Fused hybrid cell

Mammalian Cells Unlike microbial cells, mammalian cells do not continue to reproduce forever. Cancerous cells have lost this natural timing that leads to death after a few dozen generations and continue to multiply indefinitely. Hybridoma cells from the fusion of two mammalian lymphoid cells, one cancerous and the other normal, are important for mammalian cell culture. They produce monoclonal antibodies for research, for affinity methods for biological separations, and for analyses used in the diagnosis and treatment of some diseases. However, the frequency of fusion is low. If the unfused cells are not killed, the myelomas 1 overgrow the hybrid cells. The myelomas can be isolated when there is a defect in their production of enzymes involved in nucleotide synthesis. Mammahan cells can produce the necessary enzymes and thus so can the fused cells. When the cells are placed in a medium in which the enzymes are necessaiy for survival, the myelomas will not survive. The unfused normal cells will die because of their limited life span. Thus, after a period of time, the hybridomas will be the only cells left ahve. 

Figure 10.3. Schematic representation of monoclonal antibody production using immortalized hybrid cells that secrete antibodies selective for the target antigen. The mortal, immune B cells Isolated from mice immunized with a target antigen are fused with myeloma, immortal B cells that express defective antibodies. The selecting of antigen-specific, immortal hybrid cells (hybridomas) results in identification of unique clones of cells that express antibodies with high specificity and affinity (monoclonal antibodies). These cells are cloned and expanded for large-scale monoclonal antibody preparations.

The development of hybridoma technology by Milstein and Kohler in 1975 revolutionized the antibody field and radically increased the purity and specificity of antibodies used in the clinic and for diagnostic tests in the laboratory. Hybridomas consist of antibody-forming cells fused to immortal plasmacytoma cells. Hybrid cells that are stable and produce the required antibody can be subcloned for mass culture for antibody production. Large-scale fermentation facilities are now used for this purpose in the pharmaceutical industry. 

Fusion with the cells compensates for this deficiency. When fused and unfused cells are incubated in the presence of the folic acid antagonist aminopterin, the de novo synthesis of purines and pyrimidines for DNA is blocked. Cells deficient in HGPRT die, whereas hybrid cells are able to bypass aminopterin blockage by metabolism of hypoxanthine and thymidine added to the medium. In the generation of mouse hybridomas, an number of myelomas deficient in HGPRT are available, all originating from MOPC 21, a spontaneous myeloma from the BALB/c mouse strain. 

Cell Fusion Unlike antibody-secreting cells, myeloma cells, malignant tumor cells of the immune system, can be cultured continuously. Kohler and Milstein (1975) developed a method to fuse (hybridize) B-lymphocytes from the mouse spleen with mouse myeloma cells, so that the fused cell, hybrid-myeloma (or hybridoma) cell, can have the characteristic of the both cell lines that is, the production of specific antibodies and the immortality. Since the hybridoma is derived from a single B-lymphocyte, it produces only one kind of antibody, thus a monoclonal antibody. 

Once inter-species fusion was shown to be possible, the next step was an attempt to fuse myelomas with normal B lymphocytes. Only in 1975 did Kohler and Milstein propose a protocol that led to the efficient production of hybrid cells for the secretion of mAbs with a predetermined specificity, which could be perpetuated in cell cultures, the so-called hybridomas (Kohler, 1981). 

Monoclonal antibodies are produced by hybrid cells created from B lymphocytes fused with immortal B lymphocyte tumor cells. The resulting hybrids can be individually cloned, and each clone will produce antibodies directed against a single antigen type. Two new monoclonal antibody types have been created that are useful in the treatment of cancer. 

EBV), or by fusing it with another immortalized cell. The latter approach is called hybridoma technology because it involves fusion of two cells to produce a hybrid cell. This is the approach used most widely for the production of mouse monoclonal antibodies. 

The basic principle of hybridoma technology is shown in Fig. 2.23. B cells obtained from the spleens of mice immunized with the antigen of interest are fused with estabhshed mouse myeloma cells. Myeloma cells are transformed cancerous cells of the B lymphocyte lineage with infinite growth capacity. While many myeloma cell hnes continue to secrete their antibody, others lose the ability to produce antibodies. Spleen cells from immunized animals are fused with non-antibody-producing myeloma cells, resulting in a hybrid cell which inherits the properties of both the fusion partners. Thus the hybrids are capable of continued growth in culture like the myeloma cell and also maintain the antibody production of the spleen B cell. 

In 1973, Cotton et al. successfully fused cells of two plasmacytoma lines to produce hybrid cells capable of synthesizing both myeloma proteins. Subsequently, hybrid cells derived by fusion of a murine myeloma with spleen cells from appropriately immunized donors were shown to se-... 

Hybridomas (or fused cell culture lines) are produced by fusing lymphocytes isolated from the spleen of an immunized animal with the HPRT deficient malignant plasma cells. The lymphocytes. The unfused cells die, since spleen cells survive only a few days, while the malignant plasma cells lack HPRT. Fused cells, however, are HPRT positive as well as immortal these hybridomas, or hybrid cells, retain the Ab-producing characteristics of the spleen cells. Hybrid cell lines that produce a specific antibody are then cloned from the single cells and cultured. Each clone created in this manner produces antibodies of a single epitope specificity ... 

Electrofusion has been successful in all types of cells tested to date, including microbe and plant protoplasts, mammalian cells, and sea urchin ova. One can (a) fuse unlike cells to create hybrid cells (b) fuse like cells to form larger entities such as giant cells 100 to 1000 times the volume of individual unit cells and (c) help drive external objects or chemical agents such as DNA into cells. 

A EXPERIMENTAL FIGURE 12-22 The movement of human hnRNP Al protein between nuclei in a heterokaryon shows that it can cycle in and out of the cytoplasm, but human hnRNP C protein, which showed no such movement, cannot. Cultured HeLa cells and Xenopus cells were fused by treatment with polyethylene glycol, producing heterokaryons containing nuclei from each cell type. The hybrid cells were treated with cycloheximide Immediately after fusion to prevent protein synthesis. After 2 hours, the cells were fixed and stained with fluorescent-labeled antibodies specific for human hnRNP C and Al proteins. These antibodies do not bind to the homologous Xenopus proteins, (a) A fixed preparation viewed by phase-contrast microscopy Includes unfused HeLa cells (arrowhead) and Xenopus cells (dotted arrow), as well as fused heterokaryons... 

An alternative procedure is to isolate the B-cells, fuse them with a tumor line (myeloma), and separate these hybrid cells into individual clones, to produce antibody producing hybrids called hybridomas [10-12]. These separate cell lines can be screened to select a clone that produces an antibody with a single structure, called a monoclonal antibody. For analytical purposes, one advantage of a monoclonal antibody is that the cell line can be preserved so that a consistent source of antibody can be obtained. Again, usually researchers prepare their own monoclonals or obtain cell lines from other investigators who have published reports about the use of their monoclonals. Many researchers deposit their cultures with the American Type Culture Collection, Rockville, MD. 

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