Chemistry: electrical and wave energy – Processes and products – Electrophoresis or electro-osmosis processes and electrolyte...
Patent
1994-04-05
1996-12-31
Niebling, John
Chemistry: electrical and wave energy
Processes and products
Electrophoresis or electro-osmosis processes and electrolyte...
204547, 204554, 4351734, 4351735, 4351736, C25B 700
Patent
active
055890474
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to a method for the selective recovery of one or more cells expressing or bearing a defined bio-synthesised target molecule (a ligand); a method for the separation of cells of different types; and, independently but concomitantly, to a method for fusing just two such individual cells or for fusing such a cell and a vector to form one single "fusate cell". The fusion method is particularly applicable to the making of hybridomas, that is, the mammalian hybrid cells which produce monoclonal antibodies, and to other mammalian cell and plant cell constructs.
PRIOR ART
Hybridomas are binary hybrid cells made from the fusion of antibody secreting normal cells, for example B lymphocyte cells, with immortal cells which are in practice mostly non- secretors of antibody, usually a myeloma cell line. The secreting cells can be obtained by various means from an immunised animal, whilst the tumour line is grown in culture. The effect and purpose of the fusions is to produce numbers of different immortal hybrid cells each of which retains the genes for and property of producing and secreting a defined bio-synthesised target molecule such as a monoclonal antibody, specific to just one epitope of the antigen used in the immunisation. Although the commonest method of hybridoma generation involves direct bulk fusion of cells of the two types, immortalised antibody secreting cells have been produced by more exotic and more hazardous methods, such as by fusion of human B cells in the presence of transforming virus particles; this approach was developed because the direct translation of the method developed for mice cells to the human cell case had been largely fruitless. Such immortalised cells are also called hybridomas, though the usage is loose, since unlike the normal cell-myeloma cell hybridomas, the exact aetiology of these transformed cells is unknown.
Current hybridoma production methods involve a series of distinct steps (the method detailed is that used for the mouse): previously immunised to the antigen for which an antibody is being sought. Some enrichment methods may be used, post collection, to enhance the relative proportion of B-lymphocytes in the total cell count. myeloma cells and fusion between cells in the mixture is promoted by: these is used.
The short exposure to electrical fields leads to electroporation and electrofusion. These phenomena are described in, for instance, U.S. Pat. Nos. 4,441,972 and 4,832,814. fused. The fusion process produces fused cells (Fusates) resulting from single and multiple fusions of myeloma-myeloma, lymphocyte-lymphocyte and myeloma-lymphocyte partners.
If a suitable immortal fusion partner cell is chosen the myeloma-myeloma fusates can be eliminated by culturing in a special growth medium, typically a normal medium supplemented with Hypoxanthine, Aminopterin and Thymidine, ("HAT") in which these fusates cannot survive.
Normal Lymphocyte-normal lymphocyte fusates survive, and at most undergo only a limited number of cell divisions.
Some of the myeloma-lymphocyte fusates may remain viable after extended culture; these are the hybridomas--but they may not necessarily be hybridomas of interest, in that they do not necessarily make the defined bio-synthesised target molecule. of antibodies and in particular antibodies specific for the original antigen of interest. If this procedure, which can be long and laborious, reveals a "clonal" fusate which produces the required antibody, the cell can be grown in culture to manufacture "Monoclonal Antibodies" in quantity. re-cloning over an extended period of time.
The methodology employed to immortalise B cells as hybridomas has also been used to immortalise other distinct normal cell types as cell lines which secrete some defined bio-synthesised target molecule (for example, the human and mouse Interleukins) or perform some function (for example T-cell effector function or phagocytic activities) of clinical or commercial interest.
Present methods of generating cells which secrete th
REFERENCES:
patent: 4018662 (1977-04-01), Ruhenstroth-Bauer et al.
patent: 4441972 (1984-04-01), Pohl
patent: 4561961 (1985-12-01), Hofmann
patent: 4970154 (1990-11-01), Chang
patent: 5001056 (1991-03-01), Snyder et al.
Petrucci, General Chemistry: Principles and Modern Applications, 4th ed., p. 621, 1985 (no month).
Zimmermann et al., Electric Field-Induced Cell-to-Cell Fusion, The Journal of Membrane Biology, vol. 67, pp. 165-182 (1982) [no month].
Ashcroft Robert G.
Coster Hans G. L.
Mahaworasilpa Tohsak
Fucell Pty Limited
Niebling John
Wong Edna
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