures; they are usually
heterogeneous and slow growing, but are more representative of the tissue of their
origin both in cell type and properties. Once a primary culture is sub cultured, it gives
rise to cell lines, which may either die after several subcultures (such cell lines are
known as finite cell lines) or may continue to grow indefinitely (these are called
continuous cell lines). Usually, normal tissues give rise to finite cell lines, while
tumours give rise to continuous cell lines. But there are several examples of
continuous cell lines, which were derived from normal tissues and are themselves
non-tumorigenic, e.g., MDCK dog kidney, 3T3 fibroblasts, etc. The evolution of
continuous cell lines from primary cultures is supposed to involve a mutation, which
alters their properties as compared to those of finite lines.
Culture Media:
Culture of animal cells and tissue is rather more difficult than that of microorganisms
and plants because the later synthesise certain chemical constituents from inorganic
substances. However, the culture media provide the optimum growth factors (e.g.,
pH, osmotic pressure, etc) and chemical constituents (unlike microbes). There are
two types of media used for culture of animal cell and tissue, the natural media and
the synthesized media.
Natural media:
Natural media are the natural sources of nutrient sufficient for growth and
proliferation of animal cells and tissue. These are of three types: (1) coagulants or
plasma clots (it is used since long time but now available in market in the form of
liquid plasma kept in silicon ampoules or lyophilized plasma. Plasma may also be
prepared in laboratory taking out blood from male fowl and adding heparin to
prevent blood coagulation), (ii) biological fluid (it is obtained in the form of serum
from human adult blood, placental, cord blood, horse blood, calf blood or in the form
of biological fluids such as coconut water, amniotic fluid, pleural fluid, insect
haemolymph serum, culture filtrate, aqueous humour (from eyes), etc. The most
commonly used fluids are human placental, cord serum and foetal calf serum. Before
use its toxicity should be checked, and (iii) tissue extract (extract from some tissues
such as embryo, liver, spleen, leukocytes, tumour, bone marrow, etc. are also used
for culture of animal cells, where embryo extract is of most common use. Tissue
extract should be used before a week or stored at 27°C.
Synthetic media:
Synthetic media are prepared artificially by adding several nutrients (organic and
inorganic), vitamins, salts, O, and CO₂ gas phases, serum proteins, carbohydrates,
cofactors, etc. However, different types of synthetic media may be prepared for a
variety of cells and tissues to be cultured. It can be prepared for different functions.
Basically, synthetic media are of two types, serum-containing media (i.e., the media
containing serum) and serum-free media (i.e., media devoid of serum). Example of
some of the media are: minimal essential medium
Glassware, Equipment’s and Culture Media. The thoroughly washed glassware and
equipment’s and carefully prepared culture media are sterilized by heat, steam or
Millipore filter paper (Table 6.4). The sterilized materials are removed when
temperature cools down and used according to procedure adopted.
sterilization item
Autoclaving Apparatus containing glass and silicon tubing, disposable tips
for micropipettes, dispenser tubing for puppet, glass bottles
with screw caps, magnetic stirrer bars, screw caps, silicon
tubing, stopper-rubber, silicon,
Millipore filters, etc

Dry heat Glassware, glass coverslips, glass slides, instruments, Pasteur

pipettes, pipettes, test tubes, etc.

15 lb/in² (=121°C) for 20 minutes; 160°C /1 hour.

 Sterilization of liquid by different methods
Sterilization Storage solution
Autoclave* Room temperature Agar, antibiotics, EDTA, glucose,
glycerol, HEPES, lactalbumin
hydrolysate, NaHCO3, solution,
NaOH solution, phenol red, salt
solution (with glucose), tryptose,
water

Autoclave 4°C Methocel

Filter** Room temperature Glucose solution, HCl, NaHCO3,

solution, NaOH solution

Filter -20°C Collagenase, antibodies, glutamine,

sodium pyruvate solution,
transferrin, trypsin, vitamins.

Filter-stacked filters 4°C Bovine serum albumin

Filter-stacked filters -20°C Serum

Steam (30min at 4 0c Carboxyl methylcellulose

1000c)
Evolution of cell lines:
This mixture of cells is in single cell suspension which may be used as primary culture
or starter culture. The primary culture (in the form of single cell suspension) is sub
cultured 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, in spite 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 sub cultured 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
Characteristics:
Small, more rounded, less adherent cells with a higher nucleus/ cytoplasm ratio.
2. Increased growth rate; cell doubling time reduced to 12-36 hours from 36-48
hours for finite cell lines.
3. Aneuploid (between 2n and 4n) chromosome number showing considerable
variation among cells of a cell line.
4. Reduced serum dependence
5. Increased cloning efficiency.
6. Reduced dependence on substrate adhesion (anchorage) and increased tendency
to grow in suspension.
7. Loss of contact inhibition of cell mobility.
8. Many of the cell lines become malignant, but some cell lines are normal and non-
malignant. The changes in growth control involved in transformation are under
distinct positive (oncogenes) and negative (tumour suppressor genes) controls
specified by specific genes.
9. Ability to grow up to higher cell densities, i.e., growth is less dependent on cell
density.
The faster growth rate, lower serum requirement, ability to grow in suspension,
independence from cell density are distinct advantages of continuous cell lines. But
their limitations relate to the greater chromosome instability, loss of tissue-specific
markers and differences from the phenotypes of donor tissues.
Maintenance of Cell Lines
Thousands of cell lines have been developed from human and other animal tissues;
many of them are finite, while others are continuous cell lines. The continuous cell
lines themselves may have originated from normal (through transformation) or
tumour tissues. American Type Culture Collection (ATCC) maintains a large number
of cell lines acquired from their originators; these cell lines are characterised before
preservation and/or distribution on request to research workers. A cell line is
multiplied and divided into the following two types of cultures: (I) a seed stock and
(ii) a working or distribution stock culture. The seed stock culture is characterised
and then maintained in frozen state, preferably in liquid nitrogen at -196°C. Seed
stock ampoules are used to produce distribution stock cultures so that the cell line
does not change with time due to genetic instability, selection, senescence or
transformation. In contrast, working stock or distribution stock culture consists of
material multiplied from the seed stock culture and used for further studies or for
distribution.
Freeze preservation of animal cells is now routine in all cell line banks. A
cryoprotective agent like DMSO or glycerol is generally added to minimise injury to
cells during freezing and thawing. Frozen ampoules are generally stored in liquid
nitrogen refrigerators, which are rather convenient and quite safe. Following
appropriate protocols, most animal cells can be stored for quite long periods
(Appendix-6. V).
Applications
The organ cultures have been widely used for the following. Studies on the patterns
of growth, differentiation and development of organ rudiments i.e., foetal organs,
and the influences of various factors like hormones, vitamins, etc, on these
parameters.
The action of drugs, carcinogenic agents, etc. on the animal organs is studied in vitro
to at least serve as a guide for the events in whole animals. 3. Perhaps the most
dramatic application of organ cultures is to produce tissues for implantation in
patients; this is often called tissue engineering. Human skin has been successfully
produced in vitro and used for transplantation in cases of serious burns, ulcers, etc.
The ultimate objectives of tissue engineering are to reconstitute body parts in vitro
(1) for use as grafts or transplants and (2) to be used as models for studies on drug
delivery and action. It is expected that cartilage tissue developed in vitro (artificial
cartilage) will be available for human implantation in cases of injuries, arthritis, etc.:
results with rabbits have been encouraging. It is hoped that studies will permit the
culturing and constitution of bones, liver. pancreas, etc.
Separation of cell lines:
Some of tissues consist of cells which are tightly aggregated. Tissue like epithelium is
impregnated with Ca2+ and Mg2++ ions that provide integrity to it. Therefore, for
getting primary culture it is necessary that tissue must be disaggregated either
mechanically or using enzymes or chemicals so that cell suspension could be
obtained. The cells in suspension grow to produce primary culture.
(i) Physical (mechanical) disaggregation: The tissue after careful removal
from a given spot is aseptically kept in a sieve of 100 μm sieve (Fig. 6.1). It
is put in a sterile Petri dish containing buffered medium with balanced salt
solution. The cells are alternately passed through sieves of decreasing pore
size (50 µm and 20 mm mesh). If desired the process is repeated to get
more disaggregation of cells. The debris remaining on sieves are discarded
and medium containing cells is collected. The cells are counted using a
haemocytometer. If necessary, medium is diluted by serum to raise the
level of cells to 104 cells/ml. The other methods of mechanical
disaggregation of cells are forcing the cells through syringe and needle or
repeated pipetting. Although the physical method is quick and cheap yet it
damages many live cells.
(ii) Enzymatic disaggregation: Enzymes are also used for dislodging the cells of
tissue. By using enzymes, a high number of cells is obtained. Moreover, in
embryonic tissue a high number of undifferentiated cells with least
extracellular matrix is found. Therefore, disaggregation of embryonic tissue
occurs more readily than that of adult or new born. In addition, in fragile
tissue such as tumours the chances of cell death and cell recovery are
more than the normal tissues. There are two important enzymes used in
tissue disaggregation, collagenase and trypsin.