Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1991-11-01
1993-11-23
Kepplinger, Esther L.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 30, 435 711, 43524023, 435285, 435297, 435300, G01N 33569, C12Q 124, C12N 104, C12M 118
Patent
active
052643441
DESCRIPTION:
BRIEF SUMMARY
This invention relates to cell culture.
It is to be understood that the terms "growth" and "proliferation" when applied to cells are synonymous for the purposes of this disclosure.
Both prokaryotic and eukaryotic cells are capable of producing materials of immense pharmaceutical and industrial value. These materials may be monoclonal antibodies produced by eukaryotic hybridoma cells, or products of genes of eukaryotic origin expressed in prokaryotic cells and secreted or otherwise obtained therefrom, or indeed products of prokaryotic origin, properly expressed by and secreted from such organisms without the intervention of manipulative recombinant nucleic acid methodology.
The metabolic state of such cells is important in determining the production and excretion of such products.
For example, many common micro-organisms generally only produce and secrete such products when they have been induced to enter a phase of growth-arrest, which may be elicited by, amongst other things, nutrient depletion, competition with other micro-organisms either for nutrients or oxygen, environmental changes in the microbial habitat or by the production of anti-metabolites from a different strain of micro-organism growing in the vicinity.
Examples of products which growth-arrested micro-organisms produce are antibiotics, anti-tumour agents such as mitomycin C, citrates, and heat stable enzymes which can, for example, be used in washing powders.
Initial screening procedures for cultures of micro-organisms which are potentially capable of producing such products may involve inoculating a suitable growth-medium with a crude suspension of, for example, soil. The growth medium is incubated under appropriate conditions and mixed cultures of micro-organisms are obtained. Unless these are induced to enter growth-arrest they generally will not produce, or at least excrete, appreciable quantities of useful microbial products. Moreover, in such screening procedures, a considerable quantity of growth-medium and a large number of culture vessels can be wasted through colonies not being growth-arrested and thus over-growing themselves, or over-growing nearby colonies.
Conventionally, colonies of micro-organisms, including mixed colonies containing a number of strains which additionally may have different growth rates on the same medium, reach growth-arrest slowly, but once arrested yield a dense heterogeneous mass from which it is difficult, if not impossible, to identify strains of micro-organisms of interest by use of an in situ screening procedure.
According to the present invention there is provided a method of culturing cells which produce products encouraging the cells to produce the products and identifying the thereby produced products, characterised in that the cells are cultured on the surface of a quantity of growth medium, which surface has been divided into a plurality of growth areas thereby limiting the amount of growth-medium which is available to the cells so that these reach a metabolic stage in which said products are produced, and identifying the thereby produced products by an in situ screening procedure.
The growth medium available to the cells may be limited by bounding the area of surface of growth medium available to the cells.
The area of surface of growth medium may be bounded by a membrane in contact with the surface.
The membrane may comprise a plastic film material which may be substantially impermeable to oxygen.
The plastics film material may be polyethene.
The membrane may, however, comprise a metallic material.
The membrane may have a plurality of spaced apart apertures defining areas of growth medium surface on which the cells can be grown.
The membrane may comprise a backing-layer which serves to rigidify same.
The membrane may be multilaminate in which a plurality of membranes may be superimposed upon and bonded to a semipermeable sheet. Said sheet may itself be multilaminate and may be impermeable to proteins and molecules of like-size.
The membrane may be adapted to be retained in a Petri dish or any o
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Kepplinger Esther L.
Parsons Nancy J.
University of Leicester
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