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 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 high amount of Oxygen (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:

1. 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 the 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.

2. Organ culture on Agar:

Solidified culture medium with agar is also used for organ culture. The nutrient agar media 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.

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 or 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. Foe embryo culture a suitable medium prepared into watch glasses which are then placed on moist absorbent cotton wool pad in Petri dishes. For the culture of chick embryo, egg are incubated at 38 degree Celsius for 40-42 h so that a dozen of embryos could be produced. The egg shell sterlized with 70 percent ethanol is broken into pieces and transferred into 50 ml of BSS. The vetelline 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 in sterile absorbent cotton wool pad in Petri dishes. Excess of BSS is removed from medium and embryo culture of chick is incubated at 37.5 Degree Celsius for further development.

Valuable products from cell culture

From cultured animal cell several valuable products such as human monoclonal antibodies, and biochemicals can be produced on a large scale. Several million dollors have been earned from this industry in Europe, America, Africa, Japan and India. This industry has better future. More interestingly, the genetically engineered cells have revolutionized the cell culture industry. Several specific promoters of human origin are utilized for high expression of foreign genes.

For large scale production of certain biochemicals, the genetically engineered baculovirus-infected animals cells are also in use in a bioreactor. To fulfil the process several 'perfusion systems' have been developed that retain the cells in the bio reactor at the time of replacement of conditioned medium with fresh medium. This results in increase in cell density and in turn cell productivity. For commercial production of products a large scale cell density and in turn cell productivity. For commercial production of products a large scale cell culture system and scaling up of process are required. Therefore, 'master cell banks' (MCBs) are established to meet out demand. The MCBs are used to develop master working cell bank (MWCB) which meets the demand of production system. After several sub culturing, the MWCB is regularly checked for any kind of changes occurring in cells. Thus, the large scale cultures are the sources of all valuable products which are produced in a bio reactor.

Some of the important products which are produced from animal cell cultures are:

i. Enzymes (asperagenase, collagenase, urokinase, pepsin, hyaluronidase, rennin, trypsin, tyrosin, hydroxylase),

ii. Hormones (leutinizng hormone, follicle stimulating hormone, chorionic hormone and erythropoietin),

iii. Vaccines (foot and mouth disease vaccine, vaccines for influenza, measles and mumphs, rubella and rabies),

iv. Monoclonal antibodies

v. Interferons, etc.

Tolbert et al (1982) got success in producing large quantities of human interleukin-2 or T-cell growth factor by culturing a permanent lymphoblastoid T-cell line in a large batch suspension culture in a bio reactor.

Cultivation of Animal Cells En Masse in Bioreactor:

The cells are cultivated in two different phases as freely suspended cells in liquid phase and immobilized cells on solid phase.

Suspension culture:

In suspension culture the cells are dispersed in liquid medium and grow freely but not attached with any solid. The medium is agitated so that they should not form sediments. While adherent cells are permitted to get attached on solid support and they grow as immobilized culture. Therefore, it depends on choice of cells whether they are capable of growing freely in suspension or as immobilized culture (Anon, 1988)

A fully automated bioreactor is used that maintains the physicochemical and biological factors to optimum level and maintains freely the suspended cells in an agitated low viscous liquid medium. The most suitable bioreactor used is a compact-loop bioreactor that consists of marine impellers. It also relies on the integrated monitoring and control of physical, chemical, biological and biochemical parameters.

As compared to bacteria, animal cells grow slowly. This results in bringing the cells in unfavorable metabolic state due to even small changes in culture. The main carbon and energy sources are glucose and glutamine. Lactate and ammonia are their metabolic products that affect growth and productivity of cells. Therefore, on-line monitoring of glucose, glutamate and ammonia can be carried out by on-line flow injection analysis (FIA) by using gas chromatography (GC), high performance liquid chromatography (HPLC), etc.

Moreover, cell death also occurs due to certain reasons. Therefore, for measuring differences between viable and dead cells some enzyme activities are measured and correlated with cell density. Lactase dehydrogenase is used to distinguish cell density of viable cells (Fiechter, 1996).