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