“Tissue Culture”

Faculty of Veterinary and Animal Sciences,

University of Rajshahi, Rajshahi-6205.
OVERVIEW
 What is Tissue Culture?
 History of Animal Tissue Culture
 Importance of Tissue Culture
 Types of Tissue Culture
 Procedures of Animal Tissue Culture
 History of Plant tissue Culture
 Procedure of Plant Tissue Culture
 Terminologies associated with Tissue Culture
 Conclusion
WHAT IS TISSUE CULTURE?

 Tissue culture, a method of

biological research in which
fragments of tissue from an
animal or plant are transferred
to an artificial environment in
which they can continue to
survive and function.

Fig: Culture medium

 The cultured tissue may consist of
a single cell, a population of cells,
or a whole or part of an organ.
Cells in culture may multiply;
change size, form, or function;
exhibit specialized activity
(muscle cells, for example, may
contract); or interact with other
cells.

Fig: Culture medium

HISTORY OF ANIMAL TISSUE CULTURE
 1878: Claude Bernard proposed that
physiological systems of an organism can be
maintained in a living system after the death of
an organism.
 1885: Roux maintained embryonic chick cells
in a saline culture.
 1897: Loeb demonstrated the survival of cells
isolated from blood and connective tissue in
serum and plasma.
 1907: Harrison cultivated frog nerve cells in a
lymph clot and observed the growth of nerve
fibers in vitro for several weeks. He was
considered by some as the father of cell culture.
Fig: Working in lab
 1910: Burrows succeeded in long term
cultivation of chicken embryo cell in plasma
clots. He made detailed observation of mitosis.
 1911: Lewis and Lewis made the first liquid
media consisted of sea water, serum, embryo
extract, salts and peptones.
 1913: Carrel introduced strict aseptic
techniques so that cells could be cultured for
long periods.
 1916: Rous and Jones introduced proteolytic
enzyme trypsin for the subculture of
adherent cells.
 1940s: The use of the antibiotics penicillin
& streptomycin in culture medium
decreased problem of contamination in cell
culture.
 1948: Earle isolated mouse L fibroblasts Fig: Working in lab
which formed clones from single cells..
 1952: Gey established a continuous cell line
from a human cervical carcinoma known as
HeLa (Helen Lane) cells. Dulbecco
developed plaque assay for animal viruses
using confluent monolayers of cultured
 1955: Eagle studied the nutrient requirements of selected cells in culture &
established first widely used chemically defined medium.
 1965: Harris and Watkins were able to fuse human and mouse cells by the use of a
virus.
 1975: Kohler and Milstein produced the first hybridoma capable of secreting a
monoclonal antibody.
 1978: Sato established the basis for the development of serum-free media from
cocktails of hormones and growth factors.
 1982: Human insulin became the first recombinant protein to be licensed as a
therapeutic agent.
 1985: Human growth hormone produced from recombinant bacteria was accepted
for therapeutic use.

Reference: History of Tissue culture-Pulication 'Animal tissue culture' by Ms Veena D. Shriram,

Assistant Professor, Department of Physiology, B.J. Government Medical College, Pune
 IMPORTANCE OF TISSUE CULTURE

 Tissue culture has great importance in studies of plant morphogenesis, physio­logy,

biochemistry, pathology, embryology, cytology etc.
 From tissue culture studies it is possible to know bow simple cells differentiate
and become specialized to perform special functions.
 Various changes taking place in a cell can be noted from clonal culture.
 Interrelationship between two cells can be studied in tissue culture.
 With the help of phase contrast cine-photomicrography a very clear understanding
of mitosis and meiosis is possible in tissue culture.
 Haberlandt noted the importance of tissue culture in studying plant morphogenesis.
Relationship between growth and differentiation can be well understood from such
a culture.
 In vegetatively propagating plants many plantlets are formed very quickly
from callus culture or culture of explants.
 By tissue culture method new plant variants can be obtained by isolating gene­
tically unique cells.
 From callus cultures of tobacco, carrot, asparagus etc. new plants are
formed.In tissue culture, mutation can be easily induced and from such mutant
cells mutant plants may be produced.
 Tissue culture has great significance in pathological studies. The effect of
various medicine on cells infected by pathogens can be studied in tissue
culture.
 Tissue culture technique is employed in the studies of plant tumour diseases
and host parasite relationship.
 Disease free plants can be produced by tissue culture te­chnique.
 Tissue culture technique has been successfully used in nutritional research.
 Suspension culture under controlled conditions may be used to solve many
physiological or biochemical problems and also provides a system for the
production of important plant products, such as, plant alkaloids.
 From cell and organ culture under controlled environmental conditions nutri­
tional and metabolic processes can be studied.
 In tissue culture the behaviour of normal and cancer cells can be studied.
 From tissue culture studies information about some hereditary diseases of man
has been obtained. Carriers of some diseases can also be identified through
tissue cul­ture technique.

Reference: Importance of tissue culture:.https://www.biologydiscussion.com/botany/tissue-

culture/tissue-culture-definition-history-and-importance/42944
 TYPES OF TISSUE CULTURE

 Seed Culture:
Seed culture is the type of tissue
culture that is primarily used for
plants such as orchids. For this
method, explants (tissue from the
plant) are obtained from an in-vitro
derived plant and introduced in to
an artificial environment, where
they get to proliferate.

Fig:Seed Culture
 Embryo Culture:
Embryo culture is the type of
tissue culture that involves the
isolation of an embryo from a
given organism for in vitro
growth.
 Callus Culture:
Callus - This is the term used to refer to
unspecialized, unorganized and a dividing mass
of cells. A callus is produced when explants
(cells) are cultured in an appropriate medium - A
good example of this is the tumor tissue that
grows out of the wounds of differentiated
tissues/organs.
In practice, callus culture involves the growth of
a callus (composed of differentiated and non-
differentiated cells), which is the followed by a
procedure that induces organ differentiation.
 Organ culture: Organ culture is a
type of tissue culture that involves isolating
an organ for in vitro growth. Here, any
organ plant can be used as an explant for
the culture process (shoot, root, leaf, and
flower).With organ culture, or as is with
their various tissue components, the
method is used for preserve their structure
or functions, which allows the organ to still
resemble and retain the characteristics they
would have in vivo.
 Here, new growth (differentiated
structures) continues given that the organ
retains its physiological features. As such,
an organ helps provide information on
patterns of growth, differentiation as well
as development.
 There are number of methods that can be used for organ culture. These include:
 Plasma clot method - Here, the method involves the use of a clot that is composed
of plasma and chick embryo extract (or any other extract) in a watch glass. This
method is particularly used for the purposes of studying morphogenesis in
embryonic organ rudiments and more recently for studying the actions of various
hormones, vitamins and carcinogens of adult mammalian tissues.
 Raft method - For this method, the explant is placed on a raft of lens paper/rayon
acetate and floated on a serum in a watch glass.
 Agar gel method - The medium used for this method is composed of a salt
solution, serum as well as the embryo extract or a mixture of various amino acids
and vitamin with 1 percent agar. The explant has to be subcultured every 5 to 7
days. The method is largely used for the study of developmental aspects of normal
organs and tumors.
 Grid method - Grid method, as the name suggests involves the use of perforated
stainless steel sheet, on which the tissue of interest is placed before being placed in
a culture chamber containing fluid medium.
 Protoplast Culture: Protoplast -
cells without cell walls. A protoplast is the
term used to refer to cell (fungi, bacteria,
plant cells etc) in which the cell wall has
been removed, which is why they are also
referred to as naked cells.
 Protoplasts may be cultured in the
following ways:
 Hanging-drop cultures
 Micro culture chambers
 Soft agars matrix
 Once a protoplast has regenerated a cell
wall, then it goes through the process of
cell division to form a callus, which may
then be subcultured for continued growth.
Meristem Culture: It is a culture of isolated mature or immature
embryos. It ‘ is applied in micro propagation and production of virus free plants.
Anther or Pollen culture: In this technique, haploid plants are obtained
from pollen grains by replacing another or pollens into a suitable medium. This
technique is used to obtained haploid plants.
Nucellus culture: Nucellus culture has been utilized to study factors
responsible for formation of adventive embryos.

Fig:Nucellus culture
 Cell culture: The growing of individual cells that have been obtained from
an explant tissue or callus. It is applied in production of useful metabolites,
synthesis of new chemical substances.

Reference: Types of Tissue culture: https://www.microscopemaster.com/tissue-culture.html

https://www.biologydiscussion.com/tissue-cultures/tissue-culture-techniques-and-classification/24124
PROCEDURE OF ANIMAL TISSUE CULTURE

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: It consists of:

 Laminar flow cabinet

 Bio-safety cabinet.
(ii) Tissue Culture equipments: It consists of -
 Autoclave,
 Centrifuge,
 Incubator (capable of regulating the percentage of CO2),
 Water bath
 Refrigerator
 Freezer (for–20°C)
 pH meter
 Chemical balance
 Stirrer
 Bunsen burner/spirit lamp
 Culture vessels with screw capPasteur pipettes,
 Inverted microscope,
 Liquid Nitrogen freezer and storage flask
 Bench centrifuge
 Soaking bath
 Deep washing sink
 Pipette cylinder (s)
 Pipette washer
 Water purifier.
CULTURE MEDIA
A cell culture media consists of:
 PREPARATION OF TISSUE CULTURE
 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.
 The work bench, reagent bottles and growth
Fig: Cleaning of Equipments
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
Fig: Preparation of Tissue
called as wet heat. It is followed by dry
Material
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. Fig: Preparation of
Tissue Material
 WORK WITH 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
Fig cutting of tissue into small
cells present in the tissue by digesting pieces
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.

Fig: Inoculation of Tissue

 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.Tmonolaye
 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 Fig: Working in lab
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.

Reference: Procedure of tissue culture: https://madhavuniversity.edu.in/animal-

tissue-culture.html
 PLANT TISSUE CULTURE
 History of plant tissue culture:
 PROCEDURES OF PLANT TISSUE
CULTURE

The main requirements of plant tissue culture are:

 Laboratory Organisation

 Culture Media
 Aseptic Conditions
LABORATORY REQUIREMENTS
CULTURE MEDIA

 The formulation or the

medium on which the explant
is cultured is called culture
medium. It is composed of
various nutrients required for
proper culturing. Different
types of plants and organs
need different compositions
of culture media. A number
of media have been devised
for specific tissues and
organs
 STEPS OF PLANT TISSUE CULTURE

 Selection and Sterilisation of Explant

 Preparation and Sterilisation of Culture Medium
 Inoculation
 Incubation
 Sub Culturing
 Transfer of Plantlets
 Selection and Sterilisation of Explant:
Suitable explant is selected and is then excised from the donor plant.
Explant is then sterilized using disinfectants.

 Preparation and Sterilisation of Culture

Medium:
A suitable culture medium is prepared with special attention towards
the objectives of culture and type of explant to be cultured. Prepared
culture medium is transferred into sterilized vessels and then sterilized
in autoclave.
 Inoculation:
Sterilized explant is inoculated (transferred) on the culture medium under
aseptic conditions.

 Incubation:
Cultures are then incubated in the culture room where appropriate
conditions of light, temperature and humidity are provided for successful
culturing.

 Sub culturing:
Cultured cells are transferred to a fresh nutrient medium to obtain the
plantlets.
 Transfer of Plantlets:
After the hardening process (i.e., acclimatization of plantlet to the
environment), the plantlets are transferred to green house or in pots.

Referance: Plant Tissue Culture-

https://www.biologydiscussion.com/essay/plant-breeding-essay/essay-on-
 TERMINOLOGIES ASSOCIATED WITH TISSUE
CULTURE
 Totipotency: Totipotency is capability of an isolated single cell to multiply and
differentiate into multicellular organism. 'Haberlandt’ first demonstrated the
totipotency of cells in 1902.
 Micro propagation: It is in vitro multiplication of plants from a small tissue
explant.
 Re-culture : It is the Process by which a cell monolayer or a plant explant is
transferred without subdivision into a fresh medium.
 In vitro is a biochemical process or reaction taking place in a test tube (in lab).
 In vivo is a biological process or reaction taking place in a living cell or
organism.
 Allele: It is one of a number of different forms of a gene.
 Codon: A group of three nucleotides coding for an amino acid.
 Anticodon: It is triplet of nucleotides in transfer of RNA.
 RNA (ribonucleic acid): It is a molecule similar to DNA, helps in process of
decoding genetic information carried by DNA Methods used for
determination of gene sequences.
 DNA (deoxyribonucleic acid): It is the molecule encodes genetic
information.
 Double haploids:These are Individuals derived from haploid garnets and
carry two identical chromosomes.
 Microsatellite – DNAs consist of repetitions of extremely short units.
 Electroporation: It is a technique uses electric discharge to produce pores on
cell membrane for intake of recombinant DNA.
 Electrophoresis: I is a technique that separates charged molecules (DNA,
RNA, proteins) on the basis of relative migration in a appropriate matrix
(such as agarose, polyacrylamide) subjected to an electric medium.
 Embryogenesis: It is a Process of formation of somatic embryos from callus.
 Genetic Transformation: Genetic transformation is the heritable change in a cell
or organism brought about by the uptake and establishment of introduced DNA.
 Hybridization: It is crossing of two genotypically different plants.
 Somatic hybridization: It is crossing of plants through fusion of somatic cell.
 Chromomere:It is a serially aligned beads or granules of eukaryotic
chromosome from local coiling of continuous DNA thread.
 Jumping gene or Transposon:A movable genetic element. A DNA element
which has the ability to move from one chromosomal position to another. It can
insert at random into plasmids or the bacterial chromosome independently of
the host cell recombination system.
 Nucleic Acids: These are family of molecules that includes the DNA and RNA
molecules
 Genetic engineering:Change in the genetic constitution of cells by
introduction or elimination of specific genes using molecular biology
techniques is known as Genetic engineering. This is a non-sexual method of
gene transfer.
 Recombinant DNA technology:Technology of DNA molecules means the
joining or recombining of two pieces of DNA from two different species. The
techniques allow an investigator to biological purify (clone) a gene from one
species by inserting it into the DNA of another species, where it is replicated
along with the host DNA.
 GMO: a Genetically Modified or transgenic plant is a plant that has a novel
combination of genetic material obtained through the use of modern
biotechnology.A transgenic crop plant contains a gene or genes which have
been artificially introduced instead of the plant acquiring them through
pollination these genes are introduced with a view to expressing a novel trait
which is not normally found normally in the given species.
 CONCLUSION
In reality, there are numerous methods used for tissue culture given that there
are different types of tissues that require specific conditions for the culture
process yield desired results.
Both plant and animal tissue can be used for tissue culture purposes for a wide
range of purposes.
For instance, animal tissue culture may serve such purposes as preservation of
an organ/tissue, studying the tutors or given tissues or for diagnosis purposes.
On the other hand, plant tissue culture may be used for cloning purposes,
genetic modification of a given plant or simply to accelerate or increase yield
of the plant of interest.
Tissue culture is therefore of great significance in biological studies due to its
wide range of applications. The processes involved in tissue culture may be
complex, requiring a lot of care to avoid such effects as contamination.
Because of the complexities that may be involved in some of the steps, this
may not be an experiment for everyone.