MXPA00009558A - Immortalised cell lines derived from normal human skin tissues - Google Patents

Abstract

The invention concerns a cell line of human keratinocytes immortalised by at least a functional tumorigenic gene derived from a retrovirus characterised in that it is (1) non-tumorigenic,(2) preserves aptitude for differentiating and expressing proteins and enzymes which are expressed by normal differentiated keratinocytes even after a high rate of passages in tissue culture, and (3) forms a stratified and polarised epithelium comprising an ortho-keratotic stratum corneum, when cultivated in organotypical culture in a medium without serum and without feeder cell layer. The invention also concerns an improved method for immortalising human skin cells to obtain immortalised keratinocytes, characterised in that it comprises a step of infecting the keratinocytes using functional tumorigenic genes of at least two different retoviruses. The invention further concerns the use of said keratinocytes for immunologic, pharmacological, photo-toxicological and chemiotoxicological skin reactions for expressing heterologous genes and for constructing an artificial skin.

Description

IMMORTALIZED CELLULAR LINE DERIVED FROM NORMAL HUMAN LEATHER FABRICS TECHNICAL FIELD The present invention relates to new immortalized cell lines derived from normal human skin tissues, and having improved differentiation characteristics, to a new method for producing these cell lines, and to various uses thereof, particularly in the field of create artificial skin.

BACKGROUND TECHNIQUE The production of immortalized cell lines derived from normal human skin tissues has already been described. In general, the processes used for this purpose comprise the transformation of human skin cells, for example keratinocytes and melanocytes, which are cultured in vitro with agents that lead to in ortalization.

Immortalization is related to the production of cells, which can be cultured for prolonged periods of time in vitro, theoretically for an indefinite period. These cells are also designated continuous cell lines. In contrast thereto, non-immortalized cells are only capable of proliferating for a defined number of cell divisions in vitro. Immortalized cells are extremely advantageous, since they provide a stable, potentially unlimited source of cells that have defined characteristics. Typical agents for the production of immortalized cell lines and immortalized human skin cell lines comprise, in particular, for example, viruses, recombinant viruses and plasmids containing DNA sequences bearing the immortalization property. The most common process for producing immortalized human cell lines comprises probably the use of Simiesco 40 (SV 40) Virus sequences and more specifically of the Big T antigen (T-Ag) DNA of SV40 as the immortalizing agent. For example, Steimberg et al., J. Cell Phy., 123, 117-125 (1985); US4885228 (Reddel et al.); US4707448 (Major); Stoner et al., Cancer res., 51, 365-371 (1991); Chopra et al., In vitro Cell Dev. Biol., 30A, 539-546 (1994); Chopra et al., In Vitro Cell Dev. Biol., 27A, 763-765 (1991); Christian et al., Cancer Res., 47, 6066-6073 (1987); Rhi et al., Science, 227, 1250-1252 (1985); and Grubman et al., Gas trointest. Liver Physiol., 29, G1060-G1070 (1994) report the use of SV40 vectors and vectors containing the SV40 large T antigen sequence to produce immortalized human cell lines. The introduction of such sequences is usually effected by infection with the SV40 virus, or with a hybrid adenovirus 12 / SV40, or by transfection of cells with a recombinant plasmid containing the large terminal repeat units of the Rous sarcoma virus and the ori-region of SV40 regulator by coprecipitation in the presence of strontium phosphate (see B ash et al., Mol Cell. Biol., 7, 2031-2034, 1987). Another known process for the production of immortalized cell lines, and in particular of immortalized human keratinocytes, comprises the transfection or infection of cells with DNA sequences of human papillomavirus (HPV). For example, US5376542 (Schleger) describes the immortalization of human epithelial cells with the E6 and E7 genes isolated from HPV-16, 18, 31, 33 or 35, or with the E7 gene alone, to produce immortalized human non-tumorigenic cell lines. On the other hand, Barbosa et al., Oncogene, 4, 1529-1532 (1989); and Münger et al., J. Virol., 63 (10): 4417-4421 (1989) report the use of the E6 and E7 genes of HPV-16 and HPV-18 to produce immortalized human keratinocytes. On the other hand, Dürst et al., Oncogene, 1, 251-256 (1987) describes the immortalization of keratinocytes with the type 16 papillomavirus. However, although numerous groups described immortalized keratinocyte cell lines and their use in In vitro, keratinocyte cell lines immortalized in the current state of the art usually have one or more properties that render their use disadvantageous.

For example, the immortalized keratinocytes described previously exhibit one or more of the following properties: (i) reduction or loss of expression of differen- tiation markers, for example of proteins that are expressed by normal differentiated keratinocytes, (ii) modified growth properties. in tissue culture, and (iii) formation of a stratified and polarized epithelium that has a para-keratinocyte stratum corneum. To eliminate these disadvantages, EP780496 (Societé des Produits Nestle) proposes a new method to immortalize human keratinocytes or melanocytes using in their a new culture medium, the vector pLXSHD + SV40 (# 328), which is derived from the SV40 virus, or equally the vector pLXSHD + E6 / E7, which is derived from the human papillomavirus 16 (HPV-16). The immortalized cells obtained in this way conserve the capacity for differentiation and expression of proteins and enzymes, which are expressed by differentiated human keratinocytes and melanocytes, even after a high number of passages in culture. However, despite the previous description, there is still an important need in the practical field to have immortalized human keratinocytes possessing even more improved properties. Such cells would be extremely advantageous for numerous uses, particularly for analyzes that require highly differentiated skin cells.

BRIEF DESCRIPTION OF THE INVENTION For this purpose, the present invention relates to a human immortalized keratinocyte cell line, by means of at least one tumorigenic functional gene of retroviral origin, which is characterized in that: (1) it is non-tumorigenic, ( 2) retains the ability to differentiate and express proteins and enzymes expressed by normal differentiated keratinocytes after a high number of tissue culture passages, and (3) forms a stratified and polarized epithelium that has a stratum corneum of ortho-keratinocyte , if it is grown in organo-typical culture in a serum-free medium, and without a layer of feeder cells. Another object of the present invention is to provide a novel process for producing immortalized keratinocyte cell lines derived from normal skin tissues. Still another object of the present invention is to provide processes for the use of these keratinocyte cell lines according to the invention, for example for unological, armacological, photo- and chemo-toxicological skin reaction analysis, and for the expression of heterologous genes.

DESCRIPTION OF THE FIGURES - Figure 1 shows the construction of the SV40 derived retrovirus, ie the plasmid pLXSHD + SV40 (# 328), used to immortalize the keratinocytes of the present invention, Figure 2 represents the construct derived from the retrovirus of the virus 16 of the papilloma, ie the plasmid pLXSHD + E6 / E7 used to immortalize the keratinocytes of the present invention.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides immortalized, non-tumorigenic keratinocyte cell lines, ie cell lines that do not form tumors when injected under the skin of an animal, using at least 2 x 10 6 cells for each injection, for example . These cell lines also retain the ability to differentiate, and to express differentiation proteins expressed by normal keratinocytes after a high number of passages. The expression "high number of passages" indicates at least 10 passages in culture, preferably at least 20 to 30 passages, more preferably at least 50 passages, and theoretically an unlimited number of passages. For example, the immortalized keratinocytes produced according to the present invention express the differentiation proteins consisting of Kl / 10, K14 keratin, involucrin, filaggrin and loricrin even after a high number of tissue culture passages.

The immortalized keratinocytes of the present invention have a cytochrome p450 profile (CYP450) that is similar to, if not identical to, the profile of normal keratinocytes. For example, the cells of the present invention express CYP450 1A1, 2E1, 2C18 and 3A5. On the other hand, the immortalized keratinocytes of the present invention express phase II enzymes, by. example glutathione-S-transferase p (GSTp), in a manner comparable to normal immortalized keratinocytes. On the other hand, the immortalized keratinocytes of the present invention express proteins and enzymes involved in cell oxidation and in inflammatory responses, for example superoxide dismutase (SOD) and collagenase type I and tumor necrosis factor alpha (TNFa) after treatment with phorbol esters, in a manner similar or identical in the manner of normal differentiated keratinocytes. With these given characteristics, these cell lines represent an extremely interesting reproducible source for immunological, pharmacological, inflammatory, photo- and chemo-toxicological studies of cutaneous reactions. On the other hand, the immortalized keratinocyte lines of the present invention form, if they are cultured in organotypic culture in serum-free medium (for example the NR2 medium of Biofluids Inc., US, enriched with c, and CaCl 2 (1.5 mM) and without a layer of feeder cells (without fibroblasts), a stratified and polarized epithelium that has superficial keratinized layers, the so-called stratum corneum, which has an ortho-keratinocyte morphology, which means that the stratum corneum is depleted of nuclear cells, ie cells containing nucleus The preparation of a stratified and polarized epithelium with immortalized keratinocytes was previously achieved under classical culture conditions, i.e. by a technique using a medium containing calf serum and a cell layer feeders (Lechner et al., Virology, 185, 536-571, 1991) .However, the immortalized cells did not form the keratinized layers. normal superficial. For example, it is known that cell lines immortalized by virus 16 or 18 of human papilloma, or E6 / E7, form very disorganized epithelium (Blanton et al., Am. J. Pathol., 138, 673-685, 1991); Hudson et al., J. Virol., 64, 519-526, 1990; McCane et al., Proc. Nati Acad. Sci., 85, 7169-7173, 1988; Oodworth et al., Oncogene, 7, 619-626, 1992).

In contrast to this, the lines described in EP780496 (Societé des Produits Nestle), if cultured in organo-typical culture in a serum-free medium and without a layer of feeder cells, formed a stratified and polarized epithelium that had superficial keratinized layers. normal, but which nevertheless had a para-keratinocyte morphology, that is, the stratum corneum still contained cells with a nucleus. The immortalized keratinocytes of the present invention also absorb exogenous essential fatty acids (EFA) and have an unsaturated state and a prolongation of the EFA chain which corresponds perfectly to the unsaturated state and the chain extension of normal keratinocytes. In general, the immortalized keratinocytes of the present invention can be obtained according to the following process: (i) obtaining a human skin sample; (ii) prepare the skin sample to obtain primary keratinocytes capable of being cultured; (iii) culturing these primary keratinocytes in a serum-free medium, preferably in the NR-3 medium (described in EP780496) on culture plates having a coating that facilitates cell attachment and growth, the coating comprises fibronectin, SAP and type I collagen; (iv) replacing the serum free medium in a manner sufficient to obtain an optimum confluent growth of keratinocytes on the culture plates, the coating of the plate is maintained in a continuous manner; (v) separating keratinocytes in melanocyte culture and transferring the separated keratinocytes to an infection medium, preferably to the NR-3 medium, and preferably after treatment of the cells with a composition containing trypsin and EDTA using culture plates coated with a Similarly; (vi) infecting the cells with functional tumorigenic genes from at least two different retroviruses, such as the SV40 virus and the human papillomavirus; (vii) transfer immortalized keratinocytes to a serum-free proliferation medium to culture plates coated in advance in a similar manner, preferably to the NR-2 or NR-3 medium (means described in EP780496, the composition of which is incorporated herein by way of reference); and (viii) transferring immortalized keratinocytes after proliferation to a convenient differentiation medium, which has a high calcium content (1.5 mM), preferably to the NR-2 medium enriched with EGF5 (5 ng / ml) and vitamin C (38 μg / ml). In detail, step (i) usually comprises obtaining tissue samples of human skin from normal human donors, for example samples obtained during a surgical intervention or a pediatric intervention. The immortalization of a single sample of skin cells, ie of an autologous sample of skin cells, allows the production of immortalized keratinocyte cell lines having defined characteristics, for example a profile of a particular receptor that is characteristic for a private donor The skin tissue sample is subsequently prepared in step (ii) such that it is suitable for an in vitro culture. This preparation is preferably effected by initially washing the skin tissue sample, for example using the medium used for the culture. Preferably, this operation is carried out in the NR-2 medium, which is a serum free medium, the exact composition of which is described in EP780496, which proved to be advantageous for the culture of normal keratinocytes. After washing, the skin tissue sample is preferably shaved, for example by means of a dermatome, and is subsequently cut into small pieces. The resulting sections of skin are then preferably separated into dermis and epidermis. This can be achieved by physical and / or enzymatic means. For example, this can be done by treatment with trypsin, for example by flotation of the skin tissue samples in a trypsin solution (for example about 0.5%) containing EDTA (for example about 0.1%) for a while. sufficient to cause separation of the cells, for example for a time of about 30 to 60 minutes at a temperature of 37 ° C, or for example overnight at 4 ° C. The dermis is separated, and the epidermis is subsequently placed in a medium to achieve a suspension. Preferably, the means for achieving a suspension contains a solution of soybean trypsin inhibitor (SBTI) and is contacted with the cells for a sufficient time, usually about 5 minutes, to inactivate the trypsin and cause the release of the cells A tissue culture medium, preferably the NR-2 medium, devoid of serum (described in EP780496) and a filter (for example a 100 nm filter) are subsequently added to obtain the desired cells, ie, the keratinocytes. The resultant primary keratinocytes obtained in step (ii) are subsequently used to seed a serum-free medium, preferably the NR-3 medium (described in EP780496), at a suitable concentration of cells, preferably of about 1.2 × 10 4 cells / c 2, on previously coated culture plates. However, it is possible to vary this concentration of cells within wide limits. The culture plates are preferably provided with a coating containing a composition that proved to increase the binding and growth of keratinocytes, more precisely a solution of fibronectin, SAP and type I collagen. In step (iv), the culture medium is replaced as frequently as necessary to obtain optimal cell growth. Preferably, the medium is replaced each time after about two days. However, this is variable, depending on the particular sample of skin tissue. After having obtained an almost total confluence, for example a confluence of about 90%, which takes place after a period of time of about 10 to 14 days, the keratinocytes and the melanocytes are separated. This can be done by any known means that allows adequate separation of cells without any detrimental effect on melanocytes and keratinocytes. For example, this can be effected by differential treatment with trypsin. Preferably, the melanocytes and keratinocytes are treated with a solution of tripsiha / EDTA and are subsequently transferred to a selection medium. In the case of keratinocytes, the cells are preferably treated for a period of time of about 5 to 10 minutes with a trypsin / EDTA solution (0.025% / 0.01%) and are subsequently used in step (v) to be planted in the NR-3 medium on precoated plates. Keratinocytes are subsequently treated with an agent for immortalization. The cells can also be frozen until immortalization is effected, for example in liquid nitrogen. Infection and immortalization are preferably carried out using functional tumorigenic genes from at least two different retroviruses, such as the SV40 virus T-Ag and the HPV16 E6 / E7. Each of these genes can be present in an independent retroviral construct, for example in the retroviral vector pLXSD + SV40 (# 328), shown in Figure 1 and described by Stockshlaeder et al. (GeneBank, access number M64753; Human Gen. Therapy, 2, 33-39, 1991) and the retroviral vector pLXSHD + E6 / E7, which are shown in Figure 2. The retroviral vector pLXSD + SV40 (# 328) contains, among other sequences, the T-Ag sequence of SV40, the sequence of the large 5 'and 3' terminal sequences of SV40, the sequences of pBR322, which allow the replication of E. coli, a multiple cloning cycle, a polyadenylation sequence of SV40. In the location of the gene coding for the T antigen, the vector pLXSHD + E6 / E7 contains the Ncol / Cfol fragment of the E6 / E7 gene derived from the human papillomavirus 16. After immortalization, the cells are subsequently subjected to the necessary number of passages during the culture, and the resulting immortalized cells are subsequently transferred to a proliferation medium during step (vii). This transfer is preferably effected during the second passage. This proliferation medium may be a serum-free medium, preferably the NR-2 or NR-3 medium. The immortalized cells are cultured on culture plates previously coated in a continuous manner, the coating again comprises a solution of fibronectin, SAP and type I collagen. After the multiplication of the immortalized cells in a proliferation medium (preferably NR-2) the keratinocytes are transferred in step (viii) to an environment that causes the differentiation of normal and immortalized keratinocytes, preferably to an environment that stimulates the conditions prevailing in the skin, thus generating the organization of keratinocytes in a stratified and polarized epithelium that has normal superficial keratinized layers. For this purpose, the cells can be cultured in a serum free medium having a high calcium content, such as the NR-2 medium, which comprises about 1.5 mM calcium, about 5 ng / ml EGF, and about 38 μg / ml of vitamin C, the culture is carried out in plates during 2 to 3 weeks in liquid-air interface, for example on plates with 12 Falcon cavities No. 3043, each cavity has a Falcon insert No. 3180 in where the keratinocytes are developing at the air / liquid interface. The air consists of the atmosphere contained in the internal space of the insert, which is devoid of feeding medium. The liquid of the feeding medium contained in the cavity, the medium that passes through the membrane of the insert on which the keratinocytes are developing. With respect to the properties of the immortalized keratinocytes of the present invention, these are perfectly suitable for immunological, pharmacological, photo- and chemo-toxicological analyzes of skin reactions. For example, the immortalized keratinocyte cell lines of the present invention can be used for analyzes that require differentiated skin cells, for example, studies on barrier function. (keratinization) of reconstructed skin tissue, studies on the metabolism of differentiated keratinocytes (metabolism of fatty acids, antioxidant metabolism), studies related to the effects of UV irradiation on skin cells, studies related to the effects of potential irritant agents of the skin and sensitization on skin cells, studies on lipid metabolism, local treatment and / or by means of xenophobic agents (for example cosmetic oils, selecting feasible protective compounds, for example photo-protective agents), studies on inflammation and over skin irritation, etc. On the other hand, the keratinocyte cell lines produced according to the present invention are used to select potential anticancer compounds, and potential compounds for the treatment of skin diseases. This usually implies that the cell line is contacted with such compounds for a given period of time, and the determination of a potential induction of detrimental effects of any kind, eg genotoxicity, the formation of DNA adducts, mutagenicity, cellular transformation or cytotoxicity. On the other hand, the immortalized keratinocyte lines of the present invention can be used in DNA mutagenicity assays, assays for the selection of mutagenic skin agents, assays for the identification of chromosome alteration agents, studies on malignant transformation, studies on cellular biochemistry (for example assays of CYP450 activation), the selection of compounds and compositions, for example of cocktails of essential fatty acids, involved in inflammatory and allergic reactions, assays for the activation of collagenase (in connection with the inflammation), which involve TNFa, and the detection of interleukin. With respect to the fact that the cell lines according to the present invention form a stratum corneum of ortho-keratinocyte, they are particularly well adapted to participate in the preparation of pielartificial, to be proposed for studies on mutagenicity, for immunological, pharmacological studies, photo- and chemo-toxicological mentioned above. This skin can be constituted only of a keratinocyte epithelium according to the invention, but preferably it also comprises collagen, fibroblasts, even melanocytes, to achieve a better resemblance to the normal human pile, for example. On the other hand, the keratinocyte cell lines of the present invention are capable of expressing recombinant proteins, for example human polypeptides and proteins, and also produce RNA and DNA.

Among the immortalized keratinocyte cell lines produced according to the present invention, only the cell line DK7-NR was deposited under the terms of the Budapest Treaty, as an example, on March 19, 1998 in the Collection Nationale de Culture de Microorganisme (C.N.C.M.) that has the address 25, rue de Docteur Roux, 75724 Paris, France, and has received the deposit number CNCM 1-1996. This deposit was made under the terms of the Budapest Treaty. All restrictions regarding the availability of this cell line will be irrevocably suppressed after the granting of a patent corresponding to the present application or another application that claims the priority right of this request. Other features of the present invention will become apparent from the following description of the examples, which are given to illustrate the present invention, and should not be construed as limiting. When not otherwise indicated, cell manipulation, vector preparation, cell transformation and all other technical processes are performed according to the protocols described in the publication of Sambrook et al., (Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, USA, 1989).

Example 1: Preparation and characterization of cell lines Breast skin tissues were taken. After separation of the dermal and epidermal parts, the dermis was cut into small pieces of 0.2 x 0.2 mm and fixed on a 6 cm culture dish with serum. Minimal essential Dulbecco's medium (DMEM, 10% fetal calf serum) was added after 2 to 4 hours. This culture of transferred fragment is subsequently incubated until excessive growth of fibroblasts becomes visible. The cultures of confluent fibroblasts were separated and multiplied to obtain frozen stock cultures. The culture boxes were ed with primary cells coated in a continuous manner with a "cocktail" coating previously described for bronchial cells (Lechner et al., J. Tiss, Cult.Meth., 9: 43-49 (1985)). . After achieving an almost total confluence, for example of about 90% confluence, which usually occurs after a period of time of about 10 to 14 days, the keratinocytes and melanocytes separate. For this, the culture is treated with a trypsin / EDTA solution (0.025% / 0.01%) for 5 minutes, and the melanocytes are subsequently harvested, which first separated them from the keratinocytes. The primary keratinocytes are subsequently cultured to the desired number of cells, in serum-free NR-3 medium, by means of the coated culture dishes described (The NR-3 medium favors the growth of keratinocytes compared to melanocytes). Subsequently, the encapsulation cells "3T3 fibroblasts of the encapsulation cell line" are transfected with plasmids pLXSHD + SV40 (# 328) and pLXSHD + E6 / E7 according to the protocol of Pfeifer et al. (Meth. Cell Sci., 17, 83-89, 1995) with the proviso that the virus is collected after encapsulation in a cell line, which grows in DMEM medium with 10% fetal bovine serum. Subsequently, the keratinocytes are infected with the virus to cause immortalization. During infection, the serum-free medium PC-1 is also used, which is described in the publication by Pfeifer et al. After immortalization, the immortalized keratinocytes are transferred to the NR-2 or NR-3 proliferation medium, using pre-coated culture dishes. After proliferation of the cells to a desired number of cells, the cells are transferred to a differentiation medium suitable for the culture of normal and immortalized keratinocytes (NR-2). It can be shown that immortalized keratinocytes have improved cell growth in a high number of passages in culture, which can be compared to that described for the cell lines of EP780496. The expression of CYP450, 1A1, 1A2, 3A5, 2E1, 2B6, 2A6 and 2D6 is analyzed in skin cells consisting of normal keratinocytes and immortalized by the DNA polymerase chain reaction at room temperature (expression of mRNA). The profile of CYP450 expressed by immortalized keratinocytes is particularly similar, even identical, to that of normal keratinocytes. The cell lines respond to the induction agent of CYP450 which consists of benz (a) pyrene, in the same way as cells not immortalized even in a high number of passages. Differentiation markers are analyzed by means of specific antibodies against T-Ag, involucrin, filaggrin, loricrin, vimentin and keratins K4, K7, K8, K10 / 1, K13, K14, K17, K18 and K19. The greater capacity for the demonstration of differentiation capacity could be demonstrated for the DK7-NR cell line. Glutathione-S-transferase (GST) is analyzed by Western Blot and Northern Blot. All keratinocyte lines express strongly the messenger RNA for GSTp. The profile of exposure to GSTa, GSTμ and GSTp in cell lines is similar to that of normal keratinocytes. To analyze and compare the desaturation and elongation of the AGE added to the keratinocytes, immortalized keratinocytes (and normal keratinocytes) are treated with linoleic acid (LA, 15 μmol) and linolenic acid (LN, 15 μmol). For these experiments, the medium NR-2 (Biofluids Inc.), deficient in AEG, is used. The cell cultures are treated after they have reached confluence, and are transferred from the medium to an NR-2 medium having a high calcium content (1.5 M). The cells are treated for 4 days with the AGEs (renewed after 2 days). The analysis of the AGE is carried out by extraction and separation of the phospholipids by CCD (thin layer chromatography), and the quantification of the methyl esters of the fatty acids is carried out by CGL (Gas-liquid chromatography). The formation of the desaturation and the elongation of LA (20: 4n-6 and 22: 4n-6) and of LN (20: 5n-3, 22: 5n-3 and 22: 6n-3) could be demonstrated. The metabolic profile was consistent with that observed with normal keratinocytes. All cell lines were hypodiploid with the majority of chromosome counts in the diploid cell interval. Apart from the cells analyzed, no other cells were detected in the cultures. This result confirms the purity of the cell lines, and the absence of any contamination originating from other sources. The tumorigenicity of the immortalized keratinocytes was determined by subcutaneous injection (1-2 x 106 keratinocytes) to athymic mice. The lines of the keratinocytes tested, and particularly of the DK7-NR line, are not tumorigenic in athymic mice.

Example 2: Preparation of an epithelium The NR-2 medium, which contained 750,000 cells of a cell line according to Example 1, was prepared, 0. 5 ml of this medium was placed in the Falcon inserts No. 3180, these were placed in the cavities of the Falcon No. 3043 plates that already contained 2 ml of fresh NR-2 medium, the keratinocytes were cultured for 2 days under favorable conditions for the growth of the keratinocytes. On the third day the medium contained in the insert was removed, and the cells were left outdoors. The medium contained in the cavities was periodically changed every 2 days with NR-2 medium supplemented with EGF (5 ng / ml), vitamin C (38 μg / ml) and CaCl2 (1.5 mM). After 2 to 3 weeks in culture at the air-liquid interface, the epithelium formed in this manner was collected, fixed with picric acid and the morphology analyzed. The results show that the keratinocyte cell lines, in particular the DK7-NR cell line, form a stratified and polarized epithelium, the so-called stratum basale, which contains cells that have a cuboidal morphology identical to that of normal cells. The stratum corneum has an ortho-keratinocyte morphology, that is, it is devoid of cells that contain a nucleus. Formation of the ortho-keratinocyte cell layer was not possible with other immortalized cell lines known hitherto. For example, under identical conditions the line DK2-NR (EP780496) forms a para-keratinocyte stratum corneum, that is, the connnified cell layer still contains cells with a nucleus. Only the morphology of ortho-kerat inocito delstratum corneum reflects the normal situation of human skin. In fact, the stratum corneo of para-keratinocyte is characterized by an abnormal hypertrophy of the epithelium, which leads to disorders, such as psoriasis or neoplasia, for example.

Example 3: Irritation test The cell lines obtained in Example 1, in particular line DK7-NR, were cultured in the NR-2 medium. The induction of the "stress gene" TNFa (necrosis factor of alpha tumors) after treatment with skin-irritating agents, consisting of PMA (12-myrist tato-13-Forbol acetate) and UV-B (ultraviolet B radiation) was analyzed by the method of Northern and by biological tests. The results showed that the cell lines, and particularly the cell line DK7-NR respond to PMA and UV-B, and express the TNFa protein even after a high number of passages.

Example 4: Construction of artificial skin The membrane of a Falcon No. 3180 insert was replaced by a sheet of the benzylic ester of hyaluronic acid (Hyaff 11). The lower part of the insert was seeded with primary human fibroblasts (0.1 x 106 cells in 0.2 ml of medium), after 30 minutes in response, the insert was filled with the DMEM medium containing 10% fetal calf serum, incubation was carried out at 37 ° C under an atmosphere containing 5% carbon dioxide for several days, the insert was emptied and 0.5 ml of the fresh medium NR-2 containing 750,000 cells of the DK7-NR cell line, the inserts were supplied. were placed in the cavities of Falcon No. 3043 plates that already contained 2 ml of fresh NR-2 medium, and the cells were cultured for 2 days under favorable conditions for the growth of keratinocytes. On the third day, the medium contained in the insert was removed, and the cells were left outdoors. The medium in the cavities was changed periodically every 2 days, with NR-2 medium supplemented with EGF (5 ng / ml), vitamin C (38 μg / ml) and CaCl2 (1.5 M). After 2 to 3 weeks in culture at the air-liquid interface, it was possible to observe the formation of an artificial pile that presents the characteristics of normal skin.

Claims (15)

1. CLAIMS 1. A human keratinocyte cell line immortalized by means of at least one functional tumorigenic gene of retroviral origin, characterized in that it is: (1) non-tumorigenic, (2) retains the ability to differentiate and express proteins and enzymes that are expressed by normal differentiated keratinocytes even after a high proportion of tissue culture passages, and (3) it forms a stratified and polarized epithelium comprising an ortho-keratotic stratum corneum, when it is grown in organotypic culture in a medium without serum and without a layer of feeder cells.
2. 2. The human keratinocyte cell line according to claim 1, immortalized by means of functional tumorigenic genes of retroviral origin from at least two different retroviruses.
3. 3. The human keratinocyte cell line according to claim 2, characterized in that the functional tumorigenic genes are derived from the SV40 virus and the human papillomavirus 16.
4. 4. The human keratinocyte cell line according to claim 3, characterized in that each functional tumorigenic gene is contained in an independent retroviral construct.
5. 5. The human keratinocyte cell line according to claim 1, characterized in that it is the cell line DK7-NR that has the deposit number according to the Budapest Treaty CNCM I-1996.
6. 6. An improved process for immortalizing human skin cells to obtain immortalized keratinocytes, characterized in that it comprises the following steps: (i) obtaining a sample of human skin; (ii) preparing the skin sample for in vitro culture; (iii) obtaining keratinocytes from this prepared sample of skin tissue and seeding a growth medium without serum with the keratinocytes, on culture plates provided with a coating comprising fibronectin, SAB and collagen type I, which facilitates the fixation and the growth of the cells; (iv) replacing the medium in a manner sufficient to obtain optimal confluent growth of the cells in culture, by continuously maintaining the coating on the plate; (v) transferring keratinocytes to a serum-free screening medium on culture plates previously coated in a similar manner; (vi) infecting keratinocytes with functional tumorigenic genes from at least two different retroviruses; (vii) transferring the resulting immortalized keratinocytes to a proliferation medium without serum suitable for the proliferation of immortalized keratinocytes on culture plates coated in advance in a similar manner; (viii) transferring the resultant proliferated keratinocytes to a serum-free differentiation medium, which has a high calcium content, on culture dishes previously coated in a similar manner.
7. 7. The process according to claim 6, characterized in that the functional tumorigenic genes are derived from the SV40 virus and the human papillomavirus 16.
8. 8. The process according to claim 7, characterized in that each functional tumorigenic gene is contained in an independent retroviral construct.
9. 9. The process according to claim 8, characterized in that the retroviral constructs used are the vectors pLXSHD + SV40 (# 328) and pLXSHD + E6 / E7.
10. 10. The process according to claim 6, characterized in that the serum-free medium in steps (iii), (v) or (viii) is the NR-3 medium.
11. 11. The process according to claim 6, characterized in that the differentiation means in step (viii) is a modified NR-2 medium having a calcium content of at least 1.5 mM.
12. 12. Use of the keratinocytes according to claim 1, for immunological, pharmacological, photo- and chemo-toxicological analysis of skin reaction, and for the expression of heterologous genes.
13. 13. The use according to claim 12, characterized in that the line is used DK7-NR, which has the deposit number according to the Budapest Treaty CNCM 1-1996.
14. 14. Artificial skin, characterized in that it comprises a keratinocyte cell line according to claim 1.
15. 15. Artificial skin, according to claim 14, characterized in that it comprises the DK7-NR cell line having the deposit number according to the Budapest Treaty CNCM 1-1996. SUMMARY OF THE INVENTION The present invention relates to a human keratinocyte cell line immortalized by means of at least one functional tumorigenic gene of retroviral origin, characterized in that it is (1) non-tumorigenic, (2) retains the ability to differentiate and express proteins and enzymes which are expressed by normal differentiated keratinocytes even after a high proportion of passages in tissue culture, and (3) forms a stratified and polarized epithelium comprising an ortho-keratotic stratum corneum, if it is grown in organotypic culture in a medium without serum and without feeder cell layer. An improved method for immortalizing human skin cells to obtain immortalized keratinocytes is characterized in that it comprises the following steps: (i) obtaining a human skin sample; (ii) preparing the skin sample for in vitro culture; (iii) obtaining keratinocytes from this prepared sample of skin tissue and seeding a growth medium without serum with the keratinocytes, on culture plates provided with a coating comprising fibronectin, SAB and collagen type I, which facilitates the fixation and the growth of the cells; (iv) replacing the medium in a manner sufficient to obtain optimal confluent growth of the cells in culture, while maintaining the plate coating continuously; (v) transferring keratinocytes to a serum-free screening medium on culture plates previously coated in a similar manner; (vi) infecting keratinocytes with functional tumorigenic genes from at least two different retroviruses; (vii) transferring the resulting immortalized keratinocytes to a proliferation medium without serum suitable for the proliferation of immortalized keratinocytes on culture plates coated in advance in a similar manner; (viii) transferring the resultant proliferated keratinocytes to a serum-free differentiation medium, which has a high calcium content, on culture dishes previously coated in a similar manner. The use of keratinocytes for immunological, pharmacological, photo- and chemical-toxicological analysis of skin reaction and for the expression of heterologous genes, and for the construction of artificial skin. Figure 1.