Cell line immortalized keratinocytes and melanocytes (options), the way the immortalization of human skin cells to obtain immortalized keratinocytes, the way the immortalization of human skin cells to obtain immortalized melanocytes, serum-free medium (options), analysis of skin reactions

 

The invention relates to biotechnology and can be used to obtain immortalized human skin cells. The cell line of immortalized human keratinocytes DK2-NR, DK3-NR, FK2-NR and immortalized melanocytes DM2-NR receive by planting skin cells to serum-free medium NR-2, then s is transferred onto selection medium NR-3, and melanocyte - NR-4 environment. Keratinocytes and melanocytes infect retroviral construct, then an immortalized keratinocytes grown in proliferative medium NR-2 and differentiate in a medium containing 1.5 mm calcium, immortalized melanocytes proliferous in the medium M2. Immortalized keratinocytes and melanocytes are used for analysis of skin reactions. 9 C. and 3 h. p. F.-ly, 3 ill., 10 table.

The invention relates to an improved continuous (immortalized) cell lines derived from normal tissues of human skin, in particular the lines of keratinocytes and melanocytes, which retain the ability to Express proteins of differentiation characteristic of differencirovannyh melanocytes or keratinocytes, even in the case of a large number of passages. The present invention relates also to a new, not Anee described getting immortalized cell lines, obtained from the tissues of human skin. Typically, such methods include transfection or transformation of human skin cells such as keratinocytes and melanocytes in vitro agents that provide immortality.

Immortality relates to the production of cells that are capable of cultivation for a long period of time in vitro, ideally indefinitely. Such cells are also referred to as continuous cell lines. In contrast neomalthusianism, cells can grow for a finite number of divisions in vitro. A significant need in immortalized cells because they provide a stable, potentially unlimited supply of cells with certain characteristics. Conventional agents to obtain immortalized cell lines and immortalized cell lines of human skin, in particular, include, for example, viruses, recombinant viruses and plasmids which contain DNA sequences that provide immortality.

Probably the most common way of receiving the immortalized cell lines of human involves using S40 sequences, and, more specifically, SV40 large T antig the 85238, issued December 5, 1989; Major, U.S. patent 4707448, issued November 17, 1987; Stoner et al., Cancer Res., 51:365-371 (1991); Chopra et al., In Vitro Cell Dev.Biol., 30A: 539-546 (1994); Chopra et al. , Jn Vitro Cell Dev.Biol., 27A:763-765 (1991); Christian et al., Cancer Res., 47:6066-6073 (1987); Rhim et al.. Science 227:1250-1252 (1985), and Grubman et al. , Gastrointest. Liver Physiol., 29:G1060-G1070 (1994) suggest the use of SV40 vectors and SV40 large T antigen sequence containing vectors to obtain immortalized cell lines of human rights. The introduction of such sequences is usually carried out by infection using SV40 virus or hybrid adenovirus-12 SV40 hybrid virus, or by transfection of cells with a recombinant plasmid containing the long terminal repeat (repeat) of Rous sarcoma virus and Ori-SV40 early lot due to co-deposition phosphate strontium (see Brash et al., Mol. Cell Biol., 7:2031-2034 (1987).

Another known method for obtaining immortalized cell lines, in particular immortalized human keratinocytes, is the way, which include transfection or infection of cells DNA sequences of human papillomavirus. For example, in U.S. patent 5376542, issued on December 27, 1994, describes immortality epithelial human cells allocated by HPV-16, 18, 31, 33 and the IU, Barbosa et al., Oncogene, 4:1529-1532 (1989), and Hunger et al., J. Virol., 63(10):4417-4421 (1989), describe the use of HPV-16 and HPV-18 E6 and E7 genes to obtain immortalized human keratinocytes.

However, although several research groups have reported immortalized cell lines keratinocytes and their use in In vitro assays, previously obtained an immortalized cell line of keratinocytes and melanocytes usually had one or more properties, which made them use unprofitable. For example, an immortalized keratinocytes reported previously, have one or more of the following characteristic: (i) reduction or loss of expression diferenciada markers, such as proteins, which are expressed normal differentsirovannymi by keratinocytes, and (ii) modifying the growth characteristics in tissue culture.

For example, Jetten et al., J. Invest.Dermatol., 92:203-209 (1989) reported SV40 immortalized the keratinocytes obtained after a large number of passages (the number of passages is greater than 12) using the vector NHEK-SV40-T8-1, which was incapable of differentiation. Similarly, Bernard et al., Cancer Res., 45:1707-1716 (1985), report on the allocation of immortalizing cell lines keratinocytes, named as SVK14, which, as Sony K1/10 ( 53 KD and 50 KD keratin (keratin K14), proteins that are normally expressed by differentiated keratinocytes.

In addition, Steinberg et al., J. Cell Physiol., 123:117-125 (1985), reports SV40 transformed keratinocytes, which gradually lose their ability to Express keratins, which are characteristic of normal keratin cytoskeleton. This loss of normal expression of keratins occurs after about 10-15 passages. Next, Hronis et al., Cancer Res., 44: 5797-5804 (1984), describe SV40 DNA immortalized keratinocytes that have lost the ability to produce K5, K6, K14/15, C and R17 keratins and involucrin, proteins that are characteristic of normal differentiated keratinocytes. Next, Morris et al., Proc.Natl.Acad. Sci, USA, 82:8498-8502 (1985), describe SV40 immortalized keratinocytes that when a large number of passages (more than 14 passages) show a strongly reduced expression of keratins class II and class I. for example, these keratinocytes almost does not Express K13(Id).

In addition, Banks-Schlegel et al., J. Cell Biol., 96: 330-337 (1983), disclose SV40 immortalized keratinocytes, which show altered growth characteristics in tissue culture. For example, in contrast to normal keratinocytes these immortalized cells require for growth T feeder SNO used the method feeder cells (when fibroblasts usually act as "feeder" cells) and usually led culture of cells containing serum environment. For example, Sexton et al., "Stable transfection of human keratinocytes: HPV immortalization", Keratinocyte Methods, eds., Leigh I. M. et al., University Press, 179-180 (1994); Garlick et al., "Retroviral Vectors", Keratinocyte methods, eds. Leigh I. M. et al. , Cambridge University Press, 181-183 (1994), describe the use of a medium containing fetal calf serum and feeder cells in the isolation and production of immortalized keratinocytes.

The use does not contain serum medium in the isolation and production of immortalized epithelial cells has been disclosed previously. For example, Barbosa et al., Oncogene, 4:1529-1532 (1989), describes the initial cultivation of human keratinocytes, transfection by electroporation or lipofection, not containing serum medium with low calcium content to confluently.

However, regardless of that previously reported, there is still a significant need in immortalized human keratinocytes and melanocytes, which would have improved characteristics, in particular, which would preserve the potential of the normal differentiation of keratinocytes and melanocytes, and which expressed the differentiation proteins characteristic of differentiated melanocytes and keratinocytes, even after a large number of passages. These skin cells. In addition, there is a need for an improved culture medium capable of supporting primary and immortalized keratinocytes and melanocytes, as well as in improved methods of cultivation, which would require the use of feeder cells.

The purpose of the invention the present invention is to obtain improved continuous (immortalized) cell lines derived from normal tissue of human skin, especially immortalized cell lines of keratinocytes and/or melanocytes derived from normal tissue of human skin, which would preserve the ability to differentiate and to Express the proteins of differentiation even after a large number of passages. More specifically, the present invention is to obtain immortalized keratinocytes, which would preserve the ability to Express keratins, cytochromes and other proteins differentiation, for example involucrin, filaggrin and loricrin that are either weakly expressed or not expressed at all termed normal cell lines keratinocytes. The next objective of the invention to provide immortalized keratinocytes and melanocyte, especially such enzymes phase II, such as glutathione-S-transferase, as well as enzymes and/or proteins that are involved in cellular oxidation and inflammatory reactions.

Another aim of the invention is the creation of a new, not containing serum medium for the cultivation, production and maintain normal or continuous keratinocytes and/or melanocytes in tissue culture. This new, not containing serum medium can also be used for separation, stabilization and the immortalization of human skin cells to produce continuous lines of melanocytes and keratinocytes according to the method of the present invention. Thus, the specific objective of this invention is to provide a fully defined culture medium (without unknown or ill-defined additions) for cultivation of keratinocytes without feeder cells containing epinephrine, which, as it has been unexpectedly discovered is a strong potentiation growth of normal keratinocytes in not containing serum environment.

Another objective of the present invention is to provide a new method for the selection, establishment and the immortalization of human skin cells to provide a continuous cell lines melanocyte is containing a series of serum environment according to the method of the present invention and additional "cocktail", containing fibronectin, BSA and collagen type I without feeder cells (e.g. fibroblasts).

Another aim of the invention is the creation of primary keratinocytes or melanocytes obtained under conditions without serum, without the use of any feeder cells, and these primary keratinocytes and melanocytes are used as skin grafts and in ex vivo genetic therapy.

Another aim of the invention is to provide methods of using such new and improved continuous cell lines keratinocytes and melanocytes, for example, for analyses, immunological, pharmacological, photo and homotoxicological skin reactions and for the expression of heterologous genes.

A brief description of the drawings Fig.1 compares the growth (in units of number of cells) neomalthusian DKO-NR keratinocytes in three different environments, i.e., NR-3, supplemented by epinephrine, a modified MCDB MCDB 153 and 153 after 6 days.

Fig. 2A represents the SV40 retroviral design pLXSHD+SV40( 328), preferably used for the immortalization of the considered melanocytes and/or keratinocytes.

Fig.2b represents HPV16 retroviral design PLXSHD+E6/E7.

Detailed description of the invention In nastojaschemu skin, i.e., an immortalized keratinocytes and melanocyte that retain the ability to differentiate and which retain the ability to Express proteins of differentiation, which is expressed by normal keratinocytes or melanocytes, even when a large number of passages. Under a large number of passages imply, at least 10 passages in culture, preferably 20-30 passages, more preferably at least 50 passages and, ideally, an indefinite number of passages. For example, an immortalized keratinocytes produced by the method of the present invention, Express proteins differentiation keratin K1/10, keratin K14, involucrin, filaggrin and loricrin even after a large number of passages in tissue culture. This is in contrast to immortalized the keratinocytes, which were previously reported that are either not expressed these proteins differentiation, or weakly expressed these proteins differentiation.

Further, in the present invention proposed primary keratinocytes and melanocytes obtained under conditions without serum and without feeder cells that retain the ability to differentiate and Express proteins characteristic of diffel cytochrome P450 (CYP450), which is similar, if not identical, to the profile of normal keratinocytes. For example, consider the cells Express CYP450 A, but not CYP450 3A4. Moreover, the immortalized keratinocytes Express the enzymes of phase II, for example, glutathione-3-transferase (GST), and more specifically, GSTGSTand GSTcomparable to normal neomalthusianism by keratinocytes.

Next, consider an immortalized keratinocytes Express protine and enzymes involved in cellular oxidation and inflammatory reactions, such as superoxide dismutase (SOD), and collagenase type I and tumor necrotic factor(TNFafter processing esters of phorbol, similar or identical to the normal differentiated keratinocytes. Given these characteristics, these cell lines provide a highly stable, reproducible source of cells to study immunological, pharmacological, inflammatory, photo and hematoxiline skin reactions.

Next, consider an immortalized melanocytes Express endogenous proteins associated with Melanie and cell lines melanocytes under cultivation in organotypic culture form a strongly stratified and polarized epithelium, containing the corneal surface layers (stratum corneum). Only this was achieved previously in normal culture conditions, i.e. in a medium containing serum, with use of a feeder layer (see, for example, Lechner et al., Virology, 185:536-571, 1991).

Next, consider an immortalized keratinocytes and melanocytes derived from normal skin in conditions without serum and without the use of any feeder cells. Usually considered an immortalized keratinocytes and melanocytes receive, using the following stages: (i) obtaining a tissue sample of human skin; (ii) preparation of a specified sample of skin for in vitro cultivation; (iii) the receipt of keratinocytes and/or melanocytes from the specified prepared tissue sample and the sowing of these keratinocytes and/or melanocytes in not containing serum to the culture medium, preferably either in the environment NR-3, or NR-4 (melanocytes) (described earlier) on the culture plate with a coating that facilitates the attachment and growth of cells moreover, the said coating contains fibronectin, collagen type 1 and BSA; (iv) if necessary, change the environment to optimize confluent growth of cultured cells by maintaining continuous coverage on cultural plastered, in the same way pre-coated culture plates;
(vi) the infection of keratinocytes or melanocytes retroviral construct, preferably a design based on SV40 or human papilloma virus 16, for example SV40 plasmid pLXSHD+SV40( 328), which contains large T antigen (T Aq) Simian virus 40, or plasmid pLXSHD+E6/E7, which contains the E6/E7 gene of the virus papillomavirus 16 (HPV16) (see below);
(vii) transferring the received immortalized keratinocytes or melanocytes on proliferation environment suitable for proliferation of immortalized keratinocytes or melanocytes in the same way pre-coated culture plates, preferably NR 2 and NR-3 medium (described later) and melanocyte medium M2 (source M. Olsson Inst. of Dermatology, Uppsala, Sweden), and
(viii) transferring the received proliferating keratinocytes by differentiating medium, preferably NR-2 (described later), or a modified MCDB 153 medium (described below), which contains a high concentration of calcium, preferably 1.5 mm, similarly pre-coated culture drives (Boyce et al. , J. Tissue Cult. Meth., 9:83-93, 1985; Pittlekow et al., J. Invest.Dermatol., 86:410-417, 1986).

More specifically, step (i) typically comprises obtaining samples of tissue corticalization one sample of skin cells, i.e. autological sample of skin cells, allows to obtain an immortalized cell line of keratinocytes and melanocytes that exhibit certain characteristics, such as specific receptor profile that is characteristic of a particular donor.

The sample was then prepare skin for stage (ii) so that it was suitable for cultivation in vitro. This is carried out, preferably by first rinsing the sample of the skin, for example using the same medium used for cultivation. It is preferable to do this in NR-2 medium containing no serum medium, the exact composition of which is disclosed hereinafter, which were found suitable for cultivation of keratinocytes and melanocytes. After washing the sample skin shave, for example, using a dermatome, and then excised into small pieces.

Then, the skin section is preferably divided into dermal and epidermal. This can be done using physical and/or enzymatic methods. For example, this can be accomplished by trypsinization, for example, subjecting the flotation "leaves" of the skin in a solution of trypsin (e.g., about 0.5%) containing EDTA (for example, about 0.1%) for a sufficient period of the Chi in 4oC.

The dermis allocate (allocation of fibroblasts see example 2) and then the epidermis is placed in a suspension medium. Preferably, the suspension medium containing the solution, which inhibits trypsin soybean (SBTI), and that he was in contact with the cells for a sufficient period of time, usually about 5 minutes to inactivate trypsin and providing cell isolation. Then the tissue culture medium was added, preferably not containing serum NR-2 medium (described later), and filter (for example, 100 mm filter) to obtain a desired cells, such as keratinocytes and/or melanocytes.

The resulting primary culture keratinocytes/melanocytes obtained in stage (ii), then sow not containing serum medium, preferably NR-3 medium (described below), when a suitable cell concentration, preferably about 1,2x104the tile./cm2on pre-coated culture plates. However, the cell concentration can be varied within wide limits. The culture plates are preferably continuously coated with the composition, which, as it was unexpectedly discovered, improves both the attachment and growth of keratinocytes and melanocytes, especially in the solution of fibronectin BSA and collagen to generate et al., J. Tissue Cult.Meth., 9:43-48 (1985) (included here for reference).

At stage (iv) culture medium replaced as often as necessary to optimize cell growth. It is preferable to replace the medium approximately every second day. However, this may depend on the specific pattern of the skin. After achieving almost full confluently, such as confluences about 90%, which usually occurs after about 10-14 days, keratinocytes and melanocytes then share. This can be accomplished by any means that can provide the appropriate separation of cells, without harmful effects on the melanocytes and/or keratinocytes. For example, this can be done by using differential trypsinization. Preferably melanocytes or keratinocytes treated with a solution of trypsin/EDTA, and then transferred to selection medium. In the case of keratinocytes cells, it is preferable to process about 5-10 minutes with a solution of trypsin/EDTA (0,025%/0,01%), and then at stage (v) sown in NR-3 medium on plates pre-coated. It is important to note that NR-3 medium promotiom growth of keratinocytes, in contrast to the melanocytes. If melanocytes cells are preferably treated for about 2-4 minutes, a solution of the t is satisfactory coating. It is important to note that NR-4 environment specifically inhibits the growth of keratinocytes.

These cells are then treated immortalized agent. In another embodiment, cells can be frozen prior to the implementation of the immortalization, for example in liquid nitrogen. Infection and immortality preferably carried out using SV40 design, defined as pLXSHD+SV40 ( 328), which is shown in Fig. 2A and which was described Stockshlaeder et al., (GeneBank registration M; Stockslaeder et al. Human Gen.Therapy, 2, 33-39,1991), or HPV16 design, defined as pLXSHD+E6/E7, which is shown in Fig. 2b. Design pLXSHD+SV40 ( 328) contains the SV40 T-Ad sequence, 5' and 3' LTR sequences of SV40, RvR sequences that allow for replication in E. Li multiple cloning site, and the sequence of the SV40 polyadenylation, along with other sequences. Design pLXSHD+E6/E7 contains instead of T-Ad NcoI fragment/CfoI gene E6/E7 virus 16 human papilloma. Method of constructing E6/E7 plasmid based on the work of Durst et al. (Durst et al., 1987, Oncogene, 1: 251-256). After immortality, if necessary, carry out several passages of the cells during culturing and then received an immortalized cell transfer on proliferation sred) immortalized cells multiply in proliferation environment for immortalized keratinocytes or melanocytes, which contains NR 2 and NR-3 and M2-medium for melanocytes (described later). Immortalized cells are again cultivated continuously pre-coated culture plates, and the floor re-enables the solution of fibronectin, BSA and collagen type 1.

After the cells are multiplied in proliferation environment, their transfer on stage (viii) in an environment that provides differentiation of normal and immortalized keratinocytes. Preferably, this medium contained NR-2 or a modified MCDB 153 medium with high content of calcium, preferably about 1.5 mm calcium, and again the cultivation is carried out on the culture plates, constantly covered with a solution of fibronectin, BSA and collagen type 1.

As mentioned previously, it has been unexpectedly discovered that an immortalized cell line of keratinocytes and melanocytes, thus obtained, retain the ability to differentiate and to the expression of proteins of differentiation, which are characteristic of normal differentiated keratinocytes and melanocytes, even when a large number of passages in tissue culture, i.e., after at least 10 passages. For example, rassmatrivaemye immortalized keratinocytes passages, which either does not expressibility or little expressibility termed the keratinocytes SV40 reported previously.

More specifically, some immortalized cell lines keratinocytes obtained by the method of the present invention, DK2-NR, DK3-NR and FK2-NR (see tables 7 and 8 below), Express proteins differentiation K1/10, keratin K14, involucrin, filaggrin and loricrin, even after a large number of passages (more than 30 passages). In addition, immortalized keratinocytes obtained by the method of the present invention have CYP450 profile, which is similar, if not identical, to the profile of normal human keratinocytes. For example, consider an immortalized keratinocytes Express CYP450 1A1, 2C, 2E1 and A, but does not Express CYP450 1A2, A, V and 2D6, which are characteristic profile of cytochrome 450 normal keratinocytes. Was the first to demonstrate that normal and immortalized keratinocytes person Express CYP450 A, instead of CYP450 3A4.

Next, consider an immortalized cell line keratinocytes Express the enzymes of phase-II, for example, glutathione-3-transferase (GST), comparable to the normal differentiated keratinocytes. More specifically, R>/img>and GSTcompared with normal keratinocytes.

Next, consider an immortalized keratinocytes Express the enzymes and other proteins that participate in cellular oxidation and inflammatory reactions compared with normal keratinocytes. For example, an immortalized keratinocytes obtained by the method of the present invention, Express dismutase (SOD). Also, in response to esters of phorbol immortalized keratinocytes obtained by the method of the present invention, Express collagenase type 1 (mediator of inflammation) and TNF-(alpha-tumor necrosis factor).

Consider the melanocytes have the ability to Express proteins associated with melanin, and vimentin.

Moreover, consider an immortalized cell lines when grown in organotypic culture form a strongly stratified and polarized epithelium with keratinized surface layer (stratum corneum) even when a large number of passages (>20 passages). Previously reported only once for immortalized cell lines keratinocytes installed in normal culture conditions, i.e. in the environment, the soda is getting in the absence of serum and without the use of any feeder layer during cultivation.

Moreover, as will be more fully disclosed hereinafter, it has been unexpectedly discovered that epinephrine is a strong growth factor for normal keratinocytes, if it is used in an environment that does not contain serum. More specifically, NR-3 medium, described below, contains epinephrine, which, as it was discovered, enhances the growth of normal keratinocytes (see Fig.1). This is quite surprising given the fact that epinephrine, as previously reported, inhibits the growth of keratinocytes (Halprin, J. Invest.Dermatol., 81:553-557 (1983)) or has a moderate effect on the growth of cells keratinocytes (Koizumi et al., J. Invest.Dermatol., 96:234-237, 1991).

In addition, it has been unexpectedly discovered that the continuous coating of culture plates or plates used for the cultivation of primary and immortalized keratinocytes and/or melanocytes, in particular coating or "cocktail" containing fibronectin, BSA and collagen type 1 improves as the attachment of keratinocytes and melanocytes to culture plates or cups, and enhances the growth of cells. The use of such covering material has not previously been described for use with termed the keratinocytes and/or melanocytes.

As discussed, the present invention further specifically Prelate normal keratinocytes and/or melanocytes from the skin of the person in conditions without serum and without the use of a feeder layer. As was discovered, this medium enhances the growth of normal keratinocytes and allows you to set / get the normal culture of keratinocytes without any contact with serum or feeder cells.

The exact composition of the medium NR-3 can be found on page 52. This environment contains various amino acids, inorganic salts in the form of trace elements, vitamins, growth factors and other components. For example, this environment contains as growth factors epidermal growth factor (EGF recombinant), insulin, hydrocortisone, transferrin (human), bullish gibofsky extract and epinephrine. As noted, it has been unexpectedly discovered that epinephrine enhances the growth of primary keratinocytes in tissue culture.

As amino acids, this medium contains L-alanine, L-arginine-HCl, L-asparagine-N2Oh, L-aspartic acid, L-cysteine-Hcl-H2Oh, L-glutamic acid, glutamine, glycine, L-histidine-model HC1-H2Oh, L-isoleucine, L-leucine, L-lysine Hcl, L-methionine, L-phenylalanine, L-Proline, L-serine, L-threonine, L-tryptophan, L-tyrosine and L-valine.

Contained in the environment inorganic salts include ammoniummolybdate, ammoniummolybdate, calcifolic, copper sulfate (2), ferrous sulfate(2), Manihari, manganese chloride, the Wat sodium, sodium Selenite, sodium silicate, tin chloride and zinc sulfate.

Contained in the environment NR-3 vitamins include d-Biotin, d-calcipotriene, choline chloride, cyancobalamin, folic acid, i-Inositol, nicotinamide, pyridoxine and Riboflavin.

Further, the environment contains adenine, ethanolamine, phosphoethanolamine, phenol red Na, putrescine 2hcl, thiamine Hcl, thioctic acid, thymidine, glucose, HEPES and antibiotics (Fungizone, penicillin and streptomycin).

The preferred composition of NR-3 medium can be found on page 52. However, it is expected that the concentration of the substituents contained in NR-3 medium may vary within wide limits. More specifically, it is expected that the number of different substituents can vary from50 to0,1%, more preferably10 to0.1% of the concentrations presented on page 52. Moreover, it is expected that one or more of these components may be eliminated, while other components may be added, provided that such replacement components will not affect the selection and stabilization of primary cell cultures of keratinocytes or melanocytes and immortalized cell lawsuit environment NR-3, does not contain civardi is epinephrine. It was found that epinephrine is a very strong promoter of the growth of primary human keratinocytes.

The reason epinephrine enhances proliferation of keratinocytes, unclear. It was reported that human keratinocytes Express the enzymes for the synthesis of epinephrine, as well as Express a high density of beta-2-adrenergic receptors (Schallreuther et al., "Production of catecholamines in the human epidermis", Biochem. and Biophys. Res. Commun., 189:72-78 (1992)). These enzymes are involved in the scheme of the biosynthesis of catecholamine, in particular phenylethanolamine-N-methyltransferase and peopleintensive of tyrosine hydroxylase. On the contrary, such enzymatic activity cannot be defined in melanocytes and fibroblasts. Accordingly, the enzymatic activity and/or expression of the receptors may explain the ability of epinephrine to modulate the proliferation of keratinocytes.

The inventors assume that NR-3 medium enhances the release and stabilization of primary cell cultures and cell lines, as it suppresses the differentiation of cells, which leads to the enrichment of cells that supports their ability to differentiate and to Express the proteins and enzymes expressed in normal differentiated the rede, containing serum that promotes differentiation on the first passage. However, it is not profitable (during the initial period of cultivation), as differentiated cells grow poorly. This, in turn, leads to excessive growth and breeding proliferative skin cells, which have only a weak ability to differentiate. Accordingly, the number of cells that have a high capacity for differentiation, reduced if serum added to the medium prior to immortalization.

On the contrary, in the present invention keratinocytes and melanocytes cultured in not containing serum medium and under conditions which inhibit the differentiation of melanocytes and keratinocytes. In the present invention not containing serum medium preferably used throughout the entire cultivation period, both before and during immortalization, and also in the process of proliferation and differentiation.

As noted, under review NR-3, containing no serum medium, inhibits the differentiation of keratinocytes, and, thus, provides a selection of primary cell cultures of keratinocytes and derived immortalized cell lines. Moreover, this does not contain serum environment which leads to highly selective growth medium, which is conducive to obtaining cultures, which predominantly contain melanocytes and keratinocytes. Accordingly considered NR-3 medium beneficial because it inhibits the differentiation of keratinocytes and also inhibits the growth of fibroblasts.

As discussed previously, the cell suspension obtained from one sample of the skin, which contains dissociatively melanocytes, keratinocytes and fibroblasts, preferably cultured under review NR-3 environment. This is implemented by sowing these cells in culture cups that are continuously coated with the composition, which facilitates their attachment. Preferably, this coating contained a mixture of fibronectin, bovine serum albumin and collagen type 1. This prokritee, or "cocktail" coverage that was previously described for bronchial cells (Lechner et al., J. Tiss.Cult.Meth., 9:43-48 (1985)). The authors of the present invention have found that this cocktail also strengthens the attachment of keratinocytes and melanocytes to plastic culture cups. Moreover, it has been unexpectedly discovered that the continuous coating of culture plates, which are used for cultivation of primary and immortalized keratinocytes, yet enhances the growth of to the of Titov or melanocytes.

During culturing primary cell culture preferably divide when they reach, or almost reach, confluently, and then propagated to other cultural cups coated. Typically, cell culture divide approximately every 10-14 days.

After cultivation was carried out primary melanocytes and/or keratinocytes and their number increased up to the required number in NR-3 medium containing no serum, using the described cultural cups coated, they are subjected to the immortalization. Preferably, the melanocytes and keratinocytes, which show the best growth were immortality. However, in another embodiment, multiplied primary melanocytes or keratinocytes can be used to immortalization, for example in the analysis, when the facial skin or in gene therapy.

The immortality as melanocytes and keratinocytes can be done by using a vector that provides for expression of SV40 T-antigen or the expression of E6/E7 gene of the virus 16 human papillomavirus (HPV16). It is preferable to carry out the immortality due to infection of melanocytes or keratinocytes retroviral design that provides e is 17:83-89, 1995 (except that the virus is obtained from Packed cell lines sent in DMEM, 10% fetal calf serum). During infection use containing serum environment. However, melanocytes and keratinocytes preferred not containing serum medium, such as preferred PC-1 medium disclosed in the article Pfeifer et al., Meth.Cell.Sci., 14, 83-89 (1995), which is incorporated here by reference. It is more preferable to carry out the immortality using retroviral construct, called pLXSHD+SV40 ( 328), shown in Fig.2A and based on the design Pfeifer et al. and Stockshlaeder et al. (Spevak, registration M64753), or called pLXSHD+E6/E7 shown in Fig.2b and based on the design of Durst et al., 1987, Oncogene 1, 251-256.

After the immortalization an immortalized cell line is transferred to proliferation environment, preferably NR 2 and NR-3, or M2 (melanocytes), using culture plates with pre-coated. After the cells have proliferated to the desired number of cells migrated in differentiating the environment suitable for culturing normal and immortalized keratinocytes. It is preferable to include NR-2 or a modified MCDB 153 high is i.i.d. coating (same BSA, collagen type 1, fibronectine floor).

As mentioned earlier, differentiated immortalized cell lines and keratinocytes melanocytes obtained by the method of the present invention demonstrate improved properties that make these cell lines suited for use in the analyses in which the desired differentiated cells of human skin. In particular, these cell lines were found, Express proteins characteristic of normal differentiated melanocytes and keratinocytes even after a large number of passages.

For example, if an immortalized keratinocytes obtained by the method of the present invention, analyzed by Western blotting and RT-PCR, they possess cytochrome p450 (CYP450) profile, similar, if not identical, normal keratinocytes. More specifically, an immortalized keratinocytes obtained by the method of the present invention, Express CYP450 1A1, 2C, 2E1, A and does not Express CYP450 1A2, A, V and 2D6. Such CYP450 profile matches the profile of normal keratinocytes. This scheme metabolism has not been previously described for immortalized keratinocytes. Indeed, the first prod immortalized keratinocytes, obtained by the method of the present invention, in the analysis using antibodies specific for markers of differentiation were found, Express other proteins differentiation even after a large number of passages. More specifically, the examined cell lines Express proteins differentiation K1/10, keratin K14, involucrin, filaggrin and loricrin even after a large number of passages, for example after 10 passages, and after significantly more passages.

Consider an immortalized keratinocytes and melanocytes capture the exogenous essential fatty acids (EFA) and demonstrate the desaturation and elongation of the chain EFA substantially corresponding to normal melanocytes and keratinocytes.

Further, as described later in more detail, consider an immortalized keratinocytes Express TNFand a mediator of inflammation collagenase type 1 when processing esters of phorbol compared with normal keratinocytes. Next, consider an immortalized keratinocytes Express dismutase, an enzyme involved in cellular oxidation similar to normal differentsirovannym the keratinocytes.

Next, immortalizes the emer, theophylline and tyrosine) and inhibitors of melanogenesis (kociewie (kojic) acid) react similarly to the reactions of normal melanocytes.

Given these properties, rassmatrivaemye immortalized keratinocytes and melanocytes, are well suited for immunological, pharmacological, photo and genotoxicological studies of skin reactions.

For example, consider an immortalized cell line of keratinocytes and melanocytes and primary melanocytes and keratinocytes can be used in assays that require differentiated skin cells, for example in the study of barrier functions (cornification) restored the skin, in the studies of the metabolism of differentiated keratinocytes (fatty acid metabolism, metabolism of antioxidants), while studies on the effects of ultraviolet radiation on skin cells, while studies on the effects
potential skin irritants (chemicals that cause skin irritation and sensitizing agents on the cells of the skin, in the studies that measure the effect of compounds on the production of melanin, the studies of the metabolism of lipids, the surface treatment with xenobiotics (e.g., cosmetices is, the ri study of inflammation and irritation of the skin, and so on

Next, an immortalized cell line of keratinocytes and melanocytes and primary melanocytes and keratinocytes obtained by the method of the present invention, can be used for screening potential anti-cancer agents and compounds useful in the treatment of skin diseases. Such studies usually involve the exposure of cell lines or primary cells such compounds within a certain period of time and determining cause if they have any harmful effects such as genotoxicity, the formation of the DNA adduct, mutagenesis, transformation of cells or cytotoxicity.

Next, consider cell lines melanocytes and keratinocytes can be used for expression of recombinant proteins such as human proteins and polypeptides, as well as to obtain RNA and DNA.

Next, consider an immortalized cell lines have the potential for ex vivo genetic therapy. The examined cell lines should provide a useful tool for genetic tagging and development of genetically engineered cells that expressional cells. Moreover, given the fact that this cell line closely mimic the normal cells of the skin, they should be well suitable for the analysis of specific biological sensitivity.

Next, primary keratinocytes and melanocytes obtained by the method of the present invention, considering the fact that they receive in the absence of serum, can be used in gene therapy. It is essential that, as these cells were not exhibited serum such as bovine serum or serum of any other animal (except for the time of viral infection and storage in liquid nitrogen), they should be less susceptible to potential contamination in the form of viruses or other pathogenic agents. Therefore, their use should minimize the risk of transmission of these pathogenic cells or infectious factors in the process of gene therapy. This ex vivo gene therapy promising in the treatment of diseases such as bullosa bullosa (violation associated with a mutation of the keratin), vitiligo (violation, including genes for melanin synthesis), carcinoma and melanoma, allergic disorders, and disorders associated with inflammation. As for therapeutic treatment, the only potential source is or human fibronectin and transferrin.

Moreover, the immortalized cell line of melanocytes and keratinocytes, and primary melanocytes and keratinocytes used in the analysis of DNA mutagenesis, in assays for screening for skin mutagen, in assays to identify agents that damage chromosomes, in studies of transformations of malignant tumors, studies of the biochemistry of cells (for example, in the analysis of activation CYP450), when skanirovaniya to identify compounds and compositions, such as "cocktails" essential fatty acids, which are involved in inflammatory and allergic reactions, in the analysis of the activation of collagenase (associated with inflammation, including TNFdetection of interleukin.

Significant potential application of primary keratinocytes or melanocytes obtained by the method of the present invention, in view of their availability and how to obtain, is their use for skin transplantation. As these primary keratinocytes and melanocytes receive in the absence of serum, they pose minimal risk to be infected by pathogens (e.g. viruses) and infectious agents. Moreover, as we consider the melanocytes and keratinocytes or eliminate the risk of transplant rejection, or the risk of other harmful immunological reactions, and also to minimize the risk of infection.

Examples of specific immortalized cell lines keratinocytes obtained by the method of the present invention are FK2-NR, DK2-NR and DK3-NR, which were deposited on 5 October 1995 in the DSM-Deutsche Sammlung von Mikroorganismen und ZeIlKulturen GmbH address Mascheroder Weg D-38124 Branschweig Germany, and given the registration number DSM ACC2240, DSM ACC2238 and DSM ASS respectively. In addition, the specific instances immortalized line of human melanocytes obtained by the method of the present invention, represent DM2-NR, which is deposited on December 11, 1996 at Institut Pasteur no address at 25 rue du Docteur Roux 75724 Paris France under the registration number CNCM I-1796. These deposits are made in accordance with the Budapest Treaty. All restrictions on the availability of these cell lines will be removed immediately after obtaining a patent on this application or to another application with an earlier priority in relation to the question.

Other features of the present invention will become apparent during consideration of preferred options, which are presented here only to illustrate the invention and should not ogranize, who were treated for viral infection. Selected keratinocytes, which showed the best growth of cells were used for immortalization.

Fibroblasts isolated from skin samples of FKO-NR, GKO-NR, DKO-NR. After separation of the dermal and epidermal parts of the dermis is cut into small pieces of size 0,2x0,2 mm and fixed at 6 cm culture plate with serum. 2-4 hours add minimal support environment Dulbecco (DMEM, 10% FCS ).

This explanatory environment incubated until such time as the proliferation of fibroblasts does not become noticeable. Confluent fibroblast culture divide and multiply to get frozen stocks.

EXAMPLE 2.

1) Characterization of keratinocyte growth in medium without serum
Culture of primary cells cultured in modified MCDB 153 (Boyce et al., J. Tissue Cult. Meth., 9:83-93 (1985); and Pittlekow et al., J. Invest.Dermatol., 86:410-417, 1986) and NR-3 environment. The best cell growth observed in NR-3 medium (Fig.1). Improved cell growth was also observed in a fully qualified NR-3 medium (NR-3 without bullish giovingo extract TIME) compared with a modified MCDB 153 no TIME.

In Fig. 1 presents a comparison of cell growth in NR-3 and supplemented by epinephrine is seeking to a modified MCDB 153. Keratinocytes are harvested in trypsin/EDTA (0,05%/0,01%) and counting are using hemocytometer. The results obtained are shown in Fig.3, are the average of three repetitions.

2) the Effect of the coating on the attachment of cells and cell growth
It was found that the coating on the culture plate improves the attachment of the cells and the cell growth of normal keratinocytes. In particular, are presented in the table 2 results, compare the growth of keratinocytes in culture plates coated with coating or without it. On a plate 3.5 cm, containing NR-3 medium, seeded 100000 keratinocytes.

EXAMPLE 3.

1) Immortality keratinocytes
The suspension of cells obtained from skin samples described in example 1, which contains dissociatively melanocytes, keratinocytes and fibroblasts cultured under review NR-3 environment. This is implemented by sowing these cells in culture Cup, which is constantly covered with a "cocktail"coating previously described for bronchial cells (Lechner et al., J. Tiss. Cult. Meth. , 9:43-48 (1985). During culturing of primary cell cultures when they reach or almost reach confluently, cells treated for 4 minutes with trypsin/EDTA (0,025%/0,01%). In primary melanocytes and keratinocytes share at this stage. After primary keratinocytes were cultivated and multiplied to the desired number of cells in NR-3 medium containing no serum, using the described cultural cups coated (promotiom growth of keratinocytes against melanocytes), they are subjected to the immortalization. The immortality of keratinocytes is performed using retroviral constructs pLXSHD+SV40 ( 328), which provides the expression of the SV40 T-antigen (see Pfeifer et al. , Meth.Cell Sci., 17:83-89, 1995; except that the virus is obtained from Packed cell lines provided in DMEM, 10% fetal calf serum). During infection using PC-1 medium containing no serum, as described in article Pfeifer et al., Meth.Cell Sci., 14, 83-89 (1995). After the immortalization an immortalized cell line is transferred to NR 2 and NR-3 proliferation environment using culture plates with pre-coated. After the proliferation of the cells to the desired number of cells migrated to the differentiating medium suitable for culturing normal and immortalized keratinocytes.

2) Cell proliferation of immortalized keratinocytes with a large number of passages
Immortalized keratinocytes have been shown to demonstrate uluchshenie doubling time of a population (PDT: time, required for a doubling of the population during the logarithmic phase of growth). Method: keratinocytes are harvested in trypsin/EDTA (0,05%/ 0,01%) and counted using hemocytometer. The results presented represent the average of three repetitions.

3) SUR-expression in immortalized lines of human keratinocytes
CYP4501A1, 1A2, A, 2E1, V, A and 2D6 analyze the expression in cell lines of normal and immortalized the keratinocytes of the skin using Western blotting (protein expression) and RT-PCR (mRNA-expression, see table 4). Downregulation of CYP450 immortalized in the keratinocytes are similar to normal keratinocytes. The rate expression is slightly reduced. However D2-NR-line shows an almost normal rate of expression of CYP450. Method: RT-PCR (reverse transcriptase-polymerase chain reaction with specific primers for CYP450 (Mace et al., forthcoming).

4) Reaction to SUR-inductor
Cell lines respond to SUR-inductor benzo(a)pyrene like neomalthusianism cells even when a large number of passages (see table 5). This induction has not been described for T-Hell immortalized keratinocytes.

5) Diferenciacija cells
Markers of differentiation analyze, ispoure ability can be demonstrated in DK2-NR and DK1-NR clones (tables 7 and 8).

6) Expression of glutathione-3-transferase
The phase II enzyme glutathione-3-transferase (GST) analyzed by Northern-blotting and Western blotting. All lines keratinocytes strongly Express mRNA for GSTbut not GSTand not GST(table 9, method: Northern-blotting). The expression profile of protein GSTGSTand GSTcell lines are similar to the profile for normal keratinocytes.

7) the Metabolism of essential fatty acids (EPA)
For analysis and comparison of desaturation and elongation added EFA to keratinocytes, immortalized (DK1-NR, FK2-NR) and normal keratinocytes treated with linoleic acid (LA, 15 μm) and-linolenic acid (LN, 15 μm). For these experiments use EFA-deficient NR-2 (Biofluids Inc. ). Cell culture process after reaching confluently and shifting NR-2 high content of calcium (1.5 mm). Cells treated for 4 days EFA (updating after 2 days).

EFA analysis is carried out using the extraction and separation of phospholipids by TLC (thin layer chromatography) and quantify the methyl complex EF the LA (20:4n-6 and 22:4n-6) and LN (20:5N-3, 22:5N-3 and 22:6N-3) can be demonstrated. This profile metabolism corresponds to the profile observed for normal keratinocytes.

8) Karyotyping
All cell lines were hypodiploidy with a number of most chromosomes in the diploid range (except DK2-NR with the number of chromosomes in hypothyroidsim interval). Cells that are different from the cells was analyzed cell lines in cultures was not determined. This result confirms the purity of the cell lines and the absence of cellular contamination from other sources.

9) Characterization of in vivo
Carcinogenesis immortalized keratinocytes determine, through subcutaneous injection (1-2 m & e keratinocytes) naked mice. Tested keratinocyte line DK2-NR, DK3-NR, FK2-NR turned out to be non-carcinogenic for naked mice (4 months incubation period), DK3-NR, however, was not carcinogenic for 6 animals out of 10 after 5 months of incubation period.

10) Reaction to skin irritation
The induction of "stress gene" TNF(alpha-tumor necrosis factor) after treatment skin irritant PMA (phorbol-12-myristate-13-acetate), SDS (sodium dodecyl sulphate), DMSO (dimethylsulfoxide), IL-1(Intel analyses (see table 10). Keratinocyte line responsive to PMA and UV-B and Express TNFeven when a large number of passages.

After processing immortalized keratinocytes complex with phorbol esters (PMA) observed an increased expression of collagenase (type I).

11) Organotypic culture
Also did the cultivation of human keratinocytes in organotypic conditions (keratinocytes were grown with access of air to the collagen gel with feeder cells). All keratinocyte lines show hyperproliferative morphology compared with normal keratinocytes. These studies were carried out in culture medium without serum. Hyperproliferative cell growth is reduced in conditions without the presence of serum (NR-2 with a high content of calcium (1.5 mm) to the culture cups with plastic inserts without collagen gel and feeder cells).

EXAMPLE 4.

1) Immortality melanocytes
The cell suspension obtained from a sample of skin DKO-NR described in example 1, which contains dissociatively melanocytes, keratinocytes and fibroblasts cultured under review NR-3 environment. This is implemented by sowing these cells in culture Cup to the two culturing primary cell cultures, when they reach or almost reach confluently, cells treated for 4 minutes with trypsin/EDTA (0,025%/0,01%). During this treatment, the melanocytes are separated from the culture of keratinocytes and are collected separately. Primary melanocytes and keratinocytes, thus, share at this stage. Then the collected primary melanocytes were seeded in NR-4, not containing serum environment, which specifically inhibits the growth of keratinocytes. After culturing primary melanocytes and their propagation to the desired number of cells in NR-4, not containing serum environment, using the described coating of the culture they are subjected to the immortalization. The immortality of melanocytes is performed using retroviral constructs pLXSHD+SV40( 328), which provides the expression of the SV40 T-antigen (see Pfeifer et al., Meth. Cell Sci. , 17:83-89, 1995; except that the virus is obtained from Packed cell lines provided in DMEM, 10% fetal calf serum). During infection using RS-1 that does not contain serum environment, described in the article Pfeifer et al., Meth.Cell Sci., 14, 88-89 (1995). After the immortalization an immortalized cell line is transferred to M2 proliferating and differentiating the DMEM/F12 medium, Biofluids, 148; you can also buy alaninol proteins immortalized melanocytes, obtained by the method of the present invention (especially DM2-NR), compared with normal melanocytes. Demonstrated that immortalized cells Express associated with melanin proteins associated with melanoma antigen (MAA) and NPS, similar to normal cells, although with a lower level of expression.

3) the Induction of melanin synthesis
Melanogenesis T-Ad expressing melanocyte cell lines (especially line DM2-NR) compared with normal melanocytes. Melanocytes treated with inducers of melanogenesis - tyrosine and theophylline, an inhibitor of melanogenesis - kociewie acid. Melanocyte lines (especially DM2-NR) responsive to a modulator of melanogenesis compared with normal cells. It is also shown that the induction/inhibition of melanogenesis is dose-dependent manner.

EXAMPLE 5.

Strain DKO-NR described in example 1 is subjected to the immortalization, as described previously, using a retroviral construct pLXSHD+E6/E7 on the basis of HPV16, which is shown in Fig.2b. Select multiple continuous lines keratinocytes. The results of the analysis of these lines from the point of view of product differentiation (cytokeratins, GST, TNF, involucrin, filaggrin, loricrin, the composition of the new NR-3 medium of the present invention and some others, not containing serum media of the present invention, i.e., NR-1, NR-2 and NR-4.

1) the Composition of the medium NR-1 (see below)
Amino - NR-1 (mg/liter)
L-alanine - 9,0000
L-arginine, model HC1 - 316,0000
Asparagine, N2On - 15,0000
L-aspartic acid - 4,0000
L-cysteine, model HC1, H2On - 42,0000
L-glutamic acid - 14,8000
Glutamine - 877,0000
Glycine - 7,6000
L-histidine, model HC1, H2On - 50,4000
L-isoleucine - 98,4000
L-leucine - 131,2000
L-lysine, model HC1 - 36,6000
L-methionine - 13,4000
L-phenylalanine - 14,9000
L-Proline - 34,6000
L-serine - 126,2000
L-threonine - 23,8000
L-tryptophan - 9,2000
L-tyrosine - 13,6000
L-valine - 70,2000
Inorganic salts
The ammonium metavanadate (NH4VO3) - 0,0006
The ammonium molybdate [(NH4)6Mo7About24x4H2O] - 0,0010
Calcium chloride [CAS2HN2O] - 16,2000
Copper sulphate (2) [CuSO4x5H2O] - 0,0025
Sulphate of iron (2) [FeS4x7H2O] - 1,4000
The chloride of manganese [MnCl2xH2O] is 0.0002
Magnesium chloride [MDS2HN2O] - 122,0000
The Nickel sulfate [with NISO4x6H2O] - 0,0003
Potassium chloride [KS1] - 112,0000
Acetic sodium - 301,5000
Sodium bicarbonate [Panso3] - 1088, 0000
Sodium chloride [NaCI] - 5200, 0000
Dibasic sodium phosphate [Na2HP04x7H2O] - 536,00002O] - 0,1420
The tin chloride [SnCl2x2H2O] - 0,0001
Zinc sulfate [ZnS04x7H2O] - 0,5100
Vitamins
d-Biotin - 0,0200
d-calcipotriene - 0,2600
Choline chloride - 28,0000
Cyanocobalamin (B12) - 0,4100
Folic acid - 0,7900
i-Inositol - 18,0000
Nicotinamide (B3) - 0,0400
Pyridoxine (VHN2O) - 0,0600
Riboflavin (B2) - 0,0400
Else
Adenine - 27,3000
Epidermal growth factor (EGF, human, recomb.) - 0,0010
Ethanolamine - 0,0310
Glucose - 1080, 0000
HEPES - 6000, 0000
Hydrocortisone - 0,5000
Insulin (bovine) - 5,0000
Phenol red - 1,2000
The phosphoethanolamine - 0,0710
Putrescine, NS - 0,1600
Thiamine, model HC1 - 0,3400
Thioctic acid - 0,2100
Thymidine - 0,7300
Transferrin (human) - 10,0000
Osmolarity - 280-285 mOsm/kg
2) the Composition of the medium NR-2; is the same as NR-1, but supplemented with extract of bovine pituitary (Biofluid Inc.) at 35 mg/l and contains antibiotics (Gibco BRL, Life Technologies Inc. ) fungeon (0.25 mg/l), penicillin (10000 IU) and streptomycin (10 mg/l).

3) the Composition of the medium NR-3: the same as that of the environment NR-2, but the addition of epinephrine (Biofluid Inc.) 250 µg/L.

4) the Composition of the medium NR-4: the same as that of the environment NR-2, but supplementedFGF (3 µg/l) (basic fibroblast growth factor, obtained from Sigma Inc.) and phorbol-12-myristate-13-acetate (10 m occhialini options other options in the scope of the present invention are also possible. Accordingly, no disclosure of the invention or the following claims should not be limited to the attached preferred option.


Claims

1. Cell line immortalized human keratinocytes DK2-NR (DSM ACC 2238), which retains the ability to differentiate and to Express the proteins and enzymes that are expressed in normal differentiated keratinocytes even after a large number of passages in tissue culture intended for studies of skin reactions.

2. Cell line immortalized human keratinocytes DK3-NR (DSM ACC 2239), which retains the ability to differentiate and to Express the proteins and enzymes that are expressed in normal differentiated keratinocytes even after a large number of passages in tissue culture intended for studies of skin reactions.

3. Cell line immortalized human keratinocytes FK2-NR (DSM ACC 2240), which retains the ability to differentiate and to Express the proteins and enzymes that are expressed in normal differentiated keratinous.

4. Immortalitya cell line of human melanocytes DM2-NR (CNCM 1-1796) intended for studies of skin reactions.

5. The way the immortalization of human skin cells to obtain immortalized keratinocytes, characterized in that it comprises the following stages: (i) obtaining a tissue sample of human skin, (ii) preparing the sample skin for in vitro cultivation, (iii) obtain keratinocytes prepared from the specified sample of the skin and planting these keratinocytes at not containing serum Wednesday NR-3 for growing on a culture plate, provided with a coating, including fibronectin, collagen type 1 and BSA, which facilitate the attachment of cells and their growth, (iv) replacement environment if necessary to optimize confluent growth of cultured cells with a constant coating on the plates with cultures, (v) migration of keratinocytes in not containing serum breeding environment NR-3 similarly pre-coated culture plates (vi) infection of keratinocytes retroviral design, in particular, pLXSHD+E6/E7 or pLXSHD+SV40 (N 328), (vii) transfer received immortalized keratinocytes at not containing serum proliferative environment NR-2, podgotovlenyj plates, and (viii) transfer received proliferating keratinocytes at not containing serum differentiating environment with a high content of calcium NR-2 or a modified environment MSDW containing calcium in an amount of at least 1.5 mm in the same way pre-coated culture plates.

6. The way the immortalization of human skin cells to obtain immortalized melanocytes, characterized in that it comprises the following stages: (i) obtaining a tissue sample of human skin, (ii) preparing the sample skin for in vitro cultivation, (iii) receipt of melanocytes from the specified prepared sample of the skin and planting these on melanocytes containing no serum medium NR-3 for growing on a culture plate, provided with a coating, including fibronectin, collagen type 1 and BSA, which facilitate the attachment of cells and their growth, (iv) replacement environment if necessary to optimize confluent growth of cultured cells with a constant coating on the plates with cultures, (v) migration of melanocytes in not containing serum breeding environment NR-4 similarly pre-coated culture plates (vi) infection melanocytes of Retrovir melanocytes not containing serum proliferative Wednesday M2, suitable for proliferation of immortalized melanocytes in the same way pre-coated culture plates.

7. Serum-free medium NR-3, suitable for isolation and production of melanocytes and keratinocytes person who retain the ability to differentiate and to Express the proteins and enzymes of differentiated keratinocytes and melanocytes person even after a large number of passages, which includes, mg/l medium:
Amino acids
L-alanine - 9,0000
L-arginine, model HC1 - 316,0000
Asparagine, N2On - 15,0000
L-aspartic acid - 4,0000
L-cysteine, Hcl, H2O - 42,0000
L-glutamic acid - 14,8000
Glutamine - 877,0000
Glycine - 7,6000
L-histidine, model HC1, H2On - 50,4000
L-isoleucine - 98,4000
L-leucine - 131,2000
L-lysine, model HC1 - 36,6000
L-methionine - 13,4000
L-phenylalanine - 14,9000
L-Proline - 34,6000
L-serine - 126,2000
L-threonine - 23,8000
L-tryptophan - 9,2000
L-tyrosine - 13,6000
L-valine - 70,2000
Inorganic salts
The ammonium metavanadate (NH4VO3) - 0,0006
The ammonium molybdate [(NH4)6Mo7O244H2O] - 0,0010
Calcium chloride [CaCl22H2O] - 16,2000
Copper sulphate (2) [CuSO44H2O] - is 0.0002
Magnesium chloride [MgCl26H2O] - 122,0000
The Nickel sulfate [with NISO46H2O] - 0,0003
Potassium chloride [KS1] - 112,0000
Sodium acetate - 301,5000
Sodium bicarbonate [Panso3] - 1088, 0000
Sodium chloride [NaCl] - 5200, 0000
Dibasic sodium phosphate [PA2NRA47H2O] - 536,0000
Pyruvate sodium - 55,5000
Sodium Selenite [NaSeO3] - 0,0050
Sodium silicate [NaSiO39H2O] - 0,1420
The tin chloride [SnCl22H2O] - 0,0001
Zinc sulfate [ZnSO47H2O] - 0,5100
Vitamins
d-Biotin - 0,0200
d-calcipotriene - 0,2600
Choline chloride - 28,0000
Cyanocobalamin (B12) - 0,4100
Folic acid - 0,7900
i-Inositol - 18,0000
Nicotinamide (B3) - 0,0400
Pyridoxine (B6H2O) - 0,0600
Riboflavin (B2) - 0,0400
Else
Adenine - 27,3000
Epidermal growth factor (EGF, human, recomb.) - 0,0010
Ethanolamine - 0,0310
Glucose - 1080, 0000
HEPES - 6000, 0000
Hydrocortisone - 0,5000
Insulin (bovine) - 5,0000
Phenol red - 1,2000
The phosphoethanolamine - 0,0710
Putrescine 2hcl - 0,1600
Thiam is apofiza - 35,0000
Fungizone - 0,2500
Penicillin - 10000,0000 ed
Streptomycin - 10,0000
Osmolarity - 280-285 mOsm/kg
one or more of these components may be eliminated, and the number of ingredients substituents may vary from the above values in the interval from50 toto 0.1, preferably from10 to 0.1%; and epinephrine in a concentration sufficient to enhance the growth of keratinocytes, preferably, about 250 μg/l environment.

8. Serum-free medium NR-2, designed for the selection, production and/or maintenance of immortalized keratinocytes and/or melanocytes containing, mg/l:
Amino acids
L-alanine - 9,0000
L-arginine, model HC1 - 316,0000
Asparagine, H2O - 15,0000
L-aspartic acid - 4,0000
L-cysteine, Hcl, H2On - 42,0000
L-glutamic acid - 14,8000
Glutamine - 877,0000
Glycine - 7,6000
L-histidine Hcl, H2O - 50,4000
L-isoleucine - 98,4000
L-leucine - 131,2000
L-lysine, model HC1 - 36,6000
L-methionine - 13,4000
L-phenylalanine - 14,9000
L-Proline - 34,6000
L-serine - 126,2000
L-threonine - 23,8000
L-tryptophan - 9,2000
L-tyrosine - 13,6000
L-valine - 70,2000
Inorganic salts
The ammonium metavanadate (NH4VO3) - 0,0006
Moth [l22H2O] - 16,2000
Copper sulphate (2) [CuSO45H2O] - 0,0025
Sulphate of iron (2) [FeSO47H2O] - 1,4000
The chloride of manganese [nl24H2O] is 0.0002
Magnesium chloride [MgCl26H2O] - 122,0000
The Nickel sulfate [with NISO46H2O] - 0,0003
Potassium chloride [KCl] - 112,0000
Sodium acetate - 301,5000
Sodium bicarbonate [Panso3] - 1088,0000
Sodium chloride [NaCl] - 5200,0000
Dibasic sodium phosphate [Na2HPO47H2O] - 536,0000
Pyruvate sodium - 55,5000
Sodium Selenite [NaSeO3] - 0,0050
Sodium silicate [NaSiO39H2O] - 0,1420
The tin chloride [SnCl22H2O] - 0,0001
Zinc sulfate [ZnSO47H2O] - 0,5100
Vitamins
d-Biotin - 0,0200
d-calcipotriene - 0,2600
Choline chloride - 28,0000
Cyanocobalamin (B12) - 0,4100
Folic acid - 0,7900
i-Inositol - 18,0000
Nicotinamide (B3) - 0,0400
Pyridoxine (B6H2O) - 0,0600
Riboflavin (B2) - 0,0400
Else
Adenine - 27,3000
Epidermal growth factor (EGF, Sulin (bullish) - 5,0000
Phenol red - 1,2000
The phosphoethanolamine - 0,0710
Putrescine 2hcl - 0,1600
Thiamine Hcl - 0,3400
Thioctic acid - 0,2100
Thymidine - 0,7300
Transferrin (human) - 10,0000
Extract, bovine pituitary - 35,0000
Fungizone - 0,2500
Penicillin - 10000,0000 ed
Streptomycin - 10,0000
Osmolarity - 280-285 mOsm/kg
9. Serum-free medium under item 8, characterized in that it further contains epinephrine at a concentration of 250 µg/l environment (environment NR-3).

10. Serum-free medium under item 8, characterized in that it further containsFGF concentration of 3.0 µg/l environment and orbital-12-myristate-13 acetated at a concentration of 10,000 ág/l environment (environment NR-4).

11. Analysis of skin reactions, including the use of immortalized cells keratinocytes and/or melanocytes in PP.1-4.

12. Analysis on p. 11, characterized in that the analysis is an analysis of inflammation.

 

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