Chemistry: molecular biology and microbiology – Spore forming or isolating process
Patent
1982-11-10
1985-07-23
Warden, Robert J.
Chemistry: molecular biology and microbiology
Spore forming or isolating process
435285, 435297, C12N 500, C12M 304, C12M 122
Patent
active
045309078
DESCRIPTION:
BRIEF SUMMARY
The invention relates to a method of producing stable perfusion-cultivations of animal cells in a specially designed system, thereby determining toxicity, and apparatus for performing the method.
Requirements for improved working environment, combined with the rapidly increasing number of chemical substances in working life and in society in general has increased the need for toxicological examination. Such examination is normally performed by means of animal experiments which, however, are expensive, time-consuming and require considerable resources. The determination of acute toxicity (LD.sub.50 values) on all the substances in question requires such vast resources that it is in practice difficult to achieve. The objection can also be raised that these experiments constitute cruelty to animals.
Attempts have been made to achieve mathematical models and store information in computers as to how an organ or a system of organs reacts in various situations, the computer subsequently simulating the toxic action of the substances tested, on the basis of this information. In biochemical models, the action of the chemical substance is tested on isolated biologically active substances, i.e. substances such as enzymes which control the chemical process in plants and animals which is the basis of all life. However, these mathematical and chemical models are uncertain and it is difficult to obtain reliable values of acute toxicity with them. The microbiological methods (Ames tests) are mostly used to determine the mutagenity of the substances, and thus their ability to cause cancer.
A simpler and less expensive method is thus required for determining the toxicity, and such a method has been effected according to the invention by means of stable perfusion-cultivations of animal cells in a specially designed apparatus giving laminar flow of the substrate.
Cultivation of cell cultures is performed by keeping isolated cells from animals and humans alive and cultivating them outside the body. The most usual type of cultivation is when the cells are placed in a dish or glass or plastic and surrounded by a substrate similar in composition to the body liquids. A test substance can be added to the cultivation substrate and changes in the cells be measured. The concentration of the substance which gives a certain defined alteration (in appearance, growth rate, size, O.sub.2 absorption, enzyme activity, or ratio between living and dead cells, for instance) constitutes a gauge of the toxic activity in the cell culture. A great advantage with cell cultures is that they enable tests on human material since cultures can be started from cells in blood samples, amniotic fluid or surgically removed tissues, for instance.
When a cell culture is used for studying biochemical processes, it is important that cultivation occurs in a well defined environment. Perfusion cultivation enables this, providing the cultivation equipment used allows a uniform supply of nutrient. The flow properties of the cultivation chamber are important.
If it were possible to keep the perfusion cultivations stable, they could be used for fundamental biochemical studies of various animal cells, in medical research and for determining acute toxicity, for instance. To enable cell cultures to be used for following up biochemical processes, the cultivation must take place under extremely stable conditions and so far it has not been possible to achieve this in a satisfactory manner.
According to the invention, stable perfusion cultures are achieved by means of a specially designed apparatus which gives an extremely uniformly, substantially laminar flow. In the following the method and apparatus will be described with reference to seven drawings in which
FIGS. 1A and 1B are perspective views of a perfusion device according to the invention;
FIG. 1C is a schematic cross-sectional view of a perfusion device according to the invention.
FIG. 2 shows how the apparatus is connected,
FIG. 3 shows a schematic view of oxygen-measuring equipment,
FIG. 4 shows examples of physica
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Peterson Anders
Walum Erik
Deck Randall E.
Warden Robert J.
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