Animal biotechnology
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Transcript of Animal biotechnology
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
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
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
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.
PROCEDURE FOR ORGAN CULTURE
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.
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
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
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.
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
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
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
Multicellular Tumour Spheroids
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.
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
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)
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.
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 pathwayExtrinsic or death receptor pathway