Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Method of regulating cell metabolism or physiology
Patent
1997-03-21
2000-11-14
Wityshyn, Michael G.
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Method of regulating cell metabolism or physiology
435 2, 435325, 435372, 435378, 435408, C12N 500, C12N 502, C12N 508
Patent
active
061468908
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a method and system for cultivating cells. The method and system are particularly useful for growing macrophages from human blood, and are therefore described below with respect to this application.
BACKGROUND OF THE INVENTION
Macrophages, namely phagocytic cells phagocytize and digest old and deteriorated red blood cells, bacteria and other microscopic particles. They have been found to play an important role in wound repair. They produce substances that stimulate proliferation of fibroblasts, the synthesis of collagen by fibroblasts, and other elements that are necessary for wound healing. For example, it was found that wound repair could be accelerated in old mice by application of the wounds of macrophages derived from peritoneal fluids of young mice (D. Danon, M. A. Kowatch and G. S. Roth, Proc. Natl. Acad. Sci. USA, Vol.86, pp.2018-2020, March 1989).
The present methods of preparing macrophages out of blood monocytes are complicated, expensive, time-consuming and difficult to apply routinely, because they require considerable specialized labour, expensive disposable materials, and specialized laboratory facilities. Moreover, the present techniques involve a significant risk of contamination during preparation and therefore require regular testing to assure the absence of bacterial infection in the resulting suspension of macrophages.
SUMMARY OF THE INVENTION
An object of the present invention is to provide a method and system for growing cells, particularly, macrophages, which have advantages in the above respects. More particularly, an object of the invention is to provide a method and system for growing cells, particularly macrophages, which do not require specialized labour, expensive disposable materials, or a specialized laboratory, and which reduce considerably the risk of infection.
According to one aspect of the present invention, there is provided a method of cultivating cells present in suspension by subjecting the cells to at least one sterile reagent and cultivating the cells in a sterile culture medium, comprising: placing the cells in suspension, the at least one reagent, and the culture medium in three separate sterile containers; connecting together the containers, and hermetically sealing their contents from the atmosphere, by sterile tubings having fluid flow control devices which may be opened and closed; and opening and closing the fluid flow control devices as required in order to transfer the contents of one container to another, while isolating the container contents from the external environment.
The invention is particularly useful for cultivating macrophages from blood.
Since the complete process can thus be carried out in a completely controlled atmosphere hermetically sealed from the outside atmosphere, there is little risk of infection, and therefore no need for special sterile hoods, laminary flows, and sterile disposable tools. Testing to assure the absence of bacterial infection is not crucial. In fact, not one case of infection was found in 150 preparations of macrophages in accordance with the method of the present invention where every second or third preparation was tested.
According to another aspect of the present invention, there is provided a method of cultivating macrophages, comprising: separating from an initial quantity of blood, a white blood cell fraction which includes a mixture of all kinds of white blood cells and red blood cells having usually a much higher concentration of white blood cells than in the initial quantity of blood; subjecting the white blood cell fraction (buffy coat) to an osmotic shock which is more destructive of red blood cells than white blood cells; re-establishing iso-tonicity in the white blood cell fraction; adding the white blood cell fraction to a culture medium in a container; and incubating the culture medium with the white blood cells fraction.
It has been found that subjecting the white blood cell fraction to osmotic shock and then re-establishing isotonicity therein destroys substan
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Kerr Janet M.
Wityshyn Michael G.
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