SOMATIC CELL HYBRIDIZATION

It has been known since the 60's that somatic cells from the same or different species in culture could spontaneously fuse to form polyploid cells. The product of fusion was called Homokaryon if the two parental cells come from the same species, and Heterokaryon or Somatic Cell Hybrid if the fusion was site-specific.


The hybrid cells could divide by mitosis and proliferate and thus could be maintained in culture. Cell fusion is followed by nuclear fusion to produce uninucleate hybrid cells or Synkaryons. The rate of cell fusion is very low; about one cell fuses in million of cells.


In 1962 Okada discovered that inactivated Sandai virus could greatly increase the rate of cell fusions. Since then several agents causing cell fusion has been tried among which Polyethylene Glycol (PEG) has some advantages. The exact mechanism of cell fusion is not known. In case of UV-inactivated Sandai virus, it seems that the virus absorbs to the cell surface leading to agglutination of cells. The protein coat of virus forms the connecting bridge between the cells. The membrane of two cells swells into this region and when they come in contact they get dissolved. The cell contents mix up, the nuclei fuse and a Heterokaryon is formed. When cell fusion is mediated by Polyethylene Glycol the two cell membranes directly come in contact.

Basic Hybridoma Technology

The term Hybridoma is applied to fused cells resulting due to fusion of following two types of cells: An antibody producing lymphocyte cell e.g. spleen cells of mouse. Myeloma (cancer) cells which are capable of multiplying indefinitely. These fused hybrid cells or hybridoma have the antibody producing capability inherited from lymphocytes and have the ability to grow continuously like cancer cells. Following steps are involved in the production of monoclonal antibodies (specific antibody for specific antigen).

A mouse is immunized to a specific antigen for the production of specific antibody. Tumor is produced in a mouse (it is done by injecting tumor causing antigens). From the above two types of animals, spleen cells (that produces antibodies) and Myeloma cells (that produces tumor) are isolated and cultured separately.

The Myeloma cell that is cultured is unusual in two ways, first it has stopped synthesizing antibodies and its mutant called HGPRT­- that cannot synthesize the enzyme Hypoxanthine Guanine Phosphoribosyl transferase. Now the fusion of the spleen cells to Myeloma cells is induced by using Polyethylene Glycol to produce hybridoma. The hybrid cells are grown in selective Hypoxanthine aminopterin thymidine medium (HAT).

HAT contains a drug Aminopterin which blocks one pathway for nucleotide synthesis making the cells to depend upon other pathway that needs HGPRT enzyme, which is absent in Myeloma cells. Therefore, Myeloma cells which do not fuse with the B cells will die, since they are HGPRT-. B cells which do not fuse with the Myeloma cells will die, because they lack tumorgenic property of immortal growth. Therefore HAT medium allows selection of Hybridoma cells which inherit HGPRT gene from B cells and tumorgenic property from Myeloma cells.

Now the selection of hybridoma cells is done for cloning and antibody productions. This is facilitated by preparing single cell colonies that produces antibodies. Such antibodies are called Monoclonal antibodies. The hybridoma cells are kept stored at low temperature for future use in the production of monoclonal antibodies.