Animal Tissue Culture The foundation of animal cell and tissue culture was laid by Jolly (1903) when he showed that animal cells could not only survive but could divide in culture medium. The actual beginning of animal cell culture and tissue culture was made by Harrison (1907) and later by Carrel (1912) who used frog’s tissue in tissue culture. They successfully showed that animal cells can be grown indefinitely in culture medium just like microorganisms. Later tissues from warm blooded animals like chick and mammals were used as material for tissue culture purpose.

1. B.Sc.III, Sem-VI:Unit-I
Animal Tissue Culture: History, Types and Applications
Authored By: Dr. Rajendra Chavhan, Department of Zoology, RMG College Nagbhid
Animal Tissue Culture
The foundation of animal cell and tissue culture was laid by Jolly (1903) when he showed that
animal cells could not only survive but could divide in culture medium. The actual beginning of
animal cell culture and tissue culture was made by Harrison (1907) and later by Carrel (1912) who
used frog’s tissue in tissue culture. They successfully showed that animal cells can be grown
indefinitely in culture medium just like microorganisms. Later tissues from warm blooded animals
like chick and mammals were used as material for tissue culture purpose.
What is Tissue Culture?
“Tissue culture is the technique of growing cells and tissues in an
artificial medium separate from the organism.”
Tissue culture is a technique in which fragments of plants are cultured and grown in a laboratory.
Many times the organs are also used for tissue culture. The media used for the growth of the
culture is broth and agar.
This technique is also known as micropropagation. It has proved beneficial for the production of
disease-free plants and increase plant yield in developing countries. It only requires a sterile
workplace, greenhouse, trained manpower, and a nursery.
Oil palm, banana, eggplant, pineapple, rubber tree, tomato, sweet potato have been produced by
tissue culture in the developing countries.
Types of Tissue Culture
Following are the different types of tissue culture techniques:
Seed Culture
In this culture, the explants are obtained from an in-vitro derived plant and introduced into a
laboratory where they proliferate. The explant should be sterilized to prevent it from tissue damage.
Embryo Culture
This involves the in-vitro development of an embryo. For this, an embryo is isolated from a living
organism. Both, a mature or an immature embryo can be used in the process. Mature embryos
can be obtained from ripe seeds. The immature embryos are obtained from the seeds that failed
to germinate. The ovule, seed or fruit is already sterilized, therefore, it does not need to be sterilized
again.
Callus Culture

2. A callus is an unorganized, dividing mass of cells. When the explants are cultured in a proper
medium, the callus is obtained. The growth of callus is followed by organ differentiation. The culture
is grown on a gel-like medium composed of agar and specific nutrients required for the growth of
the cells.
Organ Culture
In this, any organ of the plant such as shoot, leaf, can be used as an explant. A number of methods
can be used for the organ culture, such as plasma clot method, raft method, grid method, and agar
gel method. This method is used to preserve the structure and functions of an organism.
Protoplast Culture
It is a cell without a cell wall. A protoplast can be cultured using the hanging-drop method, or micro-
culture chambers. In protoplast culture, a number of phases can be observed: development of cell
wall, cell division, regeneration of a whole plant.
Other Types
 Pollen Culture
 Anther Culture
 Single Cell Culture
 Suspension Culture
 Somatic Embryogenesis
Steps of Tissue Culture
The steps of tissue culture are given below:
Initiation Phase
At this stage, the tissue is initiated into the culture. The tissue of interest is obtained, introduced
and sterilized to prevent the process from any contamination.
Multiplication Phase
In this stage, the sterilized explant is introduced into the medium composed of growth regulators
and appropriate nutrients. They are responsible for the multiplication of cells. This undifferentiated
mass of cells is known as a callus.
Root Formation
The roots start forming. Plant growth hormones are added to initiate the root formation.
Consequently, we obtain a complete plantlets.
Shoot Formation

3. The plant growth hormones for the formation of shoot are added and the growth is observed for a
week.
Acclimatization
When the plant starts developing, it is transferred to the greenhouse to develop under controlled
environmental conditions. It is finally transferred to the nurseries to grow under natural
environmental conditions.
Laboratory Facilities for Tissue Culture:
The laboratory facilities for animal tissue culture consist of (i) Sterile area (ii) tissue culture
equipment’s.
(i) Sterile area:
For processing the animal tissues for culture purpose a sterile or aseptic area is needed. This
working place must be free from any kind of contamination. Two types of sterile work areas are
generally recommended.
They are:
 Laminar flow cabinet
 Bio-safety cabinet.
Laminar flow cabinet:
It is a specially designed chamber inside which animal tissue for culture purpose is being handled
in an aseptic condition. It is completely open in front to allow the researcher to work comfortably
and handle the equipment’s present inside the laminar flow cabinet. A motor blows air into the
laminar flow cabinet through a coarse filter, where large dust particles are separated.
This air then passes through a 0.3 μm HF.PA (High Efficiency Particulate Air). This keeps all
contaminants away from the work surface. Such arrangement does not give protection to
researcher against pathogenic organisms. Hence, laminar flow cabinet cannot be used in any cell
or tissue culture which may contain a human pathogen (disease causing organism).
Bio-safety cabinet:
Bio-safety cabinet provides a sterile environment for tissue culture in addition to making provision
for the safety of researcher against human pathogens.
(ii) Tissue culture equipments:
The equipments required for the animal tissue culture are the followings:
 Autoclave,
 Centrifuge,
 Incubator (capable of regulating the percentage of CO2),
 Water bath,
 Refrigerator,
 Freezer (for–20°C),
 pH meter,
 Chemical balance,

4.  Stirrer,
 Bunsen burner/spirit lamp,
 Culture vessels with screw cap
 Pasteur pipettes,
 Inverted microscope,
 Liquid Nitrogen freezer,
 Liquid Nitrogen storage flask,
 Bench centrifuge
 Soaking bath,
 Deep washing sink,
 Pipette cylinder (s),
 Pipette washer,
 Water purifier.
Culture Media for Cells and Tissues:
Culture medium is the most important factor for culturing cells and tissues. It provides optimum
conditions of factors like pH, osmotic pressure etc. It also provides all nutritional requirements
needed by growing cells in the culture.
The media used in animal cell and tissue culture are of two types:
(i) Natural and
(ii) Artificial medium.
The natural media include fluids of biological origin, such as plasma clots and serum. Plasma clot
is prepared by treating the blood of an animal with an anticoagulant such as heparin. Serum is the
clear fluid part of the blood, formed after blood coagulation when fibrin separates from the plasma.
It is considered as an ideal growth medium for animal cells as if is formed of hundreds of proteins
and hormones.
The artificial culture media primarily consist of balanced salt solution (BSS) which provide essential
inorganic ions, correct osmolarity, required pH (7.0-7.3), energy (= glucose) and a pH indicator
(such as phenol red). However, BSS lacks essential amino acids and vitamins. Earle’s balanced
salt solution and Hanks’ balanced salt solution come under this category. These media cannot
support growth of cells and tissues but can keep them alive for a period of 12 hours.
For longer survival of the culture, serum may be used or the balanced salt solution may be
supplemented with amino acids, oxygen, vitamins and serum protein. One such medium was
developed and modified by Eagle (1955), which is described as minimum essential medium
(MFM). Other more complex, synthetic media include (i) CMRL 1066 (ii) RPMI 1640, (iii) F12 etc.
The use of serum in culture media has several disadvantages, which led to the development of
many serum free media.
Culture Procedures:
The procedure for tissue culture and cell culture involve preparation and sterilization of glassware,
equipment, reagents and media. As the tissue culture is being carried out in highly aseptic
condition, all the usable, media and solutions are required to be sterilized thoroughly. Some of the
methods of sterilization include swabbing, capping, flaming, dry at, wet heat, radiation, filter
sterilization etc.

5. The work bench, reagent bottles and growth media are swabbed (= cleaned) with 70% ethanol
before and after operation. Culture and reagent bottles are capped with deep screw caps. The
reagents bottles are capped immediately alter using the reagent. The necks of all bottles and the
screw caps are flamed before and after opening and closing. This is a common practice to prevent
infection and maintaining aseptic condition.
Glassware, stainless steel instruments, plastic containers, distilled water, phosphate buffered
saline and growth medium are sterilized in autoclave at 121 °C and under pressure 15 lb/square
inch for 20 minutes. This process of sterilization by means of an autoclave is called as wet heat. It
is followed by dry heat. During dry heating, glass ware and dissecting instruments are placed within
a hot air oven at 160°C for one hour. The culture room or laminar How cabinet is fitted with a UV
lamp.
The lamp is switched on half an hour before the operation of the work for the purpose of
sterilization. Some heat labile constituents of the growth medium (such as polypeptides, hormones
etc.) are sterilized not by heating method but by another method called filter sterilization. In this
method liquid substances are passed through a micro filter (0.2 μm) which removes everything
except mycoplasma (50%) and bacterial endotoxins.
Preparation of Starting Material:
The starting material is an isolated tissue or a body part. The isolated tissue or the body part
consists of numerous cells cemented together by proteinous substances. To start the cell culture,
the starting tissue is to be dissociated into cells by two methods: (i) mechanical method, (ii)
enzymatic method.
In mechanical method the isolated tissue is cut into small pieces in BSS (Balanced Salt Solution)
and these are then cultured in suitable vessels. In enzymatic method, the enzyme trypsin is
generally used to dissociate cells present in the tissue by digesting the proteinous cementing
material.
Following dissociation of starting tissue into cells by the mechanical or enzymatic method, the
dissociated cells are placed in flat bottomed culture vessel (either made of glass or high grade
plastic) containing culture medium (Fig. 7.9). The culture vessel should have optimum number of
cells and it should be incubated at 37°C. The inner surface of the culture vessel should have
negatively charged (SO-3) group.
The transfer of dissociated cells into the culture medium is called as inoculation. A culture
established directly from differentiated tissue (isolated tissue or body part) is known as primary
culture. After sometime the bottom of the culture vessel will be covered by a continuous layer of
cells, often one cell thick. This layer of cells is known as monolayer.
The cells from the primary culture can be detached from the culture vessel by trypsin or EDTA
(Ethylene Diamine Tetraacetic Acid) treatment and transferred to fresh media. The culture so
obtained is called cell lines. The process of transferring cultured cells into fresh culture vessels is
termed as sub culturing.
The cells divide at a constant rate over successive transfers. Such cells comprise a cell strain. Cell
strains do not have an infinite life. They divide 50-100 times before dying. For long term culture the
cell lines are preserved in frozen state in liquid nitrogen in presence of cryo preservative agents
and foetal calf serum.
Small-scale cultures are generally carried out either in plastic petriplates or plastic T-flasks. Large-
scale cultures involving mammalian cells are carried out in bioreactors or fermenters.

6. Applications of Tissue Culture:
 Animal cell culture was primarily aimed to study infection of animal viruses.
 Later on it was used to produce a wide range of biological products of commercial importance such
as antibodies, enzymes, hormones, immuno-regulators.
 Recently tissue culture technique has been used in the manufacture of viral vaccines, tissue
plasminogen activator, interferon-a, monoclonal antibodies and tumor specific antigens.
 Production of Foot and Mouth disease vaccines (FMD vaccines) is the most important example of
the use of large scale cell culture. There are several other vaccines including polio vaccine, bovine
leukaemia virus (BLV) vaccines, rabies vaccines etc. which are produced on commercial basis
using cell cultures
 Impact of new drugs can be evaluated using cell and tissue culture techniques.
Advantages of Tissue Culture
Following are the various advantages of tissue culture technique:
1. The plantlets are obtained in a very short time with a small amount of plant tissue.
2. The new plants produced are disease-free.
3. The plants can be grown throughout the year, irrespective of the season.
4. A large space is not required to grow plants by tissue culture technique.
5. The production of new varieties in the market place speeds up.
6. This technique is being used for the production of ornamental plants such as dahlia,
chrysanthemum, orchids, etc.
Importance of Tissue Culture
Tissue culture is very important in biology due to its wide range of applications.
Both plant and animal tissues can be used for culturing. For eg., animal tissue culture helps in
preserving an organ or tissue.
Plant tissue culture may be used for genetic modification of a plant or simply increase its yield. the
cells of the plants can be genetically altered to produce plants with desirable characteristics.
This technique utilizes the plant’s ability to rejuvenate the tissues rapidly. It produces exact copies
of itself known as clones.
It is a technique of quickly producing plants without any tubers, seeds or bulbs.
It also helps in the conservation of plant biodiversity by the production of endangered plants.