1. Organ culture
•Introduction
• Current progress
•Methods
•Factors Involving Organ Culture
•Types of Organ Culture
•Application of organ culture
•Advantage & limitation
Sankalchand Patel University, Visnagar, Gujarat
B.sc- Biotechnology
Kathan.N.Trivedi
2. Introduction
• Organ culture implies that the architecture
characteristic of the tissue in vivo is retained, at
least in part, in the culture. Toward this end the
tissue is cultured at the liquid–gas interface (on a
raft, grid, or gel), which favors the retention of a
spherical or three-dimensional shape.
• organ culture, in which whole organs, or
representative parts, are maintained as small
fragments in culture and retain their intrinsic
distribution, numerical and spatial, of participating
cells.
3. •Not whole but pieces of organs can be cultured on artificial medium. For organ
culture care
•should be taken to handle in such a way that tissue should not be damaged.
Therefore, organ culture technique demands more tactful manipulation than tissue.
•The culture media on which organ is cultured are the same as described for cell and
tissue culture.
•However, it is more easy to culture embryonic organs than the adult animals.
•Method of culturing embryonic organ and adult organs differ.
•Besides, culture of whole or part of animal organ is difficult because these require
high amount of O2 ( about 95% ).
•Special serum-free media ( e.g. T8 ) and special apparatus ( Towell’s type II culture )
are used for adult culture.
•In addition, the embryonic organs can be cultured by applying any of following
methods.
Organ Culture on Plasma Clots. (watch glass method)
Organ Culture on Agar.
Ralf Method.
Grid Method.
Organ Culture in Liquid Media.
Whole Embryo Culture.
4. Current progress
• In April 2006, scientists reported a successful trial of seven bladders grown in-vitro and given
to humans. A bladder has been cultured by Anthony Atala of the Wake Forest Institute for
Regenerative Medicine in Winston-Salem, North Carolina. A jawbone has been cultured at
Columbia University, a lung has been cultured at Yale. A beating rat heart has been cultured
by Doris Taylor at the University of Minnesota. An artificial kidney has been cultured by H.
David Humes at the University of Michigan.
• Silk cut from silkworm cocoons has been successfully used as growth scaffolding for heart
tissue production. Heart tissue does not regenerate if damaged, so producing replacement
patches is of great interest. The experiment used rat heart cells and produced functional
heart tissue. In order to further test applications to humans as a cure, a way to transform
human stem cells into heart tissue would have to be found.
• In 2015, Harald Ott was able to grow a rat forelimb. He now works at Ott Lab which focuses
on the creation of bioartificial hearts, lungs, tracheas and kidneys.
• In 2016, another test was done in which human cells were used to assemble intricately
structured hearts. The hearts ultimately proved immature but proved we were yet one step
further to making a heart from stem cells.
• In January 2017, scientists from Salk Institute for Biological Studies managed to create a pig
embryo that had part of its DNA, critical for the growth of organs, edited out. They then
introduced human stem cells inside the pig embryo to have the human DNA fill in the gaps.
5. Organ Culture on Plasma Clots.
( watch glass method )
• Gaillard (1951) described the technique of organ culture on watch
glass.
– A plasma clot is prepared by mixing five drops of embryo extract
with 15 drops of plasma in a watch glass placed on a cotton wool
pad.
– The cotton wool pad is put in petri dish.
– Time to time cotton is moistened so that excessive evaporation
should not occur.
– Thereafter, a small piece of organ tissue is placed on the top of
plasma clot present in the watch glass.
– In the modified technique the organ tissue is placed into raft of
lens paper or ryon.
– The raft makes easy to transfer the tissue, excess fluid can also be
removed.
6. • Advantage of Watch Glass Technique:
• (i) This technique is simple and cheaper.
• (ii) Materials required can be easily available.
• (iii) This is a well suited technique for growing
embryonic organ.
• Disadvantage of the Watch Glass Technique:
• (i) Usually plasma clot is not a suitable substrate, as
it liquefies in the neighbourhood of explant
so that they come to lie in the rest of the medium;
• (ii) Because of the simplicity of the medium no
biochemical investigation was possible.
7. Organ Culture on Agar
• Solidified culture medium with agar is also used for organ
culture.
• The nutrient agar media may or may not contain serum.
• When agar is used in medium, no extra mechanical support is
required.
• Agar does not allow to liquefy the support.
• The tumours obtained form adults fail to survive on agar
media, whereas embryonic organ grow well.
• The media consist of ingredients: agar (1% in basal salt
solution), chick embryo extracts and horse serum in the ratio
7:3:3.
8. Organ Culture in Liquid Media.
– The liquid media consist of
all the ingredients except
agar.
– When liquid media are used
for organ culture, generally
perforated metal gauze or
cellulose acetate or a ralf of
lens paper is used.
– These possibility provides
support.
Raft Methods
– In this approach the explant is
placed onto a raft of lens paper
or rayon acetate, which is floated
on serum in a watch glass.
– Rayon acetate rafts are made to
float on the serum by treating
their 4 corners with silicone.
– Similarly, floatability of lens
paper is enhanced by treating it
with silicone. On each raft, 4 or
more explants are usually placed.
– In a combination of raft and clot
techniques, the explants are first
placed on a suitable raft, which is
then kept on a plasma clot.
– This modification makes media
changes easy, and prevents the
sinking of explants into liquefied
plasma.
9. Grid Method
– Initially devised by Trowell in 1954, the grid method utilizes
25 mm x 25 mm pieces of a suitable wire mesh or perforated
stainless steel sheet whose edges are bent to form 4 legs of
about 4 mm height.
– Skeletal tissues are generally placed directly on the grid but
softer tissues like glands or skin are first placed on rafts,
which are then kept on the grids.
– The grids themselves are placed in a culture chamber filled
with fluid medium up to the grid; the chamber is supplied
with a mixture of O2 and CO2 to meet the high
O2 requirements of adult mammalian organs.
– A modification of the original grid method is widely used to
study the growth and differentiation of adult and embryonic
tissues.
10. Whole Embryo Culture
– During 1950s, spratt studied how metabolic inhibitors affect the development
of embryo in vitro.
– Old embryo (40 h) was studied up to another 24-48 h in vitro until died.
– For embryo culture a suitable medium prepared is poured into watch glasses
which are then placed on moist absorbent cotton wool pad in petri dishes.
– For the culture of chick embryo, eggs are incubated at 38oC for 40-42 h so that
a dozen of embryos could be produced.
– The egg shell sterilized with 70% ethanol is broken into pieces and transferred
into 50ml of BSS (balanced salt solution ).
– The vitelline membrane covering the blastoderm is removed and kept in petri
dish containing BSS.
– With the help of a forcep the adherent vitelline membrane is removed.
– The embryo is observed by using a microscope so that the developmental stage
of blastoderm could be found out.
– The blastoderm is placed on the medium in which glass placed on sterile
adsorbent cotton wool pad in petri dishes, excess of BSS is removed from
medium and embryo culture of chick is incubated at 37.5oc for further
development.
11. Factors Involving Organ Culture
1. Gas and Liquid Phase:-
• When an organ is taken as solid mass then diffusion of gases
and exchange of nutrients are impaired. When this solid mass
remains in monolayer then diffusion occurs rapidly. To get rid
of this problem cell must be incubated at liquid- gas interface.
While incubating in liquid-gas interface the following points
should be kept in mind:-
• When cells are kept in liquid- gas interface at appropriate
depth the cells tend to occupy special shape.
• If culture is done at deeper level then diffusion of gases do
not occur.
• If culture is done in shallow water then due to more surface
tension of water, outgrowth of cell will occur and no accuracy
is maintained.
• For better organ culture 02 is supplied either as 95% pure 02
or by passing hyperbaric 02.
12. 2. Structural Integrity:-
• For this culture must be interacted in such a way so
that the combined effect is same as integrated
organ and thus communication between cells take
place.
3. Blocking of Differentiation:-
• When cells are differentiated then its regularity will
be impaired. hence a system should be worked out
which block the induction of differentiation.
13. Types of Organ Culture
• The organ culture has been divided into Histotypic organ
culture and Organotypic organ culture:
• Histotypic Organ Culture:- Culture of a characterized cell line
when propagated at high density in the presence of
appropriate environment is called histotypic culture i.e., if the
explant of animal tissue maintains its structure and function
in culture, it is described as histotypic culhue. The term
histotypic culture will employ that cells have been associated
in some way to recreate a 3-dimensional matrix, such as
collagen gel.
• Organotypic Organ Culture:- When the cells of different
lineage are recombined in a specific proportion so that it
seems like an organ it is called organotypic organ culture e.g.,
epidermal keratinocytes in combined reaggregated culture
with dermal fibroblast. Here we can study the cell interaction
which is not possible in histotypic culture
14. Application of Organ Culture
• 1. Organ culture can serve as an extremely useful and appropriate
models, when the study is required on human and require species
specificity and when ethical or practical consideration prelude
using human as the experimental model.
• 2. It is most useful and better models when the infectious agents
and the pathological events that occur in infection donot depend
on host defense or serum factors, i.e., in infection when damage is
due to macrophages and neutrophils, as these elaborate tissue
destructive enzyme into the environment such as cavity formation
in infection caused by Mycobacterim tuberculosis.
• 3. Organ culture was proved to be an important tool for studying
immune responses at mucosal surface, as immune response of
intestinal mucosa was studied by using organ culture of mucosal
surface of intestine established after intestinal infection with
enteroadherent Escherichia coli. (McQueen et al. 1986). Cooper et
al. (1984) observed that organ culture from human fallopian tube
infected with gonococci elaborated substantially more IgA than
uninfected organ.
15. • 4. Organ culture of skin that contains keratinocytes at various stages of
maturation is useful to study the pathogenesis of various micro-organisms that
grow in native or maturing skin, such as herpes virus, dermatophytes and
papilloma virus etc.
• 5. Organ culture of blood vessels could prove extremely useful in studying how
micro-organism in the blood transgress endothelial barrier to enter the spinal
fluid resulting into meningitis. (Ogawa et al. 1985).
• 6. Ramphal and Pyle (1983) used tracheal organ cultures to provide evidence
that mucin and sialic acid served as receptors for attachment of Pseudomonas
aeruginosa in the lower respiratorytract. This attachment is probably an
antecedent step to pseudomonas pneumonia.
• 7. Organ cultures are ideal for investigation of the various components of the
pathologic processes, when micro-organisms produce damage by using
attachment to mucosal cells and elaboration of toxic moieties.
• 8. Organ culture has the potential to provide new information, critical for
designing vaccines that can elicit an effective immune response at selected
mucosal surfaces.
• 9. Organ culture can be used to study the morphology, physiochemical action of
hormones and drugs.
• 10. In some cases organ culture can replace the use of experimental animals for
drug testing and studying drug metabolism in tissues.
16. Advantage of Organ Culture
• (i) The explants remain comparable to the in vivo
organs both in structure and function, which makes
them suitable than cell culture for physiological
studies.
• (ii) The development of foetal organ is comparable
to that in vivo.
• (iii) In tissue explant culture hormone dependent
organs remain so, while endocrine organ secrete
specific hormone. Therefore, organ culture provides
information on the pattern of growth,
differentiation and development and on the
influence of various factors on those features.
17. Limitations of Organ Culture:
• 1. It is very expensive.
• 2. In this yield of cell is very low which is not
sufficient for molecular or biochemical assay.
• 3. Each processes require a new resource.
• 4. It is very complex
18. Reference
• https://en.wikipedia.org/wiki/Organ_culture
• A Textbook of biotechnology by Dr. R. C. DUBEY, s.
chand publishing. Page-217
• Principles of Animal Cell Cultureby Basant Kumar
Sinha and Rinesh Kumar, International Book
Distributing Co.(publishing Division) page-135
