Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of...
Reexamination Certificate
1998-11-10
2001-04-03
Lankford, Jr., Leon B. (Department: 1651)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
C435S383000, C435S384000, C435S404000, C435S405000
Reexamination Certificate
active
06210965
ABSTRACT:
The present invention relates to a process and in vitro culture medium for cells, such as primary normal or tumoral cells or cellular lines, of human or animal origin, a cellular composition based on gastro-intestinal epithelial cells and a system for the production of bioactive molecules and/or of m-RNA including such a system as well as a study model constituted by said composition.
The culture of any cells of human or animal origin always comprises a step of adherence of these cells to a support which again can be anything. This adherence step is necessary to obtain a correct survival rate of the cells whilst ensuring maintenance of their specialized functions. Conversely, an absence of adherence of the cells to a support gives rise to rapid death of said cells.
This adherence step further poses a certain number of problems that must be resolved, in particular for certain primary, normal or tumoral cells, particularly the fragile ones. Thus, it is not always known until now to maintain in primary culture pure populations of gastro-intestinal epithelial cells, whilst, in the same manner, it is known that the intestinal epithelium constitutes an extraordinary sense of bioactive peptides which could have high interest for industry such as pharmaceutical industries.
The methods described until now to maintain the culture of such populations were essentially based on the reconstitution of extra-cellular matrices and/or the utilization of predetermined media supplemented by various proteins and growth factors. Certain authors have essayed the reconstitution in vitro of the three-dimensional structure of the intestinal crypt on an endothelial “feeder-layer”. However, all these methods are seen to be inoperative in practice because of the very low adherence rate and, hence, low viability of the cells undergoing culture, by the absence of reproducibility of the results, by the too great complexity of the methods used and by the absence of maintenance of the difference functions of said cells. These methods have been used for certain normal tumoral cells which at present have the same problems.
Similarly, cell culture, in particular of cellular lines, on an industrial scale, is also very dependent on the step of adherence for different reasons from those given above. Thus, for example, optimization of the culture conditions by acceleration of the adherence and by mass adherence of the cells, constitutes a determining factor for the improvement of the output of mass cultures carried out on microballs in a fermenter, for example. As a result, the adherence must be effected under conditions such that there is obtained a very high rate of adherence of the cells to any support and this in a very short time. However, until now, the culture media used requires several hours to cause adherence of the cells to a support and moreover contain animal proteins which are in danger of being damaged in this type of medium because of the risks of contamination by virus and/or by a pathogenic agent of the prion type of the cells. The step of adherence is thus a long step, difficult and risky, in particular for the production of vaccines.
A first object of the invention is thus to provide an in vitro culture process for any type of cells which permits causing them rapidly to adhere in mass to any support to ensure their survival.
Another object of the invention is to provide an incubation medium for said simple cells to be produced and adapted to induce rapid inherence in mass of the cells to any support, this medium permitting moreover to obtain pure populations of cells which have never previously been able to be obtained.
Another object of the present invention is to provide an incubation medium for said cells chemically predetermined to be exempt from elements, in particular proteins, risking contamination of the cells undergoing culture by infectious agents of a viral nature or the like which could be carried by said elements.
Another object of the invention is to provide a cellular composition of a new type of which the cells, because of their adherence in mass to a support, can be maintained in culture under good conditions for a time up to several days.
Another object of the invention is to provide a system for the production by the epithelium of biologically active molecules and/or of ARNm coding for these molecules, these molecules being adapted to be constituted by new and inventive molecules.
Another object of the present invention is again to provide a study model in vitro of epithelial intestinal cells which permits, because of the incorporation in this model of a pure population of normal intestinal epithelial cells, an in vitro study of said cells, of the manner of regulation of their proliferation and their differentiation, of the expression of their specialized functions and of the metabolism and the toxicity of various substances which can be absorbed, particularly medicines and foodstuffs, under conditions near those which would be obtained in the case of in vivo studies.
To this end, the invention has for its object a process for the in vitro culture of cells, such as primary normal or tumoral cells and/or cellular lines, of human or animal origin, characterized in that it comprises at least one step of transitional incubation of said cells, preferably isolated, in an incubation medium in the form of a solution whose constituents are so selected that the final calcic concentration of the solution is comprised in the ratio 0-1 mM, preferably 0-100 &mgr;M, to give rise to rapid adherence in mass of said isolated cells to any support in contact with said incubation medium.
According to a preferred embodiment of the invention, the cells, once having adhered to their support, are cultivated in a culture medium whose calcic concentration is progressively increased to a predetermined value adapted to ensure the survival of the cells and to maintain their specialized functions.
The invention also relates to an incubation medium for cells, such that the primary normal or tumoral cells and/or the cellular lines, of human or animal origin, particularly for the practice of the previously-recited culture process, characterized in that it is present in the form of a solution whose constituents, particularly the salts, are selected such that the final calcic concentration of the solution is comprised in the rate 0-1 mM, preferably 0-100 &mgr;M, to give rise to the mass adherence of the incubated cells in their medium to any support in contact with said incubation medium.
The interest of such an incubation medium is its simplicity of production, its composition free from contaminants, such as proteins, in particular proteins of animal origin.
The invention also has for its object a cellular composition of a new type, characterized in that it is constituted of a pure population of gastro-intestinal epithelial cells adhering to a support, the adherence to said support having been obtained by transitional incubation of a primoculture of gastro-intestinal epithelial cells isolated from mammals in an incubation medium in the form of a solution whose constituents are so selected that the final calcic concentration of the solution of the incubation medium is comprised in the range 0-100 mM, preferably 0 to 100 &mgr;M.
When it is cultivated in these suitable culture media, this cellular composition can be used on the one hand as a system for in vitro production by the gastro-intestinal epithelium, of biologically active molecules such as growth factors, repair factors and/or ARNm coding for said molecules, on the other hand as an in vitro study model of the behavior of the intestinal epithelial cells particularly in fields such as pharmacotoxicology, immunology, in particular to assist in the production of vaccines, and genetic therapy.
REFERENCES:
patent: 4254226 (1981-03-01), Eisinger et al.
patent: 4456687 (1984-06-01), Green
patent: 5712163 (1998-01-01), Parenteau et al.
Lankford , Jr. Leon B.
Universite De Nantes
Young & Thompson
LandOfFree
Cell culture process and medium, cellular composition... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Cell culture process and medium, cellular composition..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Cell culture process and medium, cellular composition... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2542862