How have cancer cells been helpful? Hybridoma and the immortal B-cell.

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Hybridoma and the immortal B-cell.

Most people associate cancer with disease or illness that results in death, however our understanding of cancer has allowed us to do quite a bit. The definition of a cancerous cell is a non-stop proliferating cell trapped in a cycle of unlimited growth and constantly dividing itself. One way we have hijacked the abilities of these cells is in our production of monoclonal antibodies. Antibodies are like the ammunition of the immune system and are produced by activated B-cells to bind invading pathogens and target them for destruction via macrophages. However the activated B-cells that produce antibodies are short-lived and can only exist for only a few weeks while they fight infections and die off. What we call immunity exist because special B-cells called memory B-cells that can live for years and be ready to generate a new slew of antibody producing effector B-cells.

So how do immunologists generate bulk amounts of antibodies usable in clinical cures or antibodies to tag proteins with fluorescence? The answer is simply to fuse a short-lived B-cell with a cancerous cell to generate an immortal B-cell: aka the hybridoma. Fusion of the two cells into one seems like something from science fiction, but scientists have mastered this technique to generate any antibody of interest in unlimited quantities.

typical hybridoma fusion

How is this done exactly, here is one example of a typical hybridoma fusion. The first step involves immunizing an animal with the protein of interest we want to produce antibodies for, then allowing time for an immune response and for activated B-cells to develop. The next step involves dissecting the animal and removing the spleen because it is a secondary organ of the immune system with the highest quantity of B-cells. White blood cells are then isolated from other cells, spleen cell tissue, macrophages, red blood cells, etc. Myeloma cells are non-antibody producing continuously dividing cancer cells that will be used for the fusion. The cell line lacks the enzyme hypoxanthine-guanine phosphoribosyl transferase (HGPRT-) required for the salvage pathway of nucleotide biosynthesis. Thus these cells cannot grow on a growth hypoxanthine-aminopterin thymidine (HAT) medium because the salvage pathway is forced and blocks the normal biosynthetic pathways for purines and pyrimidines necessary for DNA replication. The B-cells derived from the spleen do have this enzyme and can grow on the HAT medium. The actual fusion of these cells is a sensitive procedure in which the B-cells and myeloma cells are centrifuged together to generate a good cell-to-cell contact and then mixed with polyethylene glycol (PEG). PEG disrupts the membrane of cells allowing them to become leaky and instigates potential fusions. The then fused cells are plated in the HAT medium for selection of fused cells. After time the short lived B-cells will eventually die and the unfused myeloma cells die because they are HGPRT-, however the successful fusions survive indefinitely in this medium producing antibodies of interest. We can mass culture our new fused hybridoma cells and generate all the antibodies we need.
Resources:
Goldsby, R.A., Immunology, 6th ed., Chapter 4, Antibodies: “Structure and Function”, pp. 105-107
Milstein, C. Scientific American 243 (4): 66 October 1980
Mishell, B. B., and Shiigi, S. M.Selected Methods in Cellular Immunology Chapter 17, 1980

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