CA2219038C - Immortalized human corneal epithelial cell line - Google Patents
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Abstract
Immortalized human corneal epithelial cell line Immortalized human corneal epithelial cell line capable of becoming stratified, and capable of expressing (1) metabolic markers specific for non-immortalized human epithelial cells such as vimentin, cytokeratins, connections between the cells, cytochrome P450s, a glutathione-S-transferase, Cu/Zn-superoxide dismutase, glutathione peroxidase, aldehyde reductase and catalase; (2) metabolic differentiation markers specific for non-immortalized human corneal epithelial cells such as the cytokeratin of 64 kD, the glutathione-S-transferase hGST5.8, and the profile of cytokines and growth factors comprising the compounds TNF.alpha., IL-1.beta., IL-1.alpha., IL-6, IL-8, GM CSF-.beta., IL-ra, TGF-.beta.1, TGF-.beta.2, TGF.alpha., EGF, PDGF-.beta.; (3) and markers specific for an inflammatory reaction such as collagenase I, the bradykinin, histamine and PAF receptors, and the system for transduction of an inflammatory signal by the phosphoinositides. Process for identifying the mutagenic, toxic or beneficial effect of an agent on the metabolism of the corneal cells, in which (1) an agent suspected of being a mutagenic, toxic or beneficial agent for the metabolism of the cells of the human cornea is reacted, cultured or brought into contact with a culture comprising a cell line according to the invention, and (2) the effects of the said agent on the said cell line are determined or measured.
Description
hortalized human corneal epithelial cell line The subject of the present invention is new immortalized human corneal epithelial cell lines as well as their use in processes for the identification of agents which are mutagenic, toxic or beneficial to the metabolism of the corneal cells.
State of the 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 which are sensitive to the surroundings of the human body.
The epithelial cells differ from other cells of the human body in the expression of compounds or struc tures which are found only 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), and vimentin (Richard et al., Arch. Dematol. Res., 282, 512-515, 1990).
Other compounds are also generally found in human epithelial cells, but not only in the epithelial cells, such as the cytochrome P450s (Mercurio et al., Biochem.
Biophy. Research Communications, 210, No.2, 350-355, 1995; McRinnon et al., Gut, 36, 259-267, 1995), the enzymes involved in the defence against cellular oxida-tion (Cu/Zn-superoxide dismutase, glutathione peroxidase, glutathione reductase and catalase: Albers et al., Toxicology and Applied Pharmacology, 109, 507-513, 1991) and/or in the detoxification of electrophiles (a-, ~- or n-glutathione-S-transferases: Singh et al. Exp. Eye Res., 40, 431-437, 1985; aldehyde reductase: Ellis et al., Biochemical J., 312, 535-541, 1995).
The epithelial cells of the human cornea also differ from other human epithelial cells in the expres sion of compounds which are found only in the epithelial cells of the cornea, such as for example the cytokeratin of 64kD and the glutathione-S-transferase hGST 5.8 which are found in the ocular tissues (hGST 5.8: Singhal et al., Invest. Ophthalmol. Vis. Sci., 36, 142-150, 1995;
64kD: Kiritoshi et al., Invest. Ophthalmol. vis. Sci., 32, 3073-3077, 1991).
Analysis of the expression of certain cytokines and growth factors in the epithelial cells of human cornea, without inflammatory stimulation, has already been determined by Cubitt et al., Wilson et al., Kennedy et al. and De-Quan et al., (J. Cellular Physiol, 163, 61-79, 1995; J. Clinical Investigation, 95, 82-88, 1995;
Invest. Ophtha. & Visual Sci., 33, 1756-1762, 1992;
Invest. Ophtha. & Visual Sci., 36, 330-340, 1995; Invest.
Ophtha. & Visual Sci., 34, 3199-3210, 1993). Moreover,.
Rosenbaum et al. have suggested that the cytokine profile of the corneal cells could be a means for effectively detecting the presence of certain diseases of the cornea (Invest. Ophthalmol. Vis. Sci., 36, 2151-2155, 1995). The profile of expression of certain cytokines and growth factors in the epithelial cells of the cornea therefore makes it possible to characterize and differentiate the normal corneal epithelial cells from the other cells, and in particular from the abnormal corneal epithelial cells .
Moreover, the methods of screening molecules which are noninflammatory for the eyes involve laboratory animals. To overcome the fact that there are substantial morphological and biochemical differences between the human eyes and those of these animals, Hainsworth et al.
propose to carry out these screening tests on primary epithelial cell cultures of cornea (J. Tissue Culture Meth., 13, 45-48, 1991). Unfortunately, primary cell cultures of cornea stop multiplying after one or two passages, each passage consisting in subculturing the cells in fresh medium after growing until they become confluent. Furthermore, these primary cells do not survive freezing in liquid nitrogen.
To overcome these disadvantages, human corneal epithelial cell lines have been developed by Kahn et al.
(Investigative Opht. & Visual Sci., 34, 3429-3441, 1993) and Araki-Sasaki et al. (Investigative Opht. & Visual Sci., 36, 614-621, 1995). Unfortunately, these lines are not yet fully satisfactory.
Indeed, the lines developed by Kahn et al. are not completely immortalized because they retain certain original differentiation characteristics up to about 25 successive culture passages (see Kahn et al.). These lines also release SV40 viruses into the culture medium (see the Araki-Sasaki et al. analysis). Finally, it is not known if they effectively express other differentia-tion markers, such as glutathione-S-transferase hGST5.8, and an appropriate cytokine and growth factor profile, as well as markers which are specific or necessary for an inflammatory reaction.
The immortalized lines developed by Araki-Sasaki et al. are, on the other hand, incapable of becoming stratified in corneal reconstruction trials in vivo (see Araki-Sasaki et al.), and it is not known if they express certain differentiation markers, such as glutathione-S
transferase hGST5.8, and an appropriate cytokine and growth factor profile, as well as markers which are specific or necessary for an inflammatory reaction.
The aim of the invention is to provide new human corneal epithelial cell lines which are genetically and physiologically similar to the normal epithelial cells of the human cornea, to the extent that they can be used effectively in trials for screening potentially inflam-matory molecules.
Summary of the invention To this end, the invention relates to any immor talized human corneal epithelial cell lines capable of becoming stratified, and capable of expressing metabolic markers specific for non-immortalized human epithelial cells, metabolic markers specific for non-immortalized human corneal epithelial cells, and markers specific for an inflammatory reaction.
Another aspect of the invention relates to a new process for identifying the mutagenic, toxic or benefi-cial effect of an agent on the metabolism of the cells of the cornea, in which (1) a culture comprising a cell line according to the invention is reacted with an agent suspected of being a mutagenic, toxic or beneficial agent for the metabolism of the cells of the human cornea, and (2) the effects of the said agent on the said cell line are determined.
The invention also relates to a diagnostic kit comprising the immortalized human corneal epithelial cells according to the invention, reagents for deter-mining a metabolic response of the said cells to mutagenic, toxic or beneficial agents for the said cells.
Finally, the subject of the invention is also any uses of the cell lines according to the invention, in processes for the identification of mutagenic, toxic or beneficial agents for the metabolism of the corneal cells, as well as any uses of these lines as active pharmaceutical agent.
Description of the figures Figure 1: schematic representation of the plasmid pZIPSVU19 which was used to prepare the vector pl/SV40U19 intended to immortalize the primary epithelial cells of the human cornea.
Figure 2: percentage accumulation of ['H]phos-phoinositides by primary epithelial cells of the human cornea and by the CNCM cells, as a function of the applied bradykinin concentration.
Figure 3: percentage accumulation of [3H]phos phoinositides by primary epithelial cells of the human cornea by the CNCM cells, as a function of the applied histamine concentration.
Figure 4: percentage accumulation of [3H]phos-phoinositides by primary epithelial cells of the human cornea and by the CNCM cells, as a function of the applied PAF concentration.
Detailed description of the invention Within the framework of the present invention, the expressions "normal cells", "primary cells" and non-immortalized cells" designate epithelial cells of the human cornea which can be collected from the cornea of a healthy adult not having crippling physiological or genetic deficiencies, and which can be cultured for a limited time without losing their original differentia-tion characteristics.
On the other hand, the expression "immortalized cells" designates cells which have undergone a genetic manipulation, by means of a DNA construct, which makes them capable of multiplying indefinitely, that is to say at more than 25 passages, preferably at least 60 pas-sages.
Likewise, the word "passage" designates the process consisting of taking an aliquot of a confluent or saturation culture of a cell line, in inoculating a fresh medium, and in culturing the line until confluence or saturation is obtained. The cell lines are thus tradi-tionally cultured by successive passages in fresh media.
It should be noted that the cell lines may lose their original differentiation characteristics after several successive passages. It is therefore extremely advant-ageous to be able to have a line whose characteristics are conserved even after numerous passages, preferably at least 30 to 60.passages.
Finally, the expression "original differentiation characteristics" designates both the markers found specifically on the human epithelial cells and the differentiation markers found specifically on the epithelial cells of the human cornea.
Preferably, the immortalized human corneal epithelial cell lines according to the invention do not express tumour markers, that is to say do not have carcinogenic genes or do not express messenger RNAs (mRNAs), proteins or differentiated cellular structure and characteristic of the transformation of the cells into tumour cells. The presence of these markers may be detected by hybridization or PCR of their DNA with a specific probe, by means of antibodies, and/or by electron microscopy, for example. The presence of a marker in a cell does not mean that the said cell is capable of conferring a cancer, after a few months, on a mouse without immune defence, but rather reflects a cancerous transformed state of the cells compared with the original cells from which they are derived.
Preferably, the lines according to the invention also lack viruses, that is to say are incapable of producing the viruses which initially immortalized them, as is the case for the lines developed by Kahn et al.
(see the introduction of Araki-Sasaki et al.).
The lines according to the invention should be capable of becoming stratified, that is to say capable of becoming organized into successive strata. For that, the lines according to the invention simply have to be cultured until they are confluent in a serum-free medium comprising 1.5 mM CaCl, and then it must simply be visually observed if the cells develop into strata, for example.
The lines according to the invention express, on the other hand, metabolic markers specific for normal human epithelial cells, that is to say markers generally found in the epithelial cells.
These specific markers may be a messenger RNA
(mRNA), a protein and/or a differentiated cellular structure capable of being derived from epithelial cells of the skin, the eye, the intestinal tract or the liver, for example. Preferably, the human epithelial cells according to the invention express at least two markers chosen from the group formed by vimentin, cytokeratins, connections between the cells (also called "tight junc-tions"), cytochrome P450s, glutathione-S-transferase (GST), Cu/Zn-superoxide dismutase (SOD), glutathione peroxidase (GP), glutathione reductase (GR), catalase (CA) and aldehyde reductase (AR).
It may also be noted that the lines according to the invention may express enzymes involved in cellular oxidation (SOD, GP, GR and CA) and/or the detoxification of electrophiles (GST, AR). These lines are thus particu-larly adapted to the study of the phenomena of inflamma-- 7 _ tions or irritations of the human cornea.
The cell lines according to the invention also express metabolic differentiation markers which are specific for the corneal cells, especially the epithelial cells of the human cornea (see Refiners et al., Carcinogenesis, 11, 957-963, 1990) . These differentiation markers may be an mRNA, a protein or a differentiated cellular structure. Preferably, the lines according to the invention express at least 2 differentiation markers chosen from the group formed by the cytokeratin of 64 kD, glutathione-S-transferase hGST5.8, and a cytokine and growth factor profile comprising the compounds TNFa, IL-1(3, IL-1a, IL-6, IL-8, GM CSF- f3, IL-ra, TGF-/31, TGF-,Ci2, TGFa, EGF, PDGF-~Ci.
The lines according to the invention are also capable of expressing at least one marker specific for an inflammatory reaction, that is to say a molecule which is detectable when the cell reacts to an inflammatory stimulus and/or a molecule or a structure which is necessary for an inflammatory reaction.
The inflammatory stimuli may be induced when the lines according to the invention are brought into contact with a microorganism of the Pseudomoaas aeruginosa species (Kernacki et al., J. Ocul. Pharmacol., 10, 281-288, 1994), with the platelet activating factor PAF
(Bazan et al., Exp. Eye Research, 14, 769-775, 1995), with 12-O-tetradecanoylphorbol (TPA: tumour promoting agent), with histamine (Sharif et al., J. Ocular Phar-macol., 10, 653-664, 1994), With bradykinin (Sharif et al., Neurochem. Int. 18, 89-96, 1991), or with other inflammatory compounds, for example.
Collagenase I is a marker which is easily detec-table when a line is subjected to an inflammatory stimu-lus (Bazan et al., Proc. Natl. Acad. Sci. USA, 90, 8678-8682, 1993). Other markers may also be detected, such as c-fos (see Bazan et al.), the arachidonic intermediates 12-HETE and 12-HETrE (Corners et al., Inves. Ophth. &
Visu. Sci., 36, 828-840, 1995) or a new cytokine and growth factor profile, for example.
_ g _ The lines according to the invention are also capable of expressing at.least one marker necessary for an inflammatory reaction, such as for example the bradykinin receptor, the histamine receptor, the PAF
receptor, and the system for transduction of a signal by the phosphoinositides (Phosphoinositide turnover signal transduction pathway; Sharif et al., Neurochem. Res., 12, 1313-1320, 1993).
The invention also relates, in particular, to an immortalized line according to the invention which was deposited, under the Budapest Treaty, with the Collection Nationale de Cultures de Microorganismes, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris, on 22 Octo ber 1996, where it received the deposit number CNCM I
1777.
The epithelial lines according to the invention advantageously conserve their original differentiation characteristics even after numerous passages, especially at least 25 passages, preferably at least 30 to 100 passages.
To obtain the lines according to the invention, it is possible to prepare a culture of primary epithelial cells derived from the human cornea, to infect the culture with a recombinant virus and to culture the immortalized cells in a serum-free culture medium. To this end, persons skilled in the art have available numerous serum-free culture media. As a guide, 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), Rulkani et al. (Carcinogenesis, 16, No. 10, 2515-2521, 1995), or in EP96201064.1 and US08/576483, for example. A serum-free medium which is perfectly adapted to the need of this process is the RGM medium (Clonetics, US).
Preferably, the following steps are used:
(1) a sample of epithelial tissues of a human cornea is obtained;
(2) this sample is prepared for the purpose of its culture in vitro;
_ g _ (3) the epithelial cells are inoculated into a serum-free culture medium and on cul-ture plates comprising a coating which facilitates the attachment of the cells and their growth;
(4) the medium is changed as many times as necessary in order to optimize the con-fluent growth;
(5) the cells are infected with a recombinant virus;
(6) and the immortalized cells are cultured in a serum-free culture medium.
In greater detail, stage 1) relates to the obtaining of samples of corneal cells from normal indi viduals during surgical acts. In stage 2), the sample may be washed in the culture medium, cut into pieces, and the epithelial part separated from other tissues by physical and/or chemical means. For example, the pieces of tissue may be placed in a solution comprising a protease for a time sufficient to achieve separation of the cells.
In stage 3), the epithelial cells may be inocu-lated into a serum-free culture medium, the culture plates having a coating consisting of a 0.01-1% gelatin solution, for example.
In stage 4), the culture medium containing the epithelial cells is changed as many times as necessary so as to optimize a confluent growth. Preferably, the medium is replaced every two days. After having reached a confluence of the order of 90% of the available surface area, which generally occurs 10 to 14 days after the inoculation, the cells are separated by treatments in a solution of proteases.
The separated cells are transferred in stage 5) into a fresh culture medium and then the cells are then conventionally infected with a recombinant virus. Nume rous transfection techniques are available to persons skilled in the art. By way of example, there may be mentioned the techniques described in W096/07750, by Claudia Chen et al. (Mol. and Cell. Biol., 7, 2745-2752, 1987) or by Wilson et al. (Analytical Biochemistry, 226, 212-220, 1995).
Preferably, a recombinant SV40 virus comprising the T Antigen (T-Ag), an inactivated virus replication origin and a selectable gene are used. By way of example, there may be used the DNA construct pLXSHD+SV40+
described by Stockshlaeder et al. whose sequence is available in the GeneBank databank (accession No. M64753;
Human Gen. Therapy, 2, 33-39, 1991).
Other appropriate vectors may be just as easily constructed by persons skilled in the art from commer-cially available vectors comprising the gene encoding T-Ag, a selectable gene and/or an inactivated virus replication origin. By way of example, there may be prepared the construct pl/SV40U19 by creating BamHI sites at the ends of the Bgll-Hpal fragment of SV40, and by cloning this fragment into the BamHI site of the plasmid pZIPSVU19 described in Figure 1 below, and by Jat et al., Mol. Cell. Biol., 9, 1672-1681, 1989.
In stage 6) , the epithelial cells are transferred into a fresh growth medium, on culture plates having the coating described above.
Knowing the new and original properties of the epithelial cell lines according to the invention, their application may be envisaged in immunological, pharmaco logical and toxicological studies.
The lines according to the invention are thus particularly adapted for screening mutagenic, toxic or beneficial agents for the metabolism of the cells of the cornea, for example in a process in which (1) an agent suspected of being a mutagenic, toxic or beneficial agent for the metabolism of the cells of the cornea is reacted, cultured or brought into contact with a culture compri-sing a cell line according to the invention, and (2) the effects of the said agent on the said cell line are determined or measured.
It may therefore also be envisaged to prepare a diagnostic kit comprising the epithelial cells according to the invention and reagents for determining a metabolic response of the said cells to mutagenic, toxic or benefi-cial agents.
The lines according to the invention are also adapted to the expression of recombinant proteins. The methods of transfection of foreign DNA are now well known to persons skilled in the art (see for example W094/05472).
The lines according to the invention also have a potential usefulness in gene therapy ex vivo. These lines might indeed constitute a suitable tool for developing recombinant cells expressing genes of interest for the purpose of a therapeutic application. One advantage additionally presented by the lines according to the invention is that they are not exposed to an animal serum, which considerably reduces the risks of potential contaminations by viruses or other pathogenic agents.
The present invention is described in greater detail below with the aid of the additional description which follows, which refers to examples of preparations of cell lines according to the invention. It goes without saying, however, that these examples are given by way of illustration of the subject of the invention and do not in any way constitute a limitation thereto. The culture of the cell lines, the preparation of SV40 vectors, the transfection, the preparation of DNA primers and probes and the analysis of the expressions of the markers are, unless otherwise stated, carried out according to the procedures described in the abovementioned references and in those in the manual by Sambrook et al. (Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Labora-tory Press, U.S.A., 1989). The percentages are given by weight, unless otherwise indicated.
Example 1 The plasmid pl/SV40U19 is constructed by creating BamHI sites at the ends of the SV40 Bgll-Hpal fragment and by cloning this fragment into the BamHI site of the plasmid pZIPSVU19 described by Jat et al., Mol. Cell.
Biol., 9, 1672-1681, 1989. As a guide, the vector pZIPSVU19 schematically represented in Figure 1 comprises the selectable marker neomycin and the Moloney virus LTR.
The SV40 viruses are prepared according to a modified version of the Lynch and Miller procedure (J.
Virol., 65, 3887-3890, 1991). For that, the ecotropic cell lines Psi2 (Mann et al., Cell, 33, 153-159, 1983) and the amphotropic cell lines PA317 (ATCC CRL9078) are cultured in the DMEM medium (Dulbecco, USA) comprising 10% foetal calf serum, at 37°C and under an atmosphere comprising 5o C02. These lines are conventionally transfected separately using 250 mM CaCl2 and 10 ~g of the plasmid pl/SV40U19, they are subjected to a treatment with trypsin after 48 h of incubation, they are mixed in an equal quantity, and the whole is incubated at 37°C
under an atmosphere comprising 5% C02. After growing to 80% confluence, the viruses are harvested in the serum-free medium KGM-1 (= KBM medium from Clonetics, US, comprising in addition 0.15 mM CaCl, 30 ~,g/ml of bovine pituitary gland extract, 0.5 ~,g/ml of hydrocortisone, 5 ~,g/ml of insulin, 10 ~.g/ml of transferrin, 10 ng/ml of murine epidermal growth factor (EGF), 10,000 U/ml of penicillin and 10,000 U/ml of streptomycin). After filtration (0.45 ~.m, Micropore) , the quantity of virus is determined on NIH 3T3 cells (ATCC CRL 1658).
Primary corneal epithelial cells were obtained following an eye biopsy on a 73-year old donor who died of myocardial infarction. The sample is washed in the phosphate buffer PBS, it is treated at 4°C for 24 h with 10 U/ml of dispase II (Boehringer Mannheim, No. 295825) in a buffer comprising 50% of KGM-2 medium (= KBM +
0.05 mM CaCl, 30 ~.g/ml of bovine pituitary gland extract, 10 ng/ml of murine epidermal growth factor, 0.5 ~.g/ml of hydrocortisone, 0.05 ~,g/ml of amphotericin B, 5 ~.g/ml of insulin, 10 ~.g/ml of transferrin, 50 ~.g/ml of genta-mycin). After incubation, the cells are separated manually, they are washed in DMEM medium (Dulbecco's modified Eagle medium, US) comprising 10°s bovine foetal serum, they are centrifuged, the pellets are taken up in the KGM-2 medium and they are spread on culture plates previously preincubated in a 0.1% solution of gelatin A
(Sigma G2500). The plates are incubated at 37°C under an atmosphere comprising 100$ relative humidity, 95~ oxygen and 5o carbon dioxide. The,culture medium containing the epithelial cells is changed as many times as is necessary to optimize a confluent growth.
After having reached a confluence of the order of 90o after 2 weeks of culture, the cells are conven-tionally separated by treatments in a solution of dispase II. The cells separated are then transferred into the KGM-1 medium and in culture plates preincubated with a O.lo solution of gelatin A. The cultures are then infected in the presence of 8 ~,g/ml of polybrene~ and a high recombinant SV40 virus titre (105-10~ CFU) prepared as described above. After 2 h of incubation with the virus, the cultures are washed in PBS and the cells are cultured by successive passages in fresh KGM-1 medium, taking care at each passage to successively wash the cells in an HBSS buffer (Hanks Balanced Salt Solution, Biofluids No. 325), to separate them by a treatment for 1-2 h at 37°C in a solution comprising 2.4 U/ml of dispase II, 50% HBSS buffer and 50o KBM medium, to harvest them in KBM medium, to centrifuge them and to take up the pellet in the KBM-1 medium and then to spread the cells on fresh new plates.
By successive passages, the transformed cells were purified from the primary epithelial cells. By then diluting, in a culture medium, the transformed cells previously separated by dispase II, it was possible to select 9 individualized immortalized human corneal epithelial cell lines of which one was deposited under the Budapest Treaty with the Collection Nationale de Cultures de Microorganismes, Institu~,Pasteur, 25 rue du Docteur Roux, 75724 Paris, on 22 October 1996, where it received the deposit number CNCM I-1777.
1. Analysis of the karyotyne of the strain CNCM I-1777 The analysis of the karyotype of the strain CNCM
I-1777 was made by the "Children's Hospital of Michigan", *Trade-mark 3901 Beaubien Blvd, Detroit, in accordance with the method described in the manual "Human cytogenetics", Edts: Rooney DE, Czepulkowski BH, IRL Press, Oxford, 1986. The results show that the line conserves intact in its genome one chromosome of each pair of chromosomes.
The sex of the line is XX.
2. TumoriQenicity of the strain CNCM I-1777 107 cells of the CNCM I-1777 line, taken at the 33rd passage, are injected into mice without immune defence ("nude") according to the procedure described by Stauffer et al. (The American journal of surgery, 169, 190-196, 1995). No tumour formation is visible after several months.
4. Viral contamination of the CNCM I-1777 line It is determined whether the CNCM I-1777 line is contaminated by the hepatitis C virus (HCV), the hepati-tis B virus (HHV) and the Aids virus (HIV-1). The results of the experiments described below are negative for the presence of the 3 viruses.
For the analysis of HBV, DNA is extracted from about 2x106 cells by treating in phenol and chloroform solutions followed by a precipitation in ethanol. DNA
samples are then subjected to a PCR amplification using primers specific for the pre-core region of the virus (Lynch et al., J. Virol., 65,-3887-3890). The amplifica tion products are then separated on an agarose gel and they are visualized under UV in the presence of ethidium bromide. For comparison, a dilution of a serum containing 10-105 HBV (Anawa Biomedical Services 6 Product, USA) is analysed in the same manner, in parallel.
For the analysis of HIV-1, DNA samples described above are subjected to a PCR amplification using primers specific for the GAG region of the virus. The amplifica-tion products are then separated on an agarose gel and they are visualized under UV in the presence of ethidium bromide. For comparison, a dilution of a serum containing 10-105 HIV-1 (Anawa Biomedical Services 6 Product, USA) is analysed in the same manner, in parallel.
For the analysis of HCV, the RNA is extracted from about 2x106 cells by the method of Chomczynski et al. (Anal. Biochem., 162, 156-159, 1987). A reverse transcription is carried out conventionally, and the complementary DNA obtained is subjected to a PCR amplifi-cation using primers specific for the non-coding 5' region of the virus. The amplification products are then separated on an agarose gel and they are visualized under UV in the presence of ethidium bromide. For comparison, a dilution of a serum containing 10-105 HCV (Anawa Biomedical Services 6 Product, USA) is analysed in the same manner, in parallel.
5 Markers specific for human epithelial cells for the CNCM I-1777 line 5.a. Cytokeratins: the cells of a culture of the CNCM I-1777 line, taken at the 22nd passage, are attached onto glass plates by a 100% cold methanol solution and then the plates are washed in a buffer comprising 0.05 M Tris pH 8.6, 1.8% NaCl and 0.2o polyethylene glycol 2000 (TNP
buffer). The cells are then incubated for 30 min in the presence of mouse antibodies specific for certain cytoke-ratins (anti-CH peptide 4, 7, 8, 13, 14, 17, 18, 19, 20, Sigma and Boehringer). After 3 washes in the TNP buffer comprising 0.5% HSA, the plates are incubated for 60 min with a goat anti-mouse IgG antibody comprising an immuno-fluorescent compound (1:300, FITC goat anti-mouse IgG;
Biosys). After 3 washes in the preceding buffer, the plates are fixed and they are analysed by fluorescence microscopy. All the cells are positive for the cytokera-tins 4, 7, 8, 13, 14, 17, 18, 19.
5.b. Vimentin: the cells of a culture of the CNCM I-1777 line, taken at the 22nd passage, are attached as described above. The fixed cells are then subjected to a mouse anti-vimentin antibody (DAKO, USA), and then to the goat anti-mouse IgG antibody mentioned above. By fluore-scence microscopy all the cells are positive for vimen-tin.
5.c. Connections between the cells: the cells of the CNCM
I-1777 line, taken at the 22nd passage, are cultured on a glass plate until they become confluent, they are fixed by treatment with a 2.5% glutaraldehyde solution in a 0.1 M phosphate buffer pH 7.4 for 1 h, at room tempera-ture. After two washes in the same phosphate buffer, the cells are again fixed in a 2% Os04 solution in the same buffer. The cells are then dehydrated in successive solutions of ethanol at 30, 50, 70, 90 and 1000 (Polaron Equipment Ltd., Watford, UK), and then the cells are covered by a fine gold layer (SEM coating unit E5100, Polaron). The cells are then examined by electron micro-scopy (Philips 505 SEM). The analysis shows that the cells have intercellular connections which are character-istic of the epithelial cells ("tight junctions").
5.d. CSrtochrome P450: the expression of cytochromes by the cells of the CNCM I-1777 line, taken at the 30th passage, is analysed with the aid of the known RT-PCR
technique using DNA primers specific for the different cytochrome P450s. These primers were conventionally prepared from the DNA sequences of the different cytochromes available on the GeneBank database (CYP1A1:
accession No. X02612; CYP2C: accession No. M61855 or M61858 or M61856 or MM61854; CYP2D6: accession No.
M33388; CYPlA2: accession No. Z00036).
For that, the cells are cultured until they become confluent on 35 mm dishes (Costar), the RNA is extracted with the aid of the RNAeasy~kit (Qiagen), a reverse transcription is performed (1st Strand cDNA
Synthesis Kit for RT-PCR, Boehringer, Mannheim), the complementary DNA obtained is subjected to a PCR amplifi-cation using DNA primers specific for the different cytochrome P450s, the amplification products are then separated on an agarose gel, and they are visualized under W in the presence of ethidium bromide. The results *Trade-mark show that the CNCM I-1777 cells express cytochromes CYP1A1, CYP1A2, CYP2C, CYP2D6.
5.e. Enzymes involved in the cellular oxidation and the detoxification of electro~hiles: the cells of the CNCM I-1777 line taken at the 30th passage are cultured, the RNA
is extracted by the Chomczynski et al. method (Anal.
Biochem., 162, 156-159, 1987), then a Northern-blot is performed on this RNA with DNA probes partially encoding Cu/Zn-superoxide dismutase (SOD), glutathione peroxidase (GP), catalase (CA) and ~r-glutathione-S-transferase (aGST). The DNA probes are prepared conventionally from DNA sequences of the genes encoding these enzymes which .
are available on the GeneBank~ database (~rrGST: accession No. X08058; GP: accession No. M21304; CA: accession No.
M81578; SOD: accession No. 729336). The results of these tests show that all the cells express a transcription of the genes encoding the SOD, GP, CA and ~rrGST activity.
Moreover, the expression of glutathione reductase is also confirmed by the enzymatic test described by 2~ Gondhowiardjo (Cornea, 12, 310-314, 1993). The catalase activity is also confirmed by the enzymatic test described by Aeby et al. (Method and Enzymology, 105, 121-126, 1984). Finally, the aldehyde reductase activity is also demonstrated by the enzymatic test described by Ellis et al. (Biochemical Journal, 312, 535-541, 1995).
6. Markers specific for the epithelial cells of the cornea for the CNCM I-1777 line 6.a. Expression of the cytokeratin of 64kD: as for the analysis of the cytokeratins described above, the cells are attached onto glass plates by a~100o cold methanol solution and then the plates are washed in TNP buffer (see above). The cells are then incubated for 30 min in the presence of mouse antibodies specific for the 64 kD
cytokeratin (AE5 antibody, ICM Biomedical Inc., US).
After 3 washes in the TNP buffer comprising 0.5o BSA, the plates are incubated for 30 min with a goat anti-mouse *Trade-mark IgG antibody comprising an immunofluorescent compound (1:300, FITC-goat anti-mouse IgG; Biosys) . After 3 washes in the preceding buffer, the plates are fixed and they are analysed by fluorescence microscopy. The cells are positive for the 64 kD cytokeratin.
6.b. Expression of alutathione-S-transferase hGST5.8: the expression of glutathione-S-transferase hGST5.8 is analysed by means of the test described by Singhal et al.
(Invest. Ophthalmol. Vis. Sci., 36, 142-150, 1995). In short, 10 ~.g/ml of a cytosolic extract of the CNCM I-1777 line is mixed with 0.1 mM 4-hydroxynonenal and 0.5 mM
glutathione in a 100 mM potassium phosphate buffer pH 6.5 and then the enzymatic activity is measured by the change in absorption in the mixture at 224 nm. The results show that the CNCM I-1777 line expresses a glutathione-S-transferase hGST5.8 activity.
6.c. Profile of expression of the cytokines and Qrowth factors: the cells of the CNCM I-1777 line taken at the 30th passage are cultured, the RNA is extracted and then a PCR is performed on this RNA with different primers in order to confirm the transcription of the RNAs encoding the cytokines and/or growth factors which follow: TNFa, IL-lei, IL-la, IL-6, IL-8, GM CSF-f3, IL-ra, TGF-(31, TGF-/32 , TGFa, EGF and PDGF-(3.
These primers correspond to a portion of the genes encoding these compounds, the DNA sequences of the said genes being accessible on the GeneBank database (accession No. TNFa: X02910; IL-lei: M15840; IL-la:
M15329; IL-6: M14584, Rosenbaum et al., Inv. Opth. & Vi.
Sc., 36, 2151-2155, 1995; IL-8: M28130; GM CSF-Vii: X03021;
IL-ra: M97748, Kennedy et al., J. Clinical Inv., 95, 82-88, 1995; TGF-(31: X02812, Nishida et al., Curr. Eye Res., 14, 235-241, 1995; TGF-(32: Y00083; TGFa: M22440; EGF:
L17032; PDGF-(3: X02811).
For comparison, the same analysis of the profile of expression of the cytokines and growth factors is performed on the RNA derived from epithelial tissues of the human cornea (4 different biopsies).
The results are presented in Table 1 below. The symbol * indicates that the expression of the compounds IL-1(3, TNFa, IL-la, IL-6 and IL-8 was also confirmed by ELISA tests using the CNCM I-1777 cell culture medium. , The specific antibodies against these different cytokines are commercially available (IL-lei, TNFa: BioSource Inter., US, catalogue No. KHC0012 and KHC3013; IL-la, IL-6 and IL-8: CLB corp., US, catalogue No. M1901, M1916, M1918 ) .
Table 1 CytokinesCNCM Biopsy Biopsy Biopsy Biopsy & growth I-1777 1 2 3 4 factors 1 TNFa' + +/- +/- +/- +/-IL-lei' +++ + ++ + ++
IL-la' +++ +/- ++ + +++
IL-6' ++ + + - -IL-8' +++ +/- + + ++
GM CSF-~i++ +/- +/- +/- +/-IL-ra ++ + +++ ++ +
TGF-al +++ + ++ + ++
TGF-~i2 +++ + ++ ++ ++
TGFa +++ + ++ + ++
2 EGF +++ + ++ ++ +++
PDGF-~i + +/- +/- +/- +/-(-. not detected; +/-. weakly detected; +++: abundant) 7 Markers specific for an inflammatory reaction for the CNCM I-1777 line 30 7.a. Colla~enase I: collagenase I is a molecule whose expression characterizes a normal state of inflammation of the corneal cells. The expression of collagenase can be detected by PCR, by Western-blotting or by ELISA.
To detect collagenase I by PCR, the CNCM I-1777 cells, taken at the 29th passage, are cultured in the presence of 20 ng/ml of TPA for 16 to 24 h, the RNA is extracted and the presence of collagenase I mRNA is detected by PCR with a primer derived from the collage-nase I DNA sequence (GeneBank: No. X05231).
To detect collagenase I by Western blotting, after having cultured the CNCM I-1777 cells in the presence of 20 ng/ml of TPA for 16 to 24 h, the culture medium is recovered and it is concentrated with an Amicon30~ filter, the protein is separated by electro-phoresis on an SDS-Page polyacrylamide gel, they are transferred by Western blotting onto a filter, the filter is subjected to a first anti-collagenase I antibody (Cortex Biochem, US, No. 60338P), to a second antibody coupled to a peroxidase (Pierce, US, No. 31400), and then to the peroxidase substrate.
To detect collagenase I by ELISA, it is suffi cient to use the Amersham hMMP-1 kit (catalogue, rpn 2610), following the recommendations of the supplier.
The results show that the expression of collage-nase I can be detected when the CNCM I-1777 cells are subjected to an inflammatory treatment. On the other hand, when the CNCM I-1777 cells are not brought into contact with TPA, they do not express collagenase I which is detectable by PCR, Western blotting or ELISA.
7.b. Markers necessary for an inflammatory reaction: the bradykinin, histamine and PAF receptors and the system for transduction of the phosphoinositides are markers essentially for the CNCM I-1777 line to be physiologi-cally in a normal state of inflammation. To detect the presence of these markers, the CNCM I-1777 cells or primary epithelial cells of the human cornea are incu-bated in the presence of inflammatory compounds which are supposed to interact with receptors coupled with the system for transduction of inflammatory signals by the phosphoinositides, and the concentration of inositol phosphate (IP) molecules thus produced is measured. The accumulation of IP is in fact characteristic of the existence of receptors fpr the inflammatory compounds.
The tests are based on the procedures by Sharifs et al. described in J. Ocular Pharmacol., 10, 653-664, 1994; and Neurochem Res., 12, 1313-1320, 1993. In short, on plates comprising 24 wells, about 2.5x105 CNCM I-1777 cells per well are cultured in DMEM medium in the pre-sence of [3H]myo-inositol (1 ~Ci/0.5 ml; 15-17 Ci/mM, Amersham Corp.), for 24 h at 37°C. The medium is sucked up from the wells and they are exposed for 60 min at 37°C
to DMEM medium comprising 10 mM LiCl, 15 mM HEPES buffer, and different concentrations of bradykinin (Collaborative Biomedical, US), of histamine (Sigma US) or of PAF
(BioMol Research Lab, US). To stop the reaction, the medium is then sucked up and 0.1 M formic acid is added to the wells. After that, the cells are lysed and the compounds of the lysate are separated on an ion-exchange column (AGlx8 resin, Econo~ column, Biorad, US), eluting the column with 10 ml of deionized water and 8 ml of a 50 mM ammonium formate solution, and the radioactivity released by the elution fractions is quantified with a scintillation counter.
The results are analysed according to the method described by Sharif et al. (Neurochem. Int., 18, 89-96, 1991). The EC50 value defines the concentration of compound required to stimulate 500 of the maximum acti-vity of the system for transduction of the phosphoinosi-tides.
The results, presented in Figures 2, 3 and 4, show that the CNCM I-1777 cells have bradykinin, hista-mine and PAF receptors and that they behave like primary epithelial cells of human cornea.
Example 2 As described in Example 1, the capacity of the CNCM I-1777 line taken at the 106 passage to express metabolic markers specific for non-immortalized human epithelial cells, metabolic differentiation markers specific for the non-immortalized epithelial cells of the human cornea, and markers for an inflammatory reaction, is analysed. The results are comparable to those pres-ented in Example 1.
Example 3 As described in Example 1, the capacity of the other lines selected in Example 1 to express metabolic markers specific for the non-immortalized human epithelial cells, metabolic differentiation markers specific for the non-immortalized epithelial cells of the human cornea, and markers for an inflammatory reaction, is analysed. For all these lines, the results are compa-rable to those presented in Example 1.
State of the 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 which are sensitive to the surroundings of the human body.
The epithelial cells differ from other cells of the human body in the expression of compounds or struc tures which are found only 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), and vimentin (Richard et al., Arch. Dematol. Res., 282, 512-515, 1990).
Other compounds are also generally found in human epithelial cells, but not only in the epithelial cells, such as the cytochrome P450s (Mercurio et al., Biochem.
Biophy. Research Communications, 210, No.2, 350-355, 1995; McRinnon et al., Gut, 36, 259-267, 1995), the enzymes involved in the defence against cellular oxida-tion (Cu/Zn-superoxide dismutase, glutathione peroxidase, glutathione reductase and catalase: Albers et al., Toxicology and Applied Pharmacology, 109, 507-513, 1991) and/or in the detoxification of electrophiles (a-, ~- or n-glutathione-S-transferases: Singh et al. Exp. Eye Res., 40, 431-437, 1985; aldehyde reductase: Ellis et al., Biochemical J., 312, 535-541, 1995).
The epithelial cells of the human cornea also differ from other human epithelial cells in the expres sion of compounds which are found only in the epithelial cells of the cornea, such as for example the cytokeratin of 64kD and the glutathione-S-transferase hGST 5.8 which are found in the ocular tissues (hGST 5.8: Singhal et al., Invest. Ophthalmol. Vis. Sci., 36, 142-150, 1995;
64kD: Kiritoshi et al., Invest. Ophthalmol. vis. Sci., 32, 3073-3077, 1991).
Analysis of the expression of certain cytokines and growth factors in the epithelial cells of human cornea, without inflammatory stimulation, has already been determined by Cubitt et al., Wilson et al., Kennedy et al. and De-Quan et al., (J. Cellular Physiol, 163, 61-79, 1995; J. Clinical Investigation, 95, 82-88, 1995;
Invest. Ophtha. & Visual Sci., 33, 1756-1762, 1992;
Invest. Ophtha. & Visual Sci., 36, 330-340, 1995; Invest.
Ophtha. & Visual Sci., 34, 3199-3210, 1993). Moreover,.
Rosenbaum et al. have suggested that the cytokine profile of the corneal cells could be a means for effectively detecting the presence of certain diseases of the cornea (Invest. Ophthalmol. Vis. Sci., 36, 2151-2155, 1995). The profile of expression of certain cytokines and growth factors in the epithelial cells of the cornea therefore makes it possible to characterize and differentiate the normal corneal epithelial cells from the other cells, and in particular from the abnormal corneal epithelial cells .
Moreover, the methods of screening molecules which are noninflammatory for the eyes involve laboratory animals. To overcome the fact that there are substantial morphological and biochemical differences between the human eyes and those of these animals, Hainsworth et al.
propose to carry out these screening tests on primary epithelial cell cultures of cornea (J. Tissue Culture Meth., 13, 45-48, 1991). Unfortunately, primary cell cultures of cornea stop multiplying after one or two passages, each passage consisting in subculturing the cells in fresh medium after growing until they become confluent. Furthermore, these primary cells do not survive freezing in liquid nitrogen.
To overcome these disadvantages, human corneal epithelial cell lines have been developed by Kahn et al.
(Investigative Opht. & Visual Sci., 34, 3429-3441, 1993) and Araki-Sasaki et al. (Investigative Opht. & Visual Sci., 36, 614-621, 1995). Unfortunately, these lines are not yet fully satisfactory.
Indeed, the lines developed by Kahn et al. are not completely immortalized because they retain certain original differentiation characteristics up to about 25 successive culture passages (see Kahn et al.). These lines also release SV40 viruses into the culture medium (see the Araki-Sasaki et al. analysis). Finally, it is not known if they effectively express other differentia-tion markers, such as glutathione-S-transferase hGST5.8, and an appropriate cytokine and growth factor profile, as well as markers which are specific or necessary for an inflammatory reaction.
The immortalized lines developed by Araki-Sasaki et al. are, on the other hand, incapable of becoming stratified in corneal reconstruction trials in vivo (see Araki-Sasaki et al.), and it is not known if they express certain differentiation markers, such as glutathione-S
transferase hGST5.8, and an appropriate cytokine and growth factor profile, as well as markers which are specific or necessary for an inflammatory reaction.
The aim of the invention is to provide new human corneal epithelial cell lines which are genetically and physiologically similar to the normal epithelial cells of the human cornea, to the extent that they can be used effectively in trials for screening potentially inflam-matory molecules.
Summary of the invention To this end, the invention relates to any immor talized human corneal epithelial cell lines capable of becoming stratified, and capable of expressing metabolic markers specific for non-immortalized human epithelial cells, metabolic markers specific for non-immortalized human corneal epithelial cells, and markers specific for an inflammatory reaction.
Another aspect of the invention relates to a new process for identifying the mutagenic, toxic or benefi-cial effect of an agent on the metabolism of the cells of the cornea, in which (1) a culture comprising a cell line according to the invention is reacted with an agent suspected of being a mutagenic, toxic or beneficial agent for the metabolism of the cells of the human cornea, and (2) the effects of the said agent on the said cell line are determined.
The invention also relates to a diagnostic kit comprising the immortalized human corneal epithelial cells according to the invention, reagents for deter-mining a metabolic response of the said cells to mutagenic, toxic or beneficial agents for the said cells.
Finally, the subject of the invention is also any uses of the cell lines according to the invention, in processes for the identification of mutagenic, toxic or beneficial agents for the metabolism of the corneal cells, as well as any uses of these lines as active pharmaceutical agent.
Description of the figures Figure 1: schematic representation of the plasmid pZIPSVU19 which was used to prepare the vector pl/SV40U19 intended to immortalize the primary epithelial cells of the human cornea.
Figure 2: percentage accumulation of ['H]phos-phoinositides by primary epithelial cells of the human cornea and by the CNCM cells, as a function of the applied bradykinin concentration.
Figure 3: percentage accumulation of [3H]phos phoinositides by primary epithelial cells of the human cornea by the CNCM cells, as a function of the applied histamine concentration.
Figure 4: percentage accumulation of [3H]phos-phoinositides by primary epithelial cells of the human cornea and by the CNCM cells, as a function of the applied PAF concentration.
Detailed description of the invention Within the framework of the present invention, the expressions "normal cells", "primary cells" and non-immortalized cells" designate epithelial cells of the human cornea which can be collected from the cornea of a healthy adult not having crippling physiological or genetic deficiencies, and which can be cultured for a limited time without losing their original differentia-tion characteristics.
On the other hand, the expression "immortalized cells" designates cells which have undergone a genetic manipulation, by means of a DNA construct, which makes them capable of multiplying indefinitely, that is to say at more than 25 passages, preferably at least 60 pas-sages.
Likewise, the word "passage" designates the process consisting of taking an aliquot of a confluent or saturation culture of a cell line, in inoculating a fresh medium, and in culturing the line until confluence or saturation is obtained. The cell lines are thus tradi-tionally cultured by successive passages in fresh media.
It should be noted that the cell lines may lose their original differentiation characteristics after several successive passages. It is therefore extremely advant-ageous to be able to have a line whose characteristics are conserved even after numerous passages, preferably at least 30 to 60.passages.
Finally, the expression "original differentiation characteristics" designates both the markers found specifically on the human epithelial cells and the differentiation markers found specifically on the epithelial cells of the human cornea.
Preferably, the immortalized human corneal epithelial cell lines according to the invention do not express tumour markers, that is to say do not have carcinogenic genes or do not express messenger RNAs (mRNAs), proteins or differentiated cellular structure and characteristic of the transformation of the cells into tumour cells. The presence of these markers may be detected by hybridization or PCR of their DNA with a specific probe, by means of antibodies, and/or by electron microscopy, for example. The presence of a marker in a cell does not mean that the said cell is capable of conferring a cancer, after a few months, on a mouse without immune defence, but rather reflects a cancerous transformed state of the cells compared with the original cells from which they are derived.
Preferably, the lines according to the invention also lack viruses, that is to say are incapable of producing the viruses which initially immortalized them, as is the case for the lines developed by Kahn et al.
(see the introduction of Araki-Sasaki et al.).
The lines according to the invention should be capable of becoming stratified, that is to say capable of becoming organized into successive strata. For that, the lines according to the invention simply have to be cultured until they are confluent in a serum-free medium comprising 1.5 mM CaCl, and then it must simply be visually observed if the cells develop into strata, for example.
The lines according to the invention express, on the other hand, metabolic markers specific for normal human epithelial cells, that is to say markers generally found in the epithelial cells.
These specific markers may be a messenger RNA
(mRNA), a protein and/or a differentiated cellular structure capable of being derived from epithelial cells of the skin, the eye, the intestinal tract or the liver, for example. Preferably, the human epithelial cells according to the invention express at least two markers chosen from the group formed by vimentin, cytokeratins, connections between the cells (also called "tight junc-tions"), cytochrome P450s, glutathione-S-transferase (GST), Cu/Zn-superoxide dismutase (SOD), glutathione peroxidase (GP), glutathione reductase (GR), catalase (CA) and aldehyde reductase (AR).
It may also be noted that the lines according to the invention may express enzymes involved in cellular oxidation (SOD, GP, GR and CA) and/or the detoxification of electrophiles (GST, AR). These lines are thus particu-larly adapted to the study of the phenomena of inflamma-- 7 _ tions or irritations of the human cornea.
The cell lines according to the invention also express metabolic differentiation markers which are specific for the corneal cells, especially the epithelial cells of the human cornea (see Refiners et al., Carcinogenesis, 11, 957-963, 1990) . These differentiation markers may be an mRNA, a protein or a differentiated cellular structure. Preferably, the lines according to the invention express at least 2 differentiation markers chosen from the group formed by the cytokeratin of 64 kD, glutathione-S-transferase hGST5.8, and a cytokine and growth factor profile comprising the compounds TNFa, IL-1(3, IL-1a, IL-6, IL-8, GM CSF- f3, IL-ra, TGF-/31, TGF-,Ci2, TGFa, EGF, PDGF-~Ci.
The lines according to the invention are also capable of expressing at least one marker specific for an inflammatory reaction, that is to say a molecule which is detectable when the cell reacts to an inflammatory stimulus and/or a molecule or a structure which is necessary for an inflammatory reaction.
The inflammatory stimuli may be induced when the lines according to the invention are brought into contact with a microorganism of the Pseudomoaas aeruginosa species (Kernacki et al., J. Ocul. Pharmacol., 10, 281-288, 1994), with the platelet activating factor PAF
(Bazan et al., Exp. Eye Research, 14, 769-775, 1995), with 12-O-tetradecanoylphorbol (TPA: tumour promoting agent), with histamine (Sharif et al., J. Ocular Phar-macol., 10, 653-664, 1994), With bradykinin (Sharif et al., Neurochem. Int. 18, 89-96, 1991), or with other inflammatory compounds, for example.
Collagenase I is a marker which is easily detec-table when a line is subjected to an inflammatory stimu-lus (Bazan et al., Proc. Natl. Acad. Sci. USA, 90, 8678-8682, 1993). Other markers may also be detected, such as c-fos (see Bazan et al.), the arachidonic intermediates 12-HETE and 12-HETrE (Corners et al., Inves. Ophth. &
Visu. Sci., 36, 828-840, 1995) or a new cytokine and growth factor profile, for example.
_ g _ The lines according to the invention are also capable of expressing at.least one marker necessary for an inflammatory reaction, such as for example the bradykinin receptor, the histamine receptor, the PAF
receptor, and the system for transduction of a signal by the phosphoinositides (Phosphoinositide turnover signal transduction pathway; Sharif et al., Neurochem. Res., 12, 1313-1320, 1993).
The invention also relates, in particular, to an immortalized line according to the invention which was deposited, under the Budapest Treaty, with the Collection Nationale de Cultures de Microorganismes, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris, on 22 Octo ber 1996, where it received the deposit number CNCM I
1777.
The epithelial lines according to the invention advantageously conserve their original differentiation characteristics even after numerous passages, especially at least 25 passages, preferably at least 30 to 100 passages.
To obtain the lines according to the invention, it is possible to prepare a culture of primary epithelial cells derived from the human cornea, to infect the culture with a recombinant virus and to culture the immortalized cells in a serum-free culture medium. To this end, persons skilled in the art have available numerous serum-free culture media. As a guide, 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), Rulkani et al. (Carcinogenesis, 16, No. 10, 2515-2521, 1995), or in EP96201064.1 and US08/576483, for example. A serum-free medium which is perfectly adapted to the need of this process is the RGM medium (Clonetics, US).
Preferably, the following steps are used:
(1) a sample of epithelial tissues of a human cornea is obtained;
(2) this sample is prepared for the purpose of its culture in vitro;
_ g _ (3) the epithelial cells are inoculated into a serum-free culture medium and on cul-ture plates comprising a coating which facilitates the attachment of the cells and their growth;
(4) the medium is changed as many times as necessary in order to optimize the con-fluent growth;
(5) the cells are infected with a recombinant virus;
(6) and the immortalized cells are cultured in a serum-free culture medium.
In greater detail, stage 1) relates to the obtaining of samples of corneal cells from normal indi viduals during surgical acts. In stage 2), the sample may be washed in the culture medium, cut into pieces, and the epithelial part separated from other tissues by physical and/or chemical means. For example, the pieces of tissue may be placed in a solution comprising a protease for a time sufficient to achieve separation of the cells.
In stage 3), the epithelial cells may be inocu-lated into a serum-free culture medium, the culture plates having a coating consisting of a 0.01-1% gelatin solution, for example.
In stage 4), the culture medium containing the epithelial cells is changed as many times as necessary so as to optimize a confluent growth. Preferably, the medium is replaced every two days. After having reached a confluence of the order of 90% of the available surface area, which generally occurs 10 to 14 days after the inoculation, the cells are separated by treatments in a solution of proteases.
The separated cells are transferred in stage 5) into a fresh culture medium and then the cells are then conventionally infected with a recombinant virus. Nume rous transfection techniques are available to persons skilled in the art. By way of example, there may be mentioned the techniques described in W096/07750, by Claudia Chen et al. (Mol. and Cell. Biol., 7, 2745-2752, 1987) or by Wilson et al. (Analytical Biochemistry, 226, 212-220, 1995).
Preferably, a recombinant SV40 virus comprising the T Antigen (T-Ag), an inactivated virus replication origin and a selectable gene are used. By way of example, there may be used the DNA construct pLXSHD+SV40+
described by Stockshlaeder et al. whose sequence is available in the GeneBank databank (accession No. M64753;
Human Gen. Therapy, 2, 33-39, 1991).
Other appropriate vectors may be just as easily constructed by persons skilled in the art from commer-cially available vectors comprising the gene encoding T-Ag, a selectable gene and/or an inactivated virus replication origin. By way of example, there may be prepared the construct pl/SV40U19 by creating BamHI sites at the ends of the Bgll-Hpal fragment of SV40, and by cloning this fragment into the BamHI site of the plasmid pZIPSVU19 described in Figure 1 below, and by Jat et al., Mol. Cell. Biol., 9, 1672-1681, 1989.
In stage 6) , the epithelial cells are transferred into a fresh growth medium, on culture plates having the coating described above.
Knowing the new and original properties of the epithelial cell lines according to the invention, their application may be envisaged in immunological, pharmaco logical and toxicological studies.
The lines according to the invention are thus particularly adapted for screening mutagenic, toxic or beneficial agents for the metabolism of the cells of the cornea, for example in a process in which (1) an agent suspected of being a mutagenic, toxic or beneficial agent for the metabolism of the cells of the cornea is reacted, cultured or brought into contact with a culture compri-sing a cell line according to the invention, and (2) the effects of the said agent on the said cell line are determined or measured.
It may therefore also be envisaged to prepare a diagnostic kit comprising the epithelial cells according to the invention and reagents for determining a metabolic response of the said cells to mutagenic, toxic or benefi-cial agents.
The lines according to the invention are also adapted to the expression of recombinant proteins. The methods of transfection of foreign DNA are now well known to persons skilled in the art (see for example W094/05472).
The lines according to the invention also have a potential usefulness in gene therapy ex vivo. These lines might indeed constitute a suitable tool for developing recombinant cells expressing genes of interest for the purpose of a therapeutic application. One advantage additionally presented by the lines according to the invention is that they are not exposed to an animal serum, which considerably reduces the risks of potential contaminations by viruses or other pathogenic agents.
The present invention is described in greater detail below with the aid of the additional description which follows, which refers to examples of preparations of cell lines according to the invention. It goes without saying, however, that these examples are given by way of illustration of the subject of the invention and do not in any way constitute a limitation thereto. The culture of the cell lines, the preparation of SV40 vectors, the transfection, the preparation of DNA primers and probes and the analysis of the expressions of the markers are, unless otherwise stated, carried out according to the procedures described in the abovementioned references and in those in the manual by Sambrook et al. (Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Labora-tory Press, U.S.A., 1989). The percentages are given by weight, unless otherwise indicated.
Example 1 The plasmid pl/SV40U19 is constructed by creating BamHI sites at the ends of the SV40 Bgll-Hpal fragment and by cloning this fragment into the BamHI site of the plasmid pZIPSVU19 described by Jat et al., Mol. Cell.
Biol., 9, 1672-1681, 1989. As a guide, the vector pZIPSVU19 schematically represented in Figure 1 comprises the selectable marker neomycin and the Moloney virus LTR.
The SV40 viruses are prepared according to a modified version of the Lynch and Miller procedure (J.
Virol., 65, 3887-3890, 1991). For that, the ecotropic cell lines Psi2 (Mann et al., Cell, 33, 153-159, 1983) and the amphotropic cell lines PA317 (ATCC CRL9078) are cultured in the DMEM medium (Dulbecco, USA) comprising 10% foetal calf serum, at 37°C and under an atmosphere comprising 5o C02. These lines are conventionally transfected separately using 250 mM CaCl2 and 10 ~g of the plasmid pl/SV40U19, they are subjected to a treatment with trypsin after 48 h of incubation, they are mixed in an equal quantity, and the whole is incubated at 37°C
under an atmosphere comprising 5% C02. After growing to 80% confluence, the viruses are harvested in the serum-free medium KGM-1 (= KBM medium from Clonetics, US, comprising in addition 0.15 mM CaCl, 30 ~,g/ml of bovine pituitary gland extract, 0.5 ~,g/ml of hydrocortisone, 5 ~,g/ml of insulin, 10 ~.g/ml of transferrin, 10 ng/ml of murine epidermal growth factor (EGF), 10,000 U/ml of penicillin and 10,000 U/ml of streptomycin). After filtration (0.45 ~.m, Micropore) , the quantity of virus is determined on NIH 3T3 cells (ATCC CRL 1658).
Primary corneal epithelial cells were obtained following an eye biopsy on a 73-year old donor who died of myocardial infarction. The sample is washed in the phosphate buffer PBS, it is treated at 4°C for 24 h with 10 U/ml of dispase II (Boehringer Mannheim, No. 295825) in a buffer comprising 50% of KGM-2 medium (= KBM +
0.05 mM CaCl, 30 ~.g/ml of bovine pituitary gland extract, 10 ng/ml of murine epidermal growth factor, 0.5 ~.g/ml of hydrocortisone, 0.05 ~,g/ml of amphotericin B, 5 ~.g/ml of insulin, 10 ~.g/ml of transferrin, 50 ~.g/ml of genta-mycin). After incubation, the cells are separated manually, they are washed in DMEM medium (Dulbecco's modified Eagle medium, US) comprising 10°s bovine foetal serum, they are centrifuged, the pellets are taken up in the KGM-2 medium and they are spread on culture plates previously preincubated in a 0.1% solution of gelatin A
(Sigma G2500). The plates are incubated at 37°C under an atmosphere comprising 100$ relative humidity, 95~ oxygen and 5o carbon dioxide. The,culture medium containing the epithelial cells is changed as many times as is necessary to optimize a confluent growth.
After having reached a confluence of the order of 90o after 2 weeks of culture, the cells are conven-tionally separated by treatments in a solution of dispase II. The cells separated are then transferred into the KGM-1 medium and in culture plates preincubated with a O.lo solution of gelatin A. The cultures are then infected in the presence of 8 ~,g/ml of polybrene~ and a high recombinant SV40 virus titre (105-10~ CFU) prepared as described above. After 2 h of incubation with the virus, the cultures are washed in PBS and the cells are cultured by successive passages in fresh KGM-1 medium, taking care at each passage to successively wash the cells in an HBSS buffer (Hanks Balanced Salt Solution, Biofluids No. 325), to separate them by a treatment for 1-2 h at 37°C in a solution comprising 2.4 U/ml of dispase II, 50% HBSS buffer and 50o KBM medium, to harvest them in KBM medium, to centrifuge them and to take up the pellet in the KBM-1 medium and then to spread the cells on fresh new plates.
By successive passages, the transformed cells were purified from the primary epithelial cells. By then diluting, in a culture medium, the transformed cells previously separated by dispase II, it was possible to select 9 individualized immortalized human corneal epithelial cell lines of which one was deposited under the Budapest Treaty with the Collection Nationale de Cultures de Microorganismes, Institu~,Pasteur, 25 rue du Docteur Roux, 75724 Paris, on 22 October 1996, where it received the deposit number CNCM I-1777.
1. Analysis of the karyotyne of the strain CNCM I-1777 The analysis of the karyotype of the strain CNCM
I-1777 was made by the "Children's Hospital of Michigan", *Trade-mark 3901 Beaubien Blvd, Detroit, in accordance with the method described in the manual "Human cytogenetics", Edts: Rooney DE, Czepulkowski BH, IRL Press, Oxford, 1986. The results show that the line conserves intact in its genome one chromosome of each pair of chromosomes.
The sex of the line is XX.
2. TumoriQenicity of the strain CNCM I-1777 107 cells of the CNCM I-1777 line, taken at the 33rd passage, are injected into mice without immune defence ("nude") according to the procedure described by Stauffer et al. (The American journal of surgery, 169, 190-196, 1995). No tumour formation is visible after several months.
4. Viral contamination of the CNCM I-1777 line It is determined whether the CNCM I-1777 line is contaminated by the hepatitis C virus (HCV), the hepati-tis B virus (HHV) and the Aids virus (HIV-1). The results of the experiments described below are negative for the presence of the 3 viruses.
For the analysis of HBV, DNA is extracted from about 2x106 cells by treating in phenol and chloroform solutions followed by a precipitation in ethanol. DNA
samples are then subjected to a PCR amplification using primers specific for the pre-core region of the virus (Lynch et al., J. Virol., 65,-3887-3890). The amplifica tion products are then separated on an agarose gel and they are visualized under UV in the presence of ethidium bromide. For comparison, a dilution of a serum containing 10-105 HBV (Anawa Biomedical Services 6 Product, USA) is analysed in the same manner, in parallel.
For the analysis of HIV-1, DNA samples described above are subjected to a PCR amplification using primers specific for the GAG region of the virus. The amplifica-tion products are then separated on an agarose gel and they are visualized under UV in the presence of ethidium bromide. For comparison, a dilution of a serum containing 10-105 HIV-1 (Anawa Biomedical Services 6 Product, USA) is analysed in the same manner, in parallel.
For the analysis of HCV, the RNA is extracted from about 2x106 cells by the method of Chomczynski et al. (Anal. Biochem., 162, 156-159, 1987). A reverse transcription is carried out conventionally, and the complementary DNA obtained is subjected to a PCR amplifi-cation using primers specific for the non-coding 5' region of the virus. The amplification products are then separated on an agarose gel and they are visualized under UV in the presence of ethidium bromide. For comparison, a dilution of a serum containing 10-105 HCV (Anawa Biomedical Services 6 Product, USA) is analysed in the same manner, in parallel.
5 Markers specific for human epithelial cells for the CNCM I-1777 line 5.a. Cytokeratins: the cells of a culture of the CNCM I-1777 line, taken at the 22nd passage, are attached onto glass plates by a 100% cold methanol solution and then the plates are washed in a buffer comprising 0.05 M Tris pH 8.6, 1.8% NaCl and 0.2o polyethylene glycol 2000 (TNP
buffer). The cells are then incubated for 30 min in the presence of mouse antibodies specific for certain cytoke-ratins (anti-CH peptide 4, 7, 8, 13, 14, 17, 18, 19, 20, Sigma and Boehringer). After 3 washes in the TNP buffer comprising 0.5% HSA, the plates are incubated for 60 min with a goat anti-mouse IgG antibody comprising an immuno-fluorescent compound (1:300, FITC goat anti-mouse IgG;
Biosys). After 3 washes in the preceding buffer, the plates are fixed and they are analysed by fluorescence microscopy. All the cells are positive for the cytokera-tins 4, 7, 8, 13, 14, 17, 18, 19.
5.b. Vimentin: the cells of a culture of the CNCM I-1777 line, taken at the 22nd passage, are attached as described above. The fixed cells are then subjected to a mouse anti-vimentin antibody (DAKO, USA), and then to the goat anti-mouse IgG antibody mentioned above. By fluore-scence microscopy all the cells are positive for vimen-tin.
5.c. Connections between the cells: the cells of the CNCM
I-1777 line, taken at the 22nd passage, are cultured on a glass plate until they become confluent, they are fixed by treatment with a 2.5% glutaraldehyde solution in a 0.1 M phosphate buffer pH 7.4 for 1 h, at room tempera-ture. After two washes in the same phosphate buffer, the cells are again fixed in a 2% Os04 solution in the same buffer. The cells are then dehydrated in successive solutions of ethanol at 30, 50, 70, 90 and 1000 (Polaron Equipment Ltd., Watford, UK), and then the cells are covered by a fine gold layer (SEM coating unit E5100, Polaron). The cells are then examined by electron micro-scopy (Philips 505 SEM). The analysis shows that the cells have intercellular connections which are character-istic of the epithelial cells ("tight junctions").
5.d. CSrtochrome P450: the expression of cytochromes by the cells of the CNCM I-1777 line, taken at the 30th passage, is analysed with the aid of the known RT-PCR
technique using DNA primers specific for the different cytochrome P450s. These primers were conventionally prepared from the DNA sequences of the different cytochromes available on the GeneBank database (CYP1A1:
accession No. X02612; CYP2C: accession No. M61855 or M61858 or M61856 or MM61854; CYP2D6: accession No.
M33388; CYPlA2: accession No. Z00036).
For that, the cells are cultured until they become confluent on 35 mm dishes (Costar), the RNA is extracted with the aid of the RNAeasy~kit (Qiagen), a reverse transcription is performed (1st Strand cDNA
Synthesis Kit for RT-PCR, Boehringer, Mannheim), the complementary DNA obtained is subjected to a PCR amplifi-cation using DNA primers specific for the different cytochrome P450s, the amplification products are then separated on an agarose gel, and they are visualized under W in the presence of ethidium bromide. The results *Trade-mark show that the CNCM I-1777 cells express cytochromes CYP1A1, CYP1A2, CYP2C, CYP2D6.
5.e. Enzymes involved in the cellular oxidation and the detoxification of electro~hiles: the cells of the CNCM I-1777 line taken at the 30th passage are cultured, the RNA
is extracted by the Chomczynski et al. method (Anal.
Biochem., 162, 156-159, 1987), then a Northern-blot is performed on this RNA with DNA probes partially encoding Cu/Zn-superoxide dismutase (SOD), glutathione peroxidase (GP), catalase (CA) and ~r-glutathione-S-transferase (aGST). The DNA probes are prepared conventionally from DNA sequences of the genes encoding these enzymes which .
are available on the GeneBank~ database (~rrGST: accession No. X08058; GP: accession No. M21304; CA: accession No.
M81578; SOD: accession No. 729336). The results of these tests show that all the cells express a transcription of the genes encoding the SOD, GP, CA and ~rrGST activity.
Moreover, the expression of glutathione reductase is also confirmed by the enzymatic test described by 2~ Gondhowiardjo (Cornea, 12, 310-314, 1993). The catalase activity is also confirmed by the enzymatic test described by Aeby et al. (Method and Enzymology, 105, 121-126, 1984). Finally, the aldehyde reductase activity is also demonstrated by the enzymatic test described by Ellis et al. (Biochemical Journal, 312, 535-541, 1995).
6. Markers specific for the epithelial cells of the cornea for the CNCM I-1777 line 6.a. Expression of the cytokeratin of 64kD: as for the analysis of the cytokeratins described above, the cells are attached onto glass plates by a~100o cold methanol solution and then the plates are washed in TNP buffer (see above). The cells are then incubated for 30 min in the presence of mouse antibodies specific for the 64 kD
cytokeratin (AE5 antibody, ICM Biomedical Inc., US).
After 3 washes in the TNP buffer comprising 0.5o BSA, the plates are incubated for 30 min with a goat anti-mouse *Trade-mark IgG antibody comprising an immunofluorescent compound (1:300, FITC-goat anti-mouse IgG; Biosys) . After 3 washes in the preceding buffer, the plates are fixed and they are analysed by fluorescence microscopy. The cells are positive for the 64 kD cytokeratin.
6.b. Expression of alutathione-S-transferase hGST5.8: the expression of glutathione-S-transferase hGST5.8 is analysed by means of the test described by Singhal et al.
(Invest. Ophthalmol. Vis. Sci., 36, 142-150, 1995). In short, 10 ~.g/ml of a cytosolic extract of the CNCM I-1777 line is mixed with 0.1 mM 4-hydroxynonenal and 0.5 mM
glutathione in a 100 mM potassium phosphate buffer pH 6.5 and then the enzymatic activity is measured by the change in absorption in the mixture at 224 nm. The results show that the CNCM I-1777 line expresses a glutathione-S-transferase hGST5.8 activity.
6.c. Profile of expression of the cytokines and Qrowth factors: the cells of the CNCM I-1777 line taken at the 30th passage are cultured, the RNA is extracted and then a PCR is performed on this RNA with different primers in order to confirm the transcription of the RNAs encoding the cytokines and/or growth factors which follow: TNFa, IL-lei, IL-la, IL-6, IL-8, GM CSF-f3, IL-ra, TGF-(31, TGF-/32 , TGFa, EGF and PDGF-(3.
These primers correspond to a portion of the genes encoding these compounds, the DNA sequences of the said genes being accessible on the GeneBank database (accession No. TNFa: X02910; IL-lei: M15840; IL-la:
M15329; IL-6: M14584, Rosenbaum et al., Inv. Opth. & Vi.
Sc., 36, 2151-2155, 1995; IL-8: M28130; GM CSF-Vii: X03021;
IL-ra: M97748, Kennedy et al., J. Clinical Inv., 95, 82-88, 1995; TGF-(31: X02812, Nishida et al., Curr. Eye Res., 14, 235-241, 1995; TGF-(32: Y00083; TGFa: M22440; EGF:
L17032; PDGF-(3: X02811).
For comparison, the same analysis of the profile of expression of the cytokines and growth factors is performed on the RNA derived from epithelial tissues of the human cornea (4 different biopsies).
The results are presented in Table 1 below. The symbol * indicates that the expression of the compounds IL-1(3, TNFa, IL-la, IL-6 and IL-8 was also confirmed by ELISA tests using the CNCM I-1777 cell culture medium. , The specific antibodies against these different cytokines are commercially available (IL-lei, TNFa: BioSource Inter., US, catalogue No. KHC0012 and KHC3013; IL-la, IL-6 and IL-8: CLB corp., US, catalogue No. M1901, M1916, M1918 ) .
Table 1 CytokinesCNCM Biopsy Biopsy Biopsy Biopsy & growth I-1777 1 2 3 4 factors 1 TNFa' + +/- +/- +/- +/-IL-lei' +++ + ++ + ++
IL-la' +++ +/- ++ + +++
IL-6' ++ + + - -IL-8' +++ +/- + + ++
GM CSF-~i++ +/- +/- +/- +/-IL-ra ++ + +++ ++ +
TGF-al +++ + ++ + ++
TGF-~i2 +++ + ++ ++ ++
TGFa +++ + ++ + ++
2 EGF +++ + ++ ++ +++
PDGF-~i + +/- +/- +/- +/-(-. not detected; +/-. weakly detected; +++: abundant) 7 Markers specific for an inflammatory reaction for the CNCM I-1777 line 30 7.a. Colla~enase I: collagenase I is a molecule whose expression characterizes a normal state of inflammation of the corneal cells. The expression of collagenase can be detected by PCR, by Western-blotting or by ELISA.
To detect collagenase I by PCR, the CNCM I-1777 cells, taken at the 29th passage, are cultured in the presence of 20 ng/ml of TPA for 16 to 24 h, the RNA is extracted and the presence of collagenase I mRNA is detected by PCR with a primer derived from the collage-nase I DNA sequence (GeneBank: No. X05231).
To detect collagenase I by Western blotting, after having cultured the CNCM I-1777 cells in the presence of 20 ng/ml of TPA for 16 to 24 h, the culture medium is recovered and it is concentrated with an Amicon30~ filter, the protein is separated by electro-phoresis on an SDS-Page polyacrylamide gel, they are transferred by Western blotting onto a filter, the filter is subjected to a first anti-collagenase I antibody (Cortex Biochem, US, No. 60338P), to a second antibody coupled to a peroxidase (Pierce, US, No. 31400), and then to the peroxidase substrate.
To detect collagenase I by ELISA, it is suffi cient to use the Amersham hMMP-1 kit (catalogue, rpn 2610), following the recommendations of the supplier.
The results show that the expression of collage-nase I can be detected when the CNCM I-1777 cells are subjected to an inflammatory treatment. On the other hand, when the CNCM I-1777 cells are not brought into contact with TPA, they do not express collagenase I which is detectable by PCR, Western blotting or ELISA.
7.b. Markers necessary for an inflammatory reaction: the bradykinin, histamine and PAF receptors and the system for transduction of the phosphoinositides are markers essentially for the CNCM I-1777 line to be physiologi-cally in a normal state of inflammation. To detect the presence of these markers, the CNCM I-1777 cells or primary epithelial cells of the human cornea are incu-bated in the presence of inflammatory compounds which are supposed to interact with receptors coupled with the system for transduction of inflammatory signals by the phosphoinositides, and the concentration of inositol phosphate (IP) molecules thus produced is measured. The accumulation of IP is in fact characteristic of the existence of receptors fpr the inflammatory compounds.
The tests are based on the procedures by Sharifs et al. described in J. Ocular Pharmacol., 10, 653-664, 1994; and Neurochem Res., 12, 1313-1320, 1993. In short, on plates comprising 24 wells, about 2.5x105 CNCM I-1777 cells per well are cultured in DMEM medium in the pre-sence of [3H]myo-inositol (1 ~Ci/0.5 ml; 15-17 Ci/mM, Amersham Corp.), for 24 h at 37°C. The medium is sucked up from the wells and they are exposed for 60 min at 37°C
to DMEM medium comprising 10 mM LiCl, 15 mM HEPES buffer, and different concentrations of bradykinin (Collaborative Biomedical, US), of histamine (Sigma US) or of PAF
(BioMol Research Lab, US). To stop the reaction, the medium is then sucked up and 0.1 M formic acid is added to the wells. After that, the cells are lysed and the compounds of the lysate are separated on an ion-exchange column (AGlx8 resin, Econo~ column, Biorad, US), eluting the column with 10 ml of deionized water and 8 ml of a 50 mM ammonium formate solution, and the radioactivity released by the elution fractions is quantified with a scintillation counter.
The results are analysed according to the method described by Sharif et al. (Neurochem. Int., 18, 89-96, 1991). The EC50 value defines the concentration of compound required to stimulate 500 of the maximum acti-vity of the system for transduction of the phosphoinosi-tides.
The results, presented in Figures 2, 3 and 4, show that the CNCM I-1777 cells have bradykinin, hista-mine and PAF receptors and that they behave like primary epithelial cells of human cornea.
Example 2 As described in Example 1, the capacity of the CNCM I-1777 line taken at the 106 passage to express metabolic markers specific for non-immortalized human epithelial cells, metabolic differentiation markers specific for the non-immortalized epithelial cells of the human cornea, and markers for an inflammatory reaction, is analysed. The results are comparable to those pres-ented in Example 1.
Example 3 As described in Example 1, the capacity of the other lines selected in Example 1 to express metabolic markers specific for the non-immortalized human epithelial cells, metabolic differentiation markers specific for the non-immortalized epithelial cells of the human cornea, and markers for an inflammatory reaction, is analysed. For all these lines, the results are compa-rable to those presented in Example 1.
Claims (6)
1. Immortalized human corneal epithelial cell line capable of becoming stratified, and capable of expressing as metabolic markers specific for non-immortalized human epithelial cells at least two markers chosen from the group formed by vimentin, cytokeratins, connections between the cells, cytochrome P450s, a glutathione-S-transferase, Cu/Zn-superoxide dismutase, glutathione peroxidase, glutathione reductase, aldehyde reductase and catalase, as metabolic differentiation markers specific for non-immortalized human corneal epithelial cells at least 2 markers chosen from the group formed by the cytokeratin of 64 kD, the glutathione-S-transferase hGST5.8, and the profile of cytokines and growth factors which follow: TNF.alpha., IL-1.beta., IL-1.alpha., IL-6, IL-8, GM CSF-.beta., IL-ra, TGF-.beta.1, TGF-.beta.2, TGF.alpha., EGF and PDGF-.beta., and as markers specific for an inflammatory reaction, collagenase I, the bradykinin, histamine and PAF receptors, and the system for transduction of a signal by the phosphoinositides.
2. Immortalized line according to claim 1, having the deposit number CNCM I-1777.
3. Process for identifying the mutagenic, toxic or beneficial effect of an agent on the metabolism of the cells of the cornea, in which (1) a culture comprising a cell line according to claim 1 or 2 is reacted with an agent suspected of being a mutagenic, toxic or beneficial agent for the metabolism of the cells of the human cornea, and (2) the effects of the said agent on the said cell line are determined.
4. Diagnostic kit comprising the immortalized epithelial cells of the human cornea according to claim 1 or 2 and reagents for determining a metabolic response of the said cells to mutagenic, toxic or beneficial agents.
5. Use of the immortalized epithelial cells of the human cornea according to claim 1 or 2 in processes for the identification of mutagenic, toxic or beneficial agents for the metabolism of the corneal cells.
6. Use of the immortalized epithelial cells of the human cornea according to claim 1 or 2 for developing recombinant cells expressing a gene of interest.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP96203707A EP0851028B1 (en) | 1996-12-24 | 1996-12-24 | Immortalized human epithelial cell line from cornea |
| EP96203707.3 | 1996-12-24 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2219038A1 CA2219038A1 (en) | 1998-06-24 |
| CA2219038C true CA2219038C (en) | 2007-03-27 |
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ID=8224764
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002219038A Expired - Fee Related CA2219038C (en) | 1996-12-24 | 1997-11-13 | Immortalized human corneal epithelial cell line |
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|---|---|
| US (1) | US6284537B1 (en) |
| EP (1) | EP0851028B1 (en) |
| JP (1) | JP4376978B2 (en) |
| AT (1) | ATE347587T1 (en) |
| AU (1) | AU728566B2 (en) |
| CA (1) | CA2219038C (en) |
| DE (1) | DE69636755T2 (en) |
| DK (1) | DK0851028T3 (en) |
| ES (1) | ES2277338T3 (en) |
| PT (1) | PT851028E (en) |
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| DE10050274A1 (en) * | 2000-10-09 | 2002-04-18 | Henkel Kgaa | In vitro assays for skin stress and skin ageing includes determination of spondin 2, cathepsin L, actin gamma 1 and vimentin fragments secreted by skin fibroblasts |
| EP1549255A4 (en) | 2002-09-13 | 2007-12-19 | Coopervision Inc | Devices and methods for improving vision |
| US7674456B2 (en) * | 2004-06-14 | 2010-03-09 | Charles Wiseman | Breast cancer cell lines and uses thereof |
| US20070225225A1 (en) * | 2006-02-28 | 2007-09-27 | Sharif Najam A | Use of natriuretic peptide receptor antagonists to treat ocular, otic and nasal edemetous conditions |
| US7883520B2 (en) | 2006-04-10 | 2011-02-08 | Forsight Labs, Llc | Corneal epithelial pocket formation systems, components and methods |
| US20080160366A1 (en) * | 2006-12-29 | 2008-07-03 | Allen Glenn M | Porous plate for a fuel cell |
| US20100120013A1 (en) * | 2008-11-07 | 2010-05-13 | Mb Research Laboratories, Inc. | Procedure for long term corneal culture |
| CN112126627B (en) * | 2020-09-30 | 2022-08-19 | 扬州大学 | Construction method and application of canine corneal epithelial cell immortalized cell line |
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| AU4986593A (en) | 1992-09-03 | 1994-03-29 | Quality Bakers New Zealand Limited | Prebuttered bread product |
| US5585265A (en) * | 1992-11-30 | 1996-12-17 | Gillette Company | Human corneal epithelial cell lines with extended lifespan |
| US5672498A (en) * | 1993-12-28 | 1997-09-30 | The Gillete Company | Human corneal epithelial cell lines with extended lifespan |
| JP3815794B2 (en) | 1994-09-08 | 2006-08-30 | ジェネンテク・インコーポレイテッド | Calcium phosphate transfection method |
| DE69623109T2 (en) | 1996-04-19 | 2002-12-12 | Nestle Sa | Human immortalized colon epithelial cells |
-
1996
- 1996-12-24 DK DK96203707T patent/DK0851028T3/en active
- 1996-12-24 ES ES96203707T patent/ES2277338T3/en not_active Expired - Lifetime
- 1996-12-24 DE DE69636755T patent/DE69636755T2/en not_active Expired - Lifetime
- 1996-12-24 AT AT96203707T patent/ATE347587T1/en active
- 1996-12-24 EP EP96203707A patent/EP0851028B1/en not_active Expired - Lifetime
- 1996-12-24 PT PT96203707T patent/PT851028E/en unknown
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1997
- 1997-11-13 CA CA002219038A patent/CA2219038C/en not_active Expired - Fee Related
- 1997-12-22 AU AU48539/97A patent/AU728566B2/en not_active Ceased
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| Publication number | Publication date |
|---|---|
| US6284537B1 (en) | 2001-09-04 |
| CA2219038A1 (en) | 1998-06-24 |
| DE69636755T2 (en) | 2007-10-11 |
| EP0851028B1 (en) | 2006-12-06 |
| DE69636755D1 (en) | 2007-01-18 |
| ATE347587T1 (en) | 2006-12-15 |
| PT851028E (en) | 2007-02-28 |
| JP4376978B2 (en) | 2009-12-02 |
| EP0851028A1 (en) | 1998-07-01 |
| JPH10215863A (en) | 1998-08-18 |
| AU728566B2 (en) | 2001-01-11 |
| ES2277338T3 (en) | 2007-07-01 |
| AU4853997A (en) | 1998-06-25 |
| DK0851028T3 (en) | 2007-02-26 |
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