Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
1999-03-04
2001-07-24
Naff, David M. (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S029000, C435S041000, C435S069100, C435S071100, C435S134000, C435S325000, C435S375000, C435S377000, C435S404000, C435S405000, C435S243000, C435S244000
Reexamination Certificate
active
06265180
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to methods for determining the biocompability of lipophilic substances in cell culture test, and to nanoemulsions suitable in said methods.
BACKGROUND OF THE INVENTION
Nanoemulsions, alternatively called nanoparticles, are composed of oil particles, the surfaces of which are occupied by an amphoteric emulsifier in aqueous dispersions. They can be prepared by mixing triglycerides or fatty acid esters in aqueous phase using a high pressure homogenizer (EP-B1-0 406 162—H. G. Weder).
So far, they were used for the manufacture of pharmaceutical and/or cosmetic preparations, and also for the preparation of nutrient solutions in which the nanoemulsions serve as the energy supplier in cell cultures (EP-B1-0 406 162—H. G. Weder).
The cell culture technique is a biological system having a very wide application. Controlled culturing of these cells is mainly used in immunology, in biotechnology, in toxicology, in gene technology, and in cell biology. In all said applications the interaction of substances with cells is of primary importance. The exchange of biochemical substances with the cells takes place in the culture medium which is composed of a large number of different substances. In order to create ideal conditions for the cells, in most cases blood serum is added to the culture medium. However, for many uses the complementation of said medium with blood serum shows severe disadvantages, since the blood serum has an undefined composition, is expensive, and may contain undesired components, such as viruses, prions and germs. Therefore, for many applications the creation of so-called serum-free media is of primary importance.
Working with cell cultures, a general difficulty is the application of test substances which are insoluble in water. If possible, lipophilic substances are dissolved in alcohol or dimethylsulfoxide, and then added to the culture medium. However, this results in undefined dispersions of said substances having a low bioavailability. The use of solubilizers, such as Tween 20®, results in unstable and very toxic emulsions.
Therefore, no reliable and simple methods were so far available to solve the following problems:
testing the biocompability of lipophilic substances in cell culture assays;
testing the toxicity and/or the genotoxicity of lipophilic substances in cell culture assays;
biotransformation of lipophilic substances in cell cultures;
promotion of cell differentiation in primary cell cultures;
separation of cells from lipids which were not incorporated;
adaptation of cells from a complete medium to a serum-free medium;
completion of cell culture media with lipophilic substances promoting cell growth;
completion of cell culture media with lipophilic substances promoting biosynthesis of desired substances within the cells;
defined and reproducible transport of lipophilic substances into the cells of cell cultures.
OBJECTS OF THE INVENTION
It is the primary object of the invention to solve the above mentioned problems by creating methods for delivering lipophilic substances to cell cultures, which lipophilic substances are to be tested in said cell cultures, by using nanoemulsions as vehicle.
The foregoing and further objects, advantages and features will be apparent from the following specification.
SUMMARY OF THE INVENTION
To meet these and other objects, the invention provides the following methods, and the following dispersions suitable for use in these methods:
a method of delivering a lipophilic substance into the cells of a cell culture, in a defined and reproducible manner, said method comprising the steps of: (a) preparing a dispersion consisting of a nanoemulsion containing said lipophilic substance, in aqueous phase; and (b) adding said dispersion to said cell culture, and optionally the further step of: (c) separating said cells from lipids which were not incorporated, by centrifugation and washing;
a method of testing the biocompatibility of a lipophilic substance in a cell culture assay, said method comprising the steps of: (a) preparing a dispersion consisting of a nanoemulsion containing said lipophilic substance to be tested, in aqueous phase; and (b) adding said dispersion to said cell culture, said test preferably being made on lipids used in the manufacture of cosmetics;
a method of testing the toxicity and/or the genotoxicity of a lipophilic substance in a cell culture assay, said method comprising the steps of: (a) preparing a dispersion consisting of a nanoemulsion containing said lipophilic substance to be tested, in aqueous phase; and (b) adding said dispersion to said cell culture;
a method of biotransforming a lipophilic substance by means of a cell culture, said method comprising the steps of: (a) preparing a dispersion consisting of a nanoemulsion containing the substance to be biotransformed, in aqueous phase; (b) adding said dispersion to said cell culture; and (c) collecting the biotransformed substance from said cell culture;
a method of promoting cell differentiation in a primary cell culture, said method comprising the steps of: (a) preparing a dispersion consisting of a nanoemulsion containing a substance able to promote cell differentiation, in aqueous phase; and (b) adding said dispersion to said cell culture;
a method of completing a cell culture medium with a lipophilic substance promoting the growth of cells by weight, said method comprising the steps of: (a) preparing a dispersion consisting of a nanoemulsion containing the substance promoting the growth of cells, in aqueous phase; and (b) adding said dispersion to said cell culture;
a method of completing a cell culture medium with a lipophilic substance promoting the biosynthesis of desired substances within the cells, said method comprising the steps of: (a) preparing a dispersion consisting of a nanoemulsion containing the substance promoting said biosynthesis, in aqueous phase; and (b) adding said dispersion to said cell culture;
a sterile dispersion useful for delivering a lipophilic substance into the cells of a cell culture, said dispersion consisting of a nanoemulsion, in aqueous phase, the oily particles of said dispersion containing said lipophilic substance to be delivered, and the surface of said oily particles being occupied by a non-toxic amphoteric emulsifier, wherein said oily component in which said lipophilic substance is dissolved preferably has such a low toxicity for said the cell culture that its growth is reduced for less than 20% as compared to a reference culture containing no oily component;
a sterile dispersion useful for testing a lipophilic substance by means of a cell culture, said dispersion consisting of a nanoemulsion, in aqueous phase, the oily particles of said dispersion containing said lipophilic substance to be tested, and the surface of said oily particles being occupied by a non-toxic amphoteric emulsifier, wherein said oily component in which said lipophilic substance is dissolved preferably has such a low toxicity for said cell culture that its growth is reduced for less than 20% as compared to a reference culture containing no oily component.
Preferably, said dispersions comprise as oily component a triglyceride of the fatty acids C 18:1 (oleic acid) and/or C 18:2 (linoleic acid), and comprise nanoemulsions comprising from 0.65 to 0.75 parts by weight of said amphoteric emulsifier for one part by weight of said oily component.
REFERENCES:
patent: 5152923 (1992-10-01), Weder et al.
patent: 5658575 (1997-08-01), Ribier et al.
Suter Franz
Zuelli Fred
Mibelle AG Cosmetics
Naff David M.
Tarolli, Sundheim, Covell Tummino & Szabo L.L.P.
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