Animal Cell, Tissue and Organ Culture

Organ Culture
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 culture. 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. Methods of culturing 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%). Special serum-free media (e.g.T8) and special apparatus (Towell's Type II culture chamber) are used for adult culture. In addition, the embryonic organs can be cultured by applying any of the following three methods:

Organ

Culture

on

Plasma

Clots

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 a 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.

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 from adults fail to survive on agar media, whereas embryonic organs grow well. The media consist of ingredients: agar (1% in basal salt solution), chick embryo extracts and horse serum in the ratio 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 organ culture, generally perforated metal gauze or cellulose acetate or a raft of lens paper is used. These possibility provides support.

Whole

Embryo

Culture

During 1950s, Spratt studied how metabolic inhibitors affect the development of embryo in vitro. Old embryo (40 h) was studied upto 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 38C for 40-42 h so that a dozon of embryos could be produced. The egg shell sterilized with 70 per cent ethanol is broken into pieces and transferred into 50 ml of BSS. The vitelline membrane covering the 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 that the developmental stage of blastoderm could be found out. The blastoderm is placed on the

medium in watch 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 37.5C for further development.

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 is associated 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

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.