Chemistry: molecular biology and microbiology – Apparatus – Including measuring or testing
Patent
1994-07-18
1996-11-26
Redding, David A.
Chemistry: molecular biology and microbiology
Apparatus
Including measuring or testing
4353052, 4353053, 422101, 422102, C12M 302
Patent
active
055784909
DESCRIPTION:
BRIEF SUMMARY
DESCRIPTION
A cell culture plate with a system for lateral diffusion of molecules across a barrier membrane.
In the course of biological experimentation, it is frequently necessary to culture one or several cell populations at high cell density and under low oxygen concentration. In such circumstances it is necessary to use special techniques, with the aim of supplying metabolically active cells with the necessary nutrients and of maintaining pH within physiological limits.
To date, these objectives have been achieved either by daily supplementation of cultures with fresh nutrients and with an alkaline substance which corrects the excess acidity of the culture (procedure A), or by the use of special culture plates in which the cells are cultured on a permeable membrane, the cell metabolites passing across this membrane into a reservoir of culture medium (procedure B).
The two procedures described have two major disadvantages:
Under procedure A, the metabolites produced by the cells are not removed, and thus may accumulate to levels which are toxic for the cultured cells. The procedure is very time consuming, and the need for daily handling of cultures increases the risk of contamination. The daily addition of an alkaline substance in order to correct pH leads to abrupt fluctuations in this parameter, and this may prove harmful to the cultured cells.
Under procedure B, the need to culture cells on a permeable membrane makes it difficult or impossible a) to harvest the cells (particularly cells that adhere to the membrane), b) to examine the cells under a microscope (since observation must be across both the culture medium reservoir and the membrane), and c) to carry out multistage cultures (eg. in situations in which it is necessary to maintain the cells in contact with a given substance for a given period of time).
In the culture plate 10 fitted with a lateral diffusion system (FIG. 1), two flat-bottomed open-topped wells, denominated the reservoir well 12 and the diffusion well 14, communicate via two slits 16, 18 situated in the lateral wall/s 20 of the diffusion well 14. Each of these slits 16,18 is covered by a semipermeable membrane 22 which permits the transfer of soluble substances, but not of insoluble substances or cells, between the two wells. Transfer of cell metabolic products from the diffusion well to the reservoir well, and of culture medium from the reservoir well to the diffusion well, may thus occur. In existing apparatuses based on basal diffusion, in which substances diffuse across a semipermeable membrane between an upper compartment (typically that containing the cells) and a lower compartment, both microscopic observation and cell handling are very difficult: these problems are overcome by the present invention. The present invention has two additional advantages over existing apparatuses based on basal diffusion. Firstly, the surface tension at the outside of the membrane 22 located in the lateral walls 20 of the diffusion well 14 of the present invention compensates for the hydrostatic pressure on the inside, preventing leakage of liquid from the diffusion well 14 when the reservoir well 12 is empty. This allows cells to be incubated with soluble substances for a given period, during which diffusion to the reservoir is not permitted. Secondly, in the present invention, in which the cultured cells are layered upon the plastic base of the diffusion well, the diffusion of cell metabolites and other soluble substances from the diffusion well to the reservoir well occurs more slowly than in existing apparatuses based upon basal diffusion, in which the semipermeable membrane is larger in area and in which the basal surface of the cell layer rests directly upon the membrane. In the present invention, therefore, the diffusion of substances from the diffusion well 14 to the reservoir well 12 occurs sufficiently rapidly to ensure the maintenance of physiological conditions but sufficiently slowly to delay the removal of substances necessary for cell growth or differentiation, such as growth
REFERENCES:
patent: 3221878 (1965-12-01), Brett
patent: 3985608 (1976-10-01), Saxholm
patent: 4636473 (1987-01-01), Kleinstreuer
patent: 4975377 (1990-12-01), Key
Redding David A.
Universidade de Santiago de Compostela
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