Chemistry: molecular biology and microbiology – Spore forming or isolating process
Patent
1989-12-11
1993-11-23
Wityshyn, Michael G.
Chemistry: molecular biology and microbiology
Spore forming or isolating process
435176, 435177, 435178, 435179, 435180, 435182, 435240243, 43524023, C12N 500, C12N 1102, C12N 1110, C12N 1114
Patent
active
052643590
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention relates to methods for the large scale cultivation of animal cells and for making supporting substrata for the cultivation.
BACKGROUND ART
Animal cells are classified into two categories: (1) anchorage-dependent cells, which grow or proliferate only after attachment to supporting substrata, and (2) anchorage-independent cells, which do not require attachment to supporting substrata and can grow even in a suspension state. The method for the large scale cultivation of anchorage-dependent cells differs from that for anchorage-independent cells in requiring a suitable supporting substrate for cell attachment. Various kinds of two- and three-dimensional culture methods, depending on differences in the supporting substrate structure or the substrate material used, have been proposed (Japanese national publication of translation of international application, showa 62-502936 (International publication number WO 86/05811), Cytodex (commercial name for a product from Pharmacia Fine Chemicals Co., Ltd.), and Japanese official patent provisional publication, showa 63-209581).
In the two-dimensional culture methods, animal cells attach to the surface of the supporting substrate and grow on the surface to form a monolayer of cells. A number of two-dimensional culture methods have been proposed, the purpose of which is to increase cell density of the cultured cells, enhance product secretion in cultured cells, simplify recovery and purification of cultivated products, and the like.
Some of the present inventors have proposed a large-scale cultivation method wherein anchorage-dependent animal cells are cultured three-dimensionally using collagen gels as supporting substrata (Japanese official patent provisional publication, showa 62-175172; Japanese official patent provisional publication, showa 62-171680; International publication number WO 87/04458; U.S. Ser. No. 110,749; and the European patent application, publication number 0258441). In this method, the cells are embedded in collagen gels and grow three-dimensionally in the gels. By using collagen, it is possible to make the in vitro surroundings very close to the in vivo surroundings. As such, the collagen gel culture method has made it possible to culture some kinds of cells which are unable to be cultured by monolayer culture methods and by three-dimensional culture methods using supporting substrata other than collagen. Therefore, compared to two-dimensional culture methods and methods using other three-dimensional substrata, the three-dimensional culture method using collagen gel is not only advantageous in that producibility arising from an increase in cell attachment area is increased, which is due to the fact that it is three-dimensional, and with respect to the simplicity of culturing due to cell fixation, but is also advantageous due to the increase in the kind of cells which may be cultured.
Considering these advantages, the present inventors have studied the development of a method for large-scale cultivation in which animal cells are embedded in collagen gels and cultured three-dimensionally. As a result of the present inventors' studies, it was found that this type of culture method has at least one problem among the following (1)-(3).
(1) If the collagen gel is soft, it is hard to handle and apt to be damaged.
(2) As the collagen gel shrinks with cell growth, long-term cultivation is impossible.
(3) While problems (1) and (2) may be solved when a collagen gel of high gel strength is used as a supporting substrate, if gel strength is high, new problems appear, such as a decrease in the cultivation rate of cells or a limitation of the kind of animal cells possible to be cultivated.
These problems not only adversely affect the convenience of the treatment, but also adversely affect cell growth.
Accordingly, the first object of the invention is to provide a method for the large-scale cultivation of animal cells wherein the collagen gels in which the animal cells are embedded are easily handled, shrinkage of the co
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Enami Junpei
Kondo Naohito
Suzuki Kaneo
Takano Toshikazu
Gitomer Ralph
Nitta Gelatin Inc.
Wityshyn Michael G.
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