Animal biotechnology

1. Achariya Arts And Science College
(Affilated To Pondicherry University)
Class Seminar
Department Of Biotechnology
Topic:-Organ Culture,Histotyphic Culture and Apoptosis

Presentation By
Gopi Krishna Giri

2. ORGAN CULTURE
The entire embryos or organs are excised
from the body and culture
Advantages
Normal physiological functions are
maintained.
Understanding of biochemical and
molecular functions of an organ/tissue
become easy

3. TECHNIQUES OF ORGAN
CULTURE
Optimal nutrient and gas exchange condition
required for organ culture.
This condition can be achive by keeping the
organ/tissue at gas-limited interface of following
the supports:-
-Semisolid gel of agar
-Clotted plasma
-Microporous filter

4. PROCEDURE FOR ORGAN
CULTURE
Dissection and collection of the organ
tissue.
Reduce the size of the tissue as
desired,preferably to less than 1mm in
thickness.
Place tissue on a support(listed above) at
the gas medium interface.

5. PROCEDURE FOR ORGAN
CULTURE

6. PROCEDURE FOR ORGAN
CULTURE
Incubate in a humid CO2 incubator.
Change the medium(M199 or CMRL1066)
as frequently as desired.
The organ culture can be analysed by
histology,autoradiography and
immunochemistry.

7. HISTOTYPIC CULTURES
Growth and propragation of cell lines in three-
dimensional matrix to high cell density.
 it is possible to use dispersed monolayers to
regenerate tissue like structures.
The commonly use technique are:-
Gel and sponge technique
Hollow fibers technique
Spheroids
Multicellular tumour spheroids

8. Gel and Sponge Technique
The gel (collagen) or sponges (gelatin) are
used which provides the matrix for the
morphogenesis and cell growth.
The cells penetrate these gels and
sponges while growing

9. Hollow Fibers Technique
Hollow fibers are used which helps in more
efficient nutrient and gas exchange.

In recent years,perfusion chambers with a bed of
plastic capillary fibers have been developed to
be used for histotypic type of cultures.
The cells get attached to capillary fibers and
increase in cell density to form tissue like
structures.

10. Spheroids
The re-association of dissociated cultured cells
leads to the formation of cluster of cells called
spheroids.
It is similar to the reassembling of embryonic
cells into specialized structures.
The principle followed in spheroid cultures is that
the cells in heterotypic or homotypic aggregates
have the ability to sort themselves out and form
groups which form tissue like architecture

11. Multicellular Tumour Spheroids
These are used as an in vitro proliferating
models for studies on tumour cells.
MTS have a three dimensional structure
which helps in performing experimental
studies related to drug therapy,
penetration of drugs, regulation of cell
proliferation, immune response, cell death,
and invasion and gene therapy

12. Multicellular Tumour Spheroids
A size bigger than 500 mm leads to the
development of necrosis at the centre of
the MCTS.
The monolayer of cells or aggregated
tumour is treated with trypsin to obtain a
single cell suspension.
The cell suspension is inoculated into the
medium in magnetic stirrer flasks or roller
tubes

13. Multicellular Tumour Spheroids

14. Multicellular Tumour Spheroids
After 3-5 days, aggregates of cells
representing spheroids are formed.
Spheroid growth is quantified by
measuring their diameters regularly.
They are used to study gene expression in
a three-dimensional configuration of cells.

15. APOPTOSIS
Programmed cell death
Orderly cellular self destruction.
Destroy cells that are a threat:-
Infected with virus
Turn off immune response
DNA damaged cell
Cancer

16. APOPTOSIS
What makes a cell commit suicide?
withdrawal of positive signals (growth factors, Il-
2)
receipt of negative signals (increased levels of
oxidants, DNA damage via X-ray or UV light,
chemotherapeutic drugs, accumulation of
improperly folded proteins, death activators such
as: TNF-a, TNF-b, Fas/FasL)

17. APOPTOSIS
Steps in apoptosis:
the decision to activate the pathway;
the actual "suicide" of the cell;
engulfment of the cell remains by specialized
immune cells called phagocytes;
degradation of engulfed cell.

18. Apoptosis
The actual steps in cell death require:
Condensing of the cell nucleus and breaking it into
pieces
Condensing and fragmenting of cytoplasm into
membrane bound apoptotic bodies;
Breaking chromosomes into fragments containing
multiple number of nucleosomes (a nucleosome
ladder)
Apoptosis Triggered via Two Pathways
Intrinsic or mitochondrial pathway
Extrinsic or death receptor pathway