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Animal Cell, Tissue and Organ

CultureOrgan CultureNot whole but pieces of organs can be cultured on artificialmedium. 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 tactfulmanipulation than tissue culture. The culture media onwhich organ is cultured are the same as described for celland tissue culture. However, it is more easy to cultureembryonic organs than the adult animals. Methods ofculturing embryonic organ and adult organs differ. Besides,culture of whole or part of animal organ is difficult because

these require nigh amount of O2 (about 95%). Specialserum-free media (e.g.T8) and special apparatus (Towell'sType II culture chamber) are used for adult culture. Inaddition, the embryonic organs can be cultured byapplying any of the following three methods:

Organ Culture on Plasma ClotsA plasma clot is prepared by mixing five drops of embryo

extract with 15 drops of plasma in a watch glass placed ona cotton wool pad. The cotton wool pad is put in a Petridish. Time to time cotton is moistened so that excessiveevaporation should not occur. Thereafter, a small piece oforgan tissue is placed on the top of plasma clot present inthe watch glass. In the modified technique the organtissue is placed into raft of lens paper or ryon. The raft

makes easy to transfer the tissue, excess fluid can also be

removed.Organ Culture on AgarSolidified culture medium with agar is also used for organculture. The nutrient agar media may or may not containserum. When agar is used in medium, no extra mechanicalsupport is required. Agar does not allow to liquefy thesupport. The tumours obtained from adults fail to survive

on agar media, whereas embryonic organs grow well. Themedia consist of ingredients: agar (1% in basal saltsolution), chick embryo extracts and horse serum in theratio of 7:3:3.

Organ Culture in Liquid Media

The liquid media consist of all the ingredients except agar. When liquid media are used for organculture, generally perforated metal gauze or cellulose acetate or a raft of lens paper is used. Thesepossibility provides support.

Whole Embryo CultureDuring 1950s, Spratt studied how metabolic inhibitors affect the development of embryo invitro. Old embryo (40 h) was studied upto another 24-48 h in vitro until died. For embryo culture asuitable medium prepared is poured into watch glasses which are then placed on moist absorbentcotton wool pad in Petri dishes. For the culture of chick embryo, eggs are incubated at 38C for40-42 h so that a dozon of embryos could be produced. The egg shell sterilized with 70 per centethanol is broken into pieces and transferred into 50 ml of BSS. The vitelline membrane coveringthe blastoderm is removed and kept in Petri dish containing BSS. With the help of a forcep the

adherant vitelline membrane is removed. The embryo is observed by using a microscope so thatthe developmental stage of blastoderm could be found out. The blastoderm is placed on the

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medium in watch glass placed on sterile adsorbent cotton wool pad in Petri dishes. Excess of BSSis removed from medium and embryo culture of chick is incubated at 37.5C for furtherdevelopment.

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality

rate in the United States1. The cell type of origin for ovarian cancers is still in question and might

be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube

fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model

specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively

determining the cell type of origin. This will allow development of more accurate biomarkers,

animal models with tissue-specific gene changes, and better prevention strategies targeted to

this disease.

Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their

three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules.

By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures

from a single organ can be generated, increasing the number of experiments from a single

animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which isassociated with ovarian cancer formation. Cell types such as the OSE that do not grow well on

plastic surfaces can be cultured using this method and facilitate investigation into normal cellular

processes or the earliest events in cancer formation4.

Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a

linear polysaccharide composed of repeating units of -D-mannuronic acid and -L-guluronic

acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not

damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate

provides mechanical support for tissues; however, proteins are not reactive with the alginate

matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate

cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the

architecture of the tissue growth in vitro.

A method is presented for the preparation, separation, and embedding of ovarian and oviductal

organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks.

The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture

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is also described. Finally, the growth of primary cell types is possible without genetic

immortalization from mice and permits investigators to use knockout and transgenic mice.