Describes tissue and it's types. Animal tissue culture their procedure and applications. Plant tissue culture their procedure and applications.

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Tissue culture
By:
Manish Kumar Das
Department of biotechnology

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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.
 The cultured tissue may consist of a single cell, a population of
cells, or a whole or part of an organ.

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Historical usage of tissue culture
 In 1885 Wilhelm Roux removed a section of the medullary plate
of an embryonic chicken and maintained it in a warm saline
solution for several days, establishing the basic principle of
tissue culture. In 1907 the zoologist Ross Granville Harrison
demonstrated the growth of frog embryonic cells that would give
rise to nerve cells in a medium of clotted lymph.

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Modern usage of tissue culture
 In modern usage, “tissue culture” generally refers to the growth
of cells from a tissue from a multicellular organism in vitro.
These cells may be cells isolated from a donor organism
(primary cells) or an immortalised cell line. The cells are bathed
in a culture medium, which contains essential nutrients and
energy sources necessary for the cells’ survival.[8] Thus, in its
broader sense, “tissue culture” is often used interchangeably
with “cell culture”. On the other hand, the strict meaning of
“tissue culture” refers to the culturing of tissue pieces, i.e.
explant culture.

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Classification of tissue culture:-
 Tissue culture can be classified into two types based on the
medium:
1. Plant tissue culture
2. Animal tissue culture

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Plant tissue culture
Plant tissue culture is a collection of techniques used to maintain or grow plant
cells, tissues or organs under sterile conditions on a nutrient culture medium of
known composition. It is widely used to produce clones of a plant in a method
known as micropropagation.

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Culturing plant tissues –the steps

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 The initiation phase is the first phase of tissue culture. Here, the
tissue of interest is obtained and introduced and sterilized in
order to prevent any microorganism from negatively affecting the
process. It is during this stage that the tissue is initiated in to
culture.

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STAGE 2: Multiplication stage
 The multiplication phase is the second step of tissue culture
where the in vitro plant material is redivided and then introduced
in to the medium. Here, the medium is composed of appropriate
components for growth including regulators and nutrients. These
are responsible for the proliferation of the tissue and the
production of multiple shoots.

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STAGE 3: Root formation
 It is at this phase that roots are formed. Here, hormones are
required in order to induce rooting, and consequently complete
plantlets.

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General procedure for plant tissue culture:
1. Medium preparation:
 The appropriate mixture (such as the MS mixture) is mixed with distilled
water and stirred while adding the appropriate amount of sugar and sugar
mixture. Here, sodium hydroxide or hydrochloric acid is used to adjust the
pH – Contents used here will depend on the plant to be cultured and the
number of tissues to be cultured.
 Agar is added to the mixture, heat and stirred to dissolve.
 After cooling, the warm medium is poured into polycarbonate tubes (to a
depth of about 4 cm).
 With lids sitting on the tubes, the tubes are placed in a pressure cooker and
sterilized for 20 minutes.

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2.Plant preparation:
 Cut the plant part in to small pieces (about 1cm across). On the
other hand, such parts as the African violet leaves can be used as a
whole.
 Using detergent and water, wash the plant part for about 20
minutes.
 Transfer the plant part in to sterilizing Clorox solution, shake for a
minute and leave to sock for 20 minutes.
 Using a lid, gently discard the Clorox and retain the plant part in the
container and then cap the container.

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 3.Transferring the plant material to a tissue culture medium:
 70 percent alcohol should be used for the sterilization of the equipment used and containers.
 Open the container and pour sterile water to cover half the container.
 Cover with a sterile lid again and shake the container for 2 to 3 minutes in order to wash the
tissue and remove the bleach.
 Pour the water and repeat this three times.
 Using sterilized gloves, remove the plant part from the container and on to a sterile Petri dish.
 Using a sterile blade cut the plant material to smaller pieces of about 2 to 3 mm across
avoiding the parts that have been damaged by bleach.
 Using sterile forceps, place a section of the plant in to the medium.
 Replace the lid/cap and close tightly.
 This procedure will result in the development of a callus, which then produces shoots after a
few weeks. Once the shoots develop, then the plant section may be placed in the right
environment (well lit, warmth etc) for further growth.

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4. Technique for plant Vitro culture:
 Micropropagation – This technique is used for the purposes of
developing high- quality clonal plants (a clone is a group of identical
cells). This has the potential to provide rapid and large scale
propagation of new genotypes.
 Somatic cell genetics – Used for haploid production and somatic
hybridization
 Transgenic plants – Used for expression of mammalian genes or
plant genes for various species it has proved beneficial for the
engineering of species that are resistant against viruses and
insects.

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Application of plant tissues culture:
1. Rapid clonal Propagation
 A clone is a group of individuals or cells derived from a single
parent individual or cell through asexual reproduction. All the cells
in callus or suspension culture are derived from a single explants
by mitotic division. Therefore, all plantlets regenerated from a
callus/suspension culture generally have the same genotype and
constitute a clone. These plantlets are used for rapid clonal
propagation.

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2. Soma-clonal Variation:
 Genetic variation present among plant cells of a culture is called
soma-clonal variation. The term soma-clonal variation is also
used for the genetic variation present in plants regenerated from
a single culture. This variation has been used to develop several
useful varieties.

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3. Transgenic Plants:
 A gene that is transferred into an organism by genetic
engineering is known as transgene. An organism that contains
and expresses a transgene is called transgenic organism. The
transgenes can be introduced into individual plant cells. The
plantlets can be regenerated from these cells. These plantlets
give rise to the highly valuable transgenic plants.

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4. Induction and Selection of Mutations:
 Mutagens are added to single cell liquid cultures for induction of
mutations. The cells are washed and transferred to solid culture
for raising mu ant plants. Useful mutants are selected for further
breeding. Tolerance to stress like pollutants, toxins, salts,
drought, flooding etc. can also be obtained by providing them in
culture medium in increasing dosage. The surviving healthy cells
are taken to solid medium for raising resistant plants.

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5. Resistance to Weedicides:
 It is similar to induction of mutations. Weedicides are added to
culture initially in very small concentrations. Dosage is increased
in subsequent cultures till die desired level of resistance is
obtained. The resistant cells are then regenerated to form
plantlets and plants.

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Animal tissue culture
Animal cell culture is the process of culturing animal cells extracted
from tissues or organs under in vitro aseptically controlled
laboratory environment (temperature, gases and pressure)
simulating that of in vivo system. Under the controlled environment,
the animal cells are able to survive and proliferate as under in vivo
conditions.

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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.

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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.

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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).

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Bio-sefety cabinet
 Bio-safety cabinet provides a sterile environment for tissue
culture in addition to making provision for the safety of
researcher against human pathogens.

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Tissue culture equipments:
 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 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.

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The media used in animal cell and
tissue culture are of two types:
(1) Natural medium.
(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.

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Procedure for animal tissue culture
1.Growth Requirements
 The culture media used for cell cultures are generally quite complex, and
culture condition widely varies for each cell type. However, media generally
include amino acids, vitamins, salts (maintain osmotic pressure), glucose, a
bicarbonate buffer system (maintains a pH between 7.2 and 7.4), growth
factors, hormones, O2 and CO2. To obtain best growth, addition of a small
amount of blood serum is usually necessary, and several antibiotics, like
penicillin and streptomycin are added to prevent bacterial contamination.
 Temperature varies on the type of host cell. Most mammalian cells are
maintained at 37oC for optimal growth, while cells derived from cold-
blooded animals tolerate a wider temperature range (i.e. 15oC to 26oC).
Actively growing cells of log phage should be used which divide rapidly
during culture.

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2.Process to obtain primary cell culture
 Primary cell cultures are prepared from fresh tissues. Pieces of
tissues from the organ are removed aseptically; which are
usually minced with a sharp sterile razor and dissociated by
proteolytic enzymes (such as trypsin) that break apart the
intercellular cement. The obtained cell suspension is then
washed with a physiological buffer (to remove the proteolytic
enzymes used). The cell suspension is spread out on the bottom
of a flat surface, such as a bottle or a Petri dish. This thin layer
of cells adhering to the glass or plastic dish is overlaid with a
suitable culture medium and is incubated at a suitable
temperature.

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3.Aseptic techniques
 Bacterial infections, like Mycoplasma and fungal infections,
commonly occur in cell culture creating a problem to identify and
eliminate. Thus, all cell culture work is done in a sterile
environment with proper aseptic techniques. Work should be
done in laminar flow with the constant unidirectional flow of
HEPA filtered air over the work area. All the material, solutions
and the whole atmosphere should be of contamination-free.

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4.Cryopreservation
 If a surplus of cells is available from sub-culturing, they should be
treated with the appropriate protective agent (e.g., DMSO or
glycerol) and stored at temperatures below –130°C until they are
needed. This stores cell stocks and prevents original cell from
being lost due to unexpected equipment failure or biological
contaminations. It also prevents finite cells from reaching
senescense and minimizes risks of changes in long term cultures.
 When thawing the cells, the frozen tube of cells is warmed quickly
in warm water, rinsed with medium and serum and then added into
culture containers once suspended in the appropriate media.

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Applications of Animal 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.

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Thank you