Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Culture medium – per se
Reexamination Certificate
2000-06-14
2002-06-04
Saucier, Sandra E. (Department: 1651)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Culture medium, per se
C435S405000, C435S371000
Reexamination Certificate
active
06399381
ABSTRACT:
FIELD OF THE INVENTION
The subject of the present invention is a new human epithelial colon immortalized cell line, a method for obtaining this line, as well as any use of this line, especially in processes for the identification of mutagenic, toxic or beneficial agents for the metabolism of the cells of the intestinal tract.
BACKGROUND ART
For many years, efforts have been made to develop human cell lines adapted to the study of human diseases, such as infections, inflammations or cancers, for example. Among the cells often involved in the onset of diseases, there are the epithelial cells, in particular the epithelial cells of the intestinal tract which are sensitive to the surroundings of the human body, and especially to the conditions of its diet.
The epithelial cells differ from other cells of the human body in the expression of compounds or structures which are mainly found in the epithelial cells, such as for example cytokeratins (Moll et al., Cell, 31, 11-24, 1982), connections between the cells (Gumbiner et al., Cell, 69, 385-387, 1992), alkaline phosphatase which is specific to the intestine (Dudeja et al., Gastroenterology, 105, 357-366, 1993), cytochromes P450 (Mercurio et al., Biochem. Biophy. Research Communications, 210, No. 2, 350-355, 1995; McKinnon et al., Gut, 36, 259-267, 1995), enzymes involved in cellular oxidation defense (Cu/Zn-superoxide dismutase, glutathione peroxidase and catalase; Albers et al., Toxicology and Applied Pharmacology, 109, 507-513, 1991) and/or in the detoxification of electrophiles (glutathione-S-transferase and quinone reductase; Chenevix-Trench et al., Carcinogenesis, 16, No. 7, 1655-1657, 1995), and vimentin (Richard et al., Arch. Dematol. Res., 282, 512-515, 1990),
In addition, the epithelial cells of the human intestinal tract are capable of adhering lactic acid bacteria in vitro (Bernet et al., Gut, 35, 483-489, 1994; U.S. Pat. No. 5,032,399).
Finally, the epithelial cells of the human colon also differ from other human epithelial cells in the expression of compounds or structures found mainly in the epithelial cells of the human intestine, such as, for example, surface villosities (Friedrich et al., Bioassays, 12, No. 9, 403-408, 1990), sucrose isomaltase (Gibson et al., Gut, 35, 791-797, 1994; Paul et al., Am. Pysiol. Soc., C266-C278, 1993), certain class II major histocompatibility antigens (Mayer et al., Gastroenterology, 100, 3-12, 1991), and dipeptidylpeptidase IV (DPPIV; Hauri et al., J. Cell. Biol., 101, 836-851, 1985).
The preparation of an epithelial cell line of the human colon may be carried out by the selection of human cancer cells. Stauffer et al. thus describe the selection of two lines NCM356 and NCM425 which comprise the p53 mutation and which express in particular the tumour antigen CEA (The American journal of surgery, 169, 190-196, 1995). These cells represents a tumorigenic transformation stage, and are of interest for studying the development of the tumorigenic transformation of the epithelial cells.
Other human colon epithelial cell lines isolated from a human colon adenoma are also known, such as, for example, the lines CaCO-2 (ATCC HTB37; Fogh et al., J. Natl. Cancer Inst., 58, 209-214, 1977) and HT29 (ATCC HTB38; Fogh et al., Human tumour Cells In Vitro, 145-159, 1975)
It is also possible to immortalize normal cells, that is to say make them capable of multiplying indefinitely. Indeed, normal cells do not survive more than a ten passages. For that, techniques for the transfection of cells, with the aid of specially adapted vectors, such as the SV40 vector comprising a sequence of the large T antigen (R. D. Berry et al., Br. J. Cancer, 57, 287-289, 1988), or a vector comprising DNA sequences of the human papillomavirus (U.S. Pat. No. 5,376,542), are generally used.
Sanderson et al. have thus immortalized normal cells of the foetal small intestine (Int. Arch. Allergy Immunol., 107, 396-397, 1995). However, these cells still remain physiologically distant from normal adult cells.
Up until now, no immortalization of normal epithelial cells of the adult human colon has been reported. Even if the immortalization techniques are known, it is still quite difficult to find the optimum conditions for immortalizing a human cell so that it conserves its original characteristics, and without exhibiting signs of a tumorigenic transformation.
One of the conditions to propagate immortalized cells is their growth in a particular culture medium. The culture media described in the literature are each specific for a cell type and cannot be easily applied to other cell types. By way of example, there may be mentioned the serum-free media described by Gibson et al. (Gut, 35, 791-797, 1991), Pfeifer et al. (Proc. Natl. Acad. Sci. USA, 90, 5123-5127, 1993) and Kulkani et al. (Carcinogenesis, 16, No. 10, 2515-2521, 1995).
The aim of the invention is to provide new human colon epithelial cell lines which are genetically and physiologically related to the normal epithelial cells of the human colon, to the extent that it is difficult to differentiate them. In particular, these lines do not express tumour markers. These lines express, in addition, numerous specific markers found in normal epithelial cells of the human colon.
SUMMARY OF THE INVENTION
To this end, the invention relates to any immortaized human colon epithelial cell lines which does not express tumour markers, which expresses metabolic markers specific for the non-immortalized human epithelial cells and metabolic differentiation markers specific for the non-immortalized epithelial cells of the human colon, and which is capable of adhering in vitro the CNCM-1225 strain of lactic acid bacterium.
The subject of the invention is also a serum-free culture media adapted to the culture and immortalization of epithelial cells of the human colon. This medium comprises the constituents usually found in the serum-free media used to culture animal cells, such as, for example, inorganic salts, glucose, sodium pyruvate, amino acids, bovine serum albumin (BSA), amines such as phosphorylethanolamine and ethanolamine, vitamins, and hormones. This medium comprises, in addition, a new combination of some of its constituents, namely trace elements, vitamins consisting of vitamin C and retinoic acid, hormones consisting of triiodothyronine, dexamethasone, hydrocortisone, extract of bovine pituarity gland, insuln, epidermal growth factor (EGF) and transfemn.
Another aspect of the invention relates to a new process for the immortalization of epithelial cells of the human colon, in which a culture of primary epithelial cells derived from the human colon is prepared, the culture is infected with a recombinant virus, and the immortalized cells are cultured in the serum-free culture medium described above.
Another aspect of the invention relates to a process for identifying the mutagenic, toxic or beneficial effect of an agent on the metabolism of the cells of the intestinal tract, in which (1) an agent suspected of being a mutagenic, toxic or beneficial agent for the metabolism of the cells of the intestinal tract is reacted, cultured of brought into contact with a culture comprising a cell line described above, and (2) the effects of the said agent on the said cell line are determined or measured.
The invention also relates to a diagnostic kit comprising the immortalized epithelial cells of the human colon described above, the culture medium described above, and the reagents for determining a metabolic response of the said cells to mutagenic, toxic or beneficial agents.
Finally, the subject of the invention is also any uses of the cell lines described above, in processes for the identification of mutagenic, toxic or beneficial agents for the metabolism of the cells of the intestinal tract, as well as any uses of the said lines as an active pharmaceutical agent.
REFERENCES:
patent: 4885238 (1989-12-01), Reddel et al.
patent: 5376542 (1994-12-01), Schlegal
patent: 5462870 (1995-10-01), Chopra
patent: 5529920 (1996-06-01), Cole et al.
patent: 5576206 (1996-11-01), Schlegel
patent: 5610043 (1
Blum Stéphanie
Pfeifer Andrea
Tromvoukis Yvonne
Afremova Vera
Nestac S.A.
Saucier Sandra E.
Winston & Strawn
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