Cellular line human immortalized keratinocytes (variants) and method for immortalizing human skin cells

FIELD: biology, genetic engineering.

SUBSTANCE: invention relates to preparing immortalized cellular lines from health human skin tissues and can be used in immunological, pharmacological, photo- and chemical-toxicological analysis of cutaneous response, for expression of heterologous genes and for construction of artificial skin. Keratinocytes are immortalized by infection of keratinocytes of health human. The human skin sample is isolated and prepared its for culturing in vitro. Keratinocytes are prepared from this prepared human skin sample and plated in serum-free medium for growing keratinocytes in cultural plates with cover alleviating attachment and growth of cells. In the process for culturing keratinocytes the serum-free medium is replaced to provide preparing the optimal confluent growth of cells in culture with continuous maintenance of cup cover. Keratinocytes are transferred in selective serum-free medium in cultural cups with cover and infected with vectors pLXSHD + SV40(#328) and pLXSHD + E6/E7. Then prepared immortalized keratinocytes are transferred in cultural cups with cover to useful medium for proliferation. Then prepared proliferated keratinocytes are transferred in medium with high calcium content for differentiation in cultural chambers with cover. Invention provides preparing the human keratinocyte cellular line that has no oncogenic property and retains capacity for differentiation and expression of proteins and enzymes expressing by normal differentiated keratinocytes being even after increased number of passages in culture. Also, this cellular line forms lamellar and polarized epithelium with keratinized layer (stratum corneum) consisting of ortho-keratinocytes in the process for culturing in organotypical culture in serum-free medium and without layer of feeding cells.

EFFECT: improved immortalizing method, valuable biological properties of cellular line.

7 cl, 2 dwg, 4 ex

 

The technical field to which the invention relates.

This invention relates to medicine, in particular to new immortalized cell lines derived from skin tissue of a healthy person and provides improved performance differentiation, to a new way of obtaining these cell lines and their various applications, in particular in the field of creation of artificial leather.

The level of technology

Getting immortalized cell lines derived from tissues of the skin of a healthy person, has already been described. Generally, the methods used for this purpose include the transformation of human skin cells such as keratinocytes and melanocytes, which are cultivated in vitro with agents that give immortality. Immortality has in mind obtaining cells that may be cultured for an extended period of time in vitro, theoretically, for an unlimited period. These cells are also referred to as continuous cell lines. In contrast naimportovane cells are able to multiply only within a certain number of cell divisions in vitro. Immortalized cells are extremely beneficial, as they provide a stable, potentially unlimited source of cells with certain characteristics. Typical AG is nami to obtain immortalized cell lines and immortalized cell lines of human skin are in particular, for example, viruses, recombinant viruses and plasmids containing the DNA sequence, giving the property the immortalization.

The most common way of receiving the immortalized cell lines of a person includes, perhaps, the use of sequences of simian vacuolating virus 40 (SV40), and more specifically DNA large T-antigen (T-Ad) SV40 as agent for the immortalization. For example, Steinberg et al., J. Cell Phys 123, 117-125 (1985); US patent 4885238 (Reddel et al.); patent US 4707448 (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); Rhim et al., Science, 227, 1250-1252 (1985) and Grubman et al., Gastrointest. Liver Physiol., 29, G1060-G1070 (1994) reported on the use of vectors SV-40 and vectors containing the sequence of the large T antigen of SV-40, to obtain immortalized cell lines of human rights. The introduction of such sequences are usually performed by infection with a virus SV-40 or hybrid adenovirus 12/SV-40 or transfection of cells with recombinant Plasmodium containing long terminal repeats of the rous sarcoma virus and regulatory ori-region SV-40, coprecipitate in the presence of phosphate strontium (see Brash et al., Mol. Cell Biol., 7, 2031-2034, 1987).

Another known method of obtaining immortalized cell lines and, in particular, immortalized human keratinocytes pre who sees the transfection or infection of cells with DNA sequences of human papillomavirus (HPV). For example, in patent US 5376542 (Schlegel) described immortality epithelial cells of human genes E6 and E7, isolated from HPV 16, 18, 31, 33 or 35, or one E7 gene to obtain non-carcinogenic immortalized cell lines of human rights. In addition, Barbosa et al., Oncogene, 4, 1529-1532 (1989) and Munger et al., J. Virol., 63(10); 4417-4421 (1989) reported that genes E6 and E7 of HPV-16 and HPV-18 for receiving immortalized human keratinocytes. In addition,et al., Oncogene, I, 251-256 (1987) describes the immortality of keratinocytes with human papillomavirus type 16.

Despite the fact that numerous groups have described an immortalized cell line of keratinocytes and their use in assays in vitro, these immortalized cell lines keratinocytes status currently find usually one or more properties that make their use profitable. For example, the previously described immortalone keratinocytes exhibit one or more of the following properties: (i) the reduction or loss of expression of differentiation markers, for example, proteins expressed in normal differentiated keratinocytes, (ii) properties of the modified growth in tissue culture, and (iii) the formation of layered and polarized epithelium with consisting of a pair of keratinocyte cornified layer (stratum corneum).

To eliminate e the deficiencies in the European patent ER (Societe des Produits Nestle) proposed a new method for the immortalization of keratinocytes or melanocytes person using en sus new culture medium, vector pLXSHD+SV40(#328), which is produced from virus SV-40, or equally vector pLXSHD+E6/E7, which is produced from human papilloma virus 16 (HPV-16). Immortalized cells obtained in this way, retain the ability differentiation and expression of proteins and enzymes that are expressed in normal differentiated keratinocytes and melanocytes, even after an increased number of passages in culture.

However, despite these previous descriptions, there is still an important need in the practical field to be immortalized keratinocytes person having more improved properties. Such cells would be extremely advantageous for numerous applications, in particular for analyses that require the use of highly differentiated cells of the skin.

The invention

For this purpose, the present invention relates to immortalizing cell line of human keratinocytes obtained using at least one oncogenic functional gene of retroviral origin that is characterized by the fact that she

(1) is non-carcinogenic,

(2) retains the ability to differentiate and to Express the proteins and enzymes expressed in normal differentiated keratinocytes, after an increased number of passages in culture cloth and

(3) forms a stratified and polarized epithelium with consisting of ortho-keratinocyte cornified layer (stratum corneum), under cultivation in organotypic culture in serum-free environment and without a layer of feeder cells.

Another purpose of this invention is to develop a new method of producing immortalized cell lines and keratinocytes derived from normal skin tissues.

Another purpose of this invention is to develop ways to use these cell lines keratinocytes in accordance with this invention, for example, immunological, pharmacological, photo - or chemical-Toxicological analysis of skin reactions and for the expression of heterologous genes.

Description of figures

- Figure 1 shows a design derived from a retrovirus SV40, namely plasmids pLXSHD+SV40(#328)used for the immortalization of keratinocytes of the present invention.

- Figure 2 represents a structure derived from a retrovirus papillomavirus 16, namely plasmids pLXSHD+E6/E7 used for the immortalization of keratinocytes of the present invention.

Detailed description of the invention

This invention provides non-carcinogenic, an immortalized cell line keratinocytes, i.e. cell lines that do not form tumors when injected under the skin of the animal using EmOC is emer, at least 2×106cells for each injection.

These cell lines also retain the ability to differentiate and to Express the proteins and enzymes expressed by normal keratinocytes, after an increased number of passages. The expression "increased number of passages" is referring to at least 10 passages in culture, preferably at least 20-30 passages, more preferably at least 50 passages and theoretically unlimited number of passages. For example, an immortalized keratinocytes obtained in accordance with this invention, Express proteins differentiation, consisting of keratin K1/10, keratin K14, involucrin, filaggrin and loricrin, even after an increased number of passages in tissue culture.

Immortalized keratinocytes of the present invention have a profile of cytochrome P450 (CYP450), which is similar, if not identical, with a profile of normal keratinocytes. For example, cells of the invention Express CYP450 1A1, 2E1, S and SE. In addition, immortalized keratinocytes of the present invention Express the enzymes of phase II, for example, glutathione-S-transferase π (gsttt), compared with normal, neimpartasita-EN keratinocytes.

In addition, immortalized keratinocytes of the present invention Express the proteins and enzymes involved kletochnom oxidation and inflammatory response reactions, for example, the superoxide dismutase (SOD) and collagenase type I and tumor necrosis factor alpha (TNFα)after processing forbalului esters of similar or identical way compared to the normal differentiated keratinocytes. With these specific characteristics of these cell lines are extremely interesting, reproducible source for immunological, pharmacological, associated with inflammation, photo - and chemical-Toxicological studies of skin reactions.

In addition, lines immortalized keratinocytes of the present invention is formed under cultivation in organotypic culture in serum-free medium [for example, in the environment of NO2 Biofluids Inc., U.S. enriched EGF (epidermal growth factor) (5 ng/ml), vitamin C (38 µg/ml) and l2(1.5 M] and without a layer of feeder cells (without fibroblasts), stratified and polarized epithelium with superficial keratinized (dead) layers, usually called the Horny layer (stratum corneum)with the morphology of ortho-keratinocytes, which means that this layer stratum corneum does not contain nuclear cells, i.e. cells containing a nucleus.

Obtaining stratified and polarized epithelium with termed by keratinocytes was achieved earlier in classical culture conditions, i.e. a method using a medium containing calf serum is a layer of feeder cells (Lechner et al., Virology, 185, 536-671, 1991). However, these immortalized cells do not form normal superficial keratinized (dead) layers. For example, it is known that cell lines, immortalone the human papilloma virus 16 or 18 or E6/E7, form a very disorganized (destroyed) epithelium (Blanton et al., Am. J. Pathol., 138, 673-685, 1991; Hudson et al., J. Virol., 64, 519-526, 1990; McCane et al., Proc. Natl. Acad. Sci., 85, 7169-7173, 1988; Woodworth et al., Oncogene, 7, 619-626, 1992).

In contrast, lines described in EP 780496 (Societe des Produits Nestle), under cultivation in organotypic culture in serum-free environment and without a layer of feeder cells, formed stratified and polarized epithelium with normal superficial keratinized (dead) layers, but nevertheless having the morphology of a pair of keratinocytes, i.e. the stratum corneum (Horny layer) still contained cells with nuclei.

Immortalized keratinocytes of the present invention also absorb exogenous essential fatty acids (AGE) and find unsaturated condition and extension chain AGE, completely corresponding unsaturated condition and extension chain AGE normal keratinocytes.

In General terms, an immortalized keratinocytes of the present invention can be obtained according to the following method:

(i) obtaining a sample of human skin;

(ii) preparation of that sample skin for receiving the Oia primary keratinocytes, suitable for cultivation;

(iii) the cultivation of these primary keratinocytes in serum-free medium, preferably in an environment NR-3 (described in EP 780496), cultural cups with a coating that facilitates the attachment and growth of cells, and this coating contains fibronectin, S and collagen type I;

(iv) the replacement of serum-free medium sufficient for optimal confluent keratinocyte growth on culture plates, and the floor of the Cup is maintained continuously;

(v) the Department of keratinocytes in culture from melanocytes and transfer separated keratinocytes in the environment for infection, preferably on Wednesday, NR-3, and preferably after treatment of these cells with a composition containing trypsin and EDTA, using cultural cups, covered the floor in the same way;

(vi) infection of cells functional oncogenic genes of at least two different retroviruses, such as the SV40 virus and human papilloma virus;

(vii) transfer immortalized keratinocytes in serum-free medium for proliferation in culture cups, covered with pre-coating in the same way, preferably in environment NR 2 and NR-3 (environment, described in EP 780496), the composition of which is incorporated herein by reference), and

(viii) the transfer immortalized keratinocytes after about what operacii in the conventional environment for differentiation, having a high content of calcium (1.5 M), preferably on Wednesday NR-2 enriched EGF (epidermal growth factor) (5 ng/ml) and vitamin C (38 µg/ml).

In more detail, step (i) typically involves obtaining tissue samples of human skin from healthy donors, for example, samples obtained during surgery or pediatric surgery. Immortality unique sample of skin cells, i.e. autologous samples of skin cells, allows to obtain an immortalized cell line keratinocytes detecting certain characteristics, such as the profile of a specific receptor, which is specific to the donor.

Then this sample of skin tissue prepared in stage (ii) so that it was suitable for in vitro culture.

This training is typically done by first washing the samples of skin tissue, for example, using the medium used for culture. Preferably, this operation is carried out in the environment NR-2, which is a serum-free medium, the exact composition of which is described in EP 780496, which, as shown, has advantages for the culture of normal keratinocytes. After washing the sample of skin tissue is preferably shave, for example, using a dermatome (tool for skin plasty), and then cut into small pieces.

Then receive the data slices of the skin share preferably in the dermis (the skin) and the epidermis (outer (epithelial) layer of the skin)). This can be achieved by physical and/or enzymatic methods. For example, this can be accomplished by treatment with trypsin, for example, flotation samples of skin tissue in a solution of trypsin (for example, approximately 0.5%)containing EDTA (for example, approximately 0.1%), in a period of time sufficient to initiate cell division, for example, in a period of time of approximately 30-60 minutes at a temperature of 37°or, for example, over night at 4°C.

The dermis is separated and then the epidermis is placed in a medium to obtain a suspension. Preferably medium to obtain a suspension contains a solution of soybean trypsin inhibitor (SBTI) and it is placed in contact with the cells over time (usually 5 minutes)sufficient to inactivate trypsin and release cells. Then medium for tissue culture, preferably Wednesday NR-2, lacking serum (described in EP 780496), and filter (such as filter 100 nm) are added to obtain the target cells, i.e. keratinocytes.

Then primary keratinocytes obtained in stage (ii), used for the planting of a serum-free environment, preferably medium NR-3 (described in ER), in a suitable cell concentration, preferably approximately 1.2×104cells/cm2on pre-coated culture plates. However, the concentration of cells may be varied within wide limit is H. Culture Cup is preferably provided with a coating containing composition, which, as has been shown, enhances the attachment and growth of keratinocytes, more specifically a solution of fibronectin, HEAD and collagen type I.

In stage (iv) culture medium replaced as often as is necessary for optimal cell growth. Preferably the medium replaced every time after about two days. However, this depends on the particular sample of skin tissue. After receiving almost full confluently, such as confluences approximately 90%, which occurs after approximately 10-14 days, keratinocytes and melanocytes share. This can be accomplished in any manner it chooses, allow adequate separation of cells without any damaging effect on the melanocytes and keratinocytes. For example, this can be accomplished by differential treatment with trypsin. Preferably melanocytes or keratinocytes treated with a solution of trypsin/EDTA and then transferred to selective medium. In the case of keratinocytes cells are preferably treated for about 5-10 minutes with a solution of trypsin/EDTA (0,025%/0,01%) and then used in stage (v) for planting on Wednesday NR-3 pre-coated cups.

Then keratinocytes treated with agent immortalization. Cells may be the e frozen before executing the immortalization, for example, in liquid nitrogen. Infection and immortality preferably carried out using functional oncogenic genes of at least two different retroviruses, such as genes T-the Hell of SV40 virus and E6/E7 of HPV-16 (HPV16). Each of these genes may be located in independent retroviral constructs, such as retroviral vector pLXSHD+SV40(#328), shown in figure 1 and described Stockshlaeder et al. (GeneBank, the access number M; Human Gen. Therapy, 2, 33-39, 1991) and the retroviral vector pLXSHD+E6/E7, which is shown in figure 2.

The retroviral vector pLXSHD+SV40(#328) includes, among other sequences, a sequence of T-Hell SV40, the sequence 5’- and 3’- long terminal repeats SV-40, the sequence of pBR322, which make possible the replication of E. coli, the cycle multiple cloning sequence and the polyadenylation SV-40.

In the location of the gene encoding T-antigen, a vector pLXSHD+E6/E7 contains NcoI/CfoI-fragment of the gene E6/E7 derived from human papilloma virus 16.

After the immortalization of the cells are then subjected to the required number of passages during cultivation and the resulting immortalized cells are then transferred into an environment for the proliferation stage (vii). This transfer is preferably performed during the second passage. Environment for proliferation may be serum-free medium, preferably a medium NR-2 and NR-3. Immortalized cells cultured on the coated pre continuously cultural cups, and cover again contains a solution of fibronectin, SAB and collagen type I.

After breeding immortalized cells in the medium for proliferation (preferably NR-2) keratinocytes transfer on stage (viii) on Wednesday, inducing differentiation of normal and immortalized keratinocytes, preferably in an environment that simulates (reproducing) conditions prevailing in the skin, generating thus the organization of keratinocytes in the stratified and polarized epithelium with normal superficial keratinized layers. For this purpose, cells may be cultured in serum-free medium with high content of calcium, such as NR-2, containing approximately 1.5 mm calcium, approximately 5 ng/ml EGF and about 38 μg/ml vitamin C, and the cultivation is performed on the cups within 2-3 weeks at the interface (interfaze) air/liquid, for example, on cups with 12 holes Falcon No. 3042, and each hole has a liner Falcon No. 3180 in which keratinocytes develop in the interphase of the air/liquid. The air consists of atmospheric air contained in the inner space of the liner, which is devoid of nutrient medium. Liquid nutritive medium is contained in the well, the rich this environment crosses the membrane liner, to develop keratinocytes.

With regard to the properties of immortalized keratinocytes of the present invention, they are quite suitable for immunological, pharmacological, photo - and chemical-Toxicological analysis of skin reactions. For example, an immortalized cell line keratinocytes of the present invention can be used for analyses that require differentiated skin cells, for example, for studies on barrier function (keratinization) reconstructed skin tissue, studies on the metabolism of differentiated keratinocytes (fatty acid metabolism, antioxidant metabolism), studies on the effects of UV radiation on skin cells, studies on the potential effects of irritating the skin and sensitizing the skin agents on skin cells, studies on the metabolism of lipids, through local processing and/or usage environment xenobiotics agents (e.g., cosmetic oils, choosing possible protective compounds, for example, sunscreen agents), studies on inflammation and irritation of the skin, etc.

In addition, cell lines keratinocytes obtained in accordance with this invention, are used for screening of potential anti-cancer compounds and potential connections for Leche is of skin diseases. This usually implies that this cell line is kept in contact with such compounds during a specific time period and determine the potential induction of any harmful effects, such as genotoxicity, formation of DNA adducts, mutagenicity, cell transformation or cytotoxicity.

In addition, lines immortalized keratinocytes of the present invention can be used in assays of mutagenicity of DNA analyses for selection of skin mutagenic agents, assays to identify agents of change, the chromosomal studies on malignant transformation, studies of cellular biochemistry (for example, in tests activation CYP450), in the selection of compounds and compositions, for example, mixtures of fatty acids which are involved in inflammatory and allergic reactions in tests for activation of collagenase (due to inflammation), part TNFa, and for the detection of interleukin.

With regard to the fact that the cell lines of this invention form containing ortho-keratinocyte cornified layer (stratum corneum), they are particularly well adapted for participation in the artificial leather used for the studies on mutagenicity, immunological, pharmacological, photo - and chemical-Toxicological studies mentioned above. This skin condition is ü only of epithelial keratinocytes of the present invention, but preferably it also contains, for example, collagen, fibroblasts, even melanocytes to have higher affinity with the skin of a healthy person.

In addition, cell lines keratinocytes of the present invention is able to Express the recombinant proteins, for example, polypeptides and proteins of the person, as well as to produce DNA and RNA.

Among immortalized cell lines keratinocytes obtained in accordance with this invention, only cell line DK7-NR was like, for example, deposited under the terms of the Budapest Treaty on March 19, 1998 in the Collection Nationale de Culture de Microorganismee (C.N.C.M.), having an address 25, rue de Docteur Roux, 75724 Paris, France, and received the Deposit number CNCM I-1996. This Deposit was made under the terms of the Budapest Treaty. All restrictions concerning the availability of this cell line will be permanently removed after the grant of a patent in accordance with this application or another application, claiming priority to this application.

Other features of this invention will become apparent from the following description of examples given to illustrate this invention and should not be construed as restrictive. If there are no other instructions, the manipulation of cells, preparation of vectors, transformation of cells and all other technical methods are carried out in accordance with the tvii protocols, described by Sambrook et al., (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, USA, 1989).

Example 1: isolation and characterisation of cell lines

Took the skin tissue of the breast. After separation of the dermal and epidermal parts of the dermis is cut into small pieces 0,2×0.2 mm and fixed on culture 6 see the Cup with whey. Minimum essential medium, Dulbecco (DMEM, 10% fetal calf serum) is added after 2-4 hours. This culture of Explant then incubated until then, until it becomes visible overgrowth of fibroblasts. Culture confluent fibroblasts separated and propagated to obtain frozen backup crops.

The culture chamber seeded with primary cells covered continuously "cocktail" (a mixture of) the coverage described previously for bronchial cells (Lechner et al., J.Tiss. Cult. Meth., 9:43-49 (1985)). After achieving almost full confluently, for example, approximately 90% of confluences, which usually takes place after about 10-14 days, keratinocytes and melanocytes share. For this purpose, the culture is treated with a solution of trypsin/EDTA (0,025%/0,01%) for 5 minutes and then collect the melanocytes, which are separated yourself from keratinocytes. Then primary keratinocytes cultured until the desired number of cells in the environment NR-3 without serum, with use of the described having a coating cultural the different cameras (Wednesday NR-3 favors the preferential growth of keratinocytes compared with melanocytes).

Then cells to encapsulate "ZTZ-fibroblasts packaging cell line" transferout plasmids pLXSHD+SV40(#328) and pLXSHD+E6/E7 in accordance with the Protocol Pfeifer et al. (Meth. Cell Sci., 17, 83-89, 1995) with the condition that the virus harvested after encapsulating it in a cell line that grows in DMEM medium containing 10% fetal calf serum. Then keratinocytes infected with this virus to cause immortality. During infection also use the medium without serum RS-1, which is described by Pfeifer et al.

After the immortalization immortalone keratinocytes is transferred into the environment for the proliferation NR 2 and NR-3 using pre-coated with coating cultural cameras. After cell proliferation until the desired number of cells, the cells are transferred into a medium for differentiation, suitable for the culture of normal and immortalized keratinocytes (NR-2).

It can be shown that immortalized keratinocytes exhibit improved growth of cells with an increased number of passages in culture, which can be comparable to the growth described for cell lines in EP 780496.

The expression of CYP450, 1A1, 1A2, A, 2E1, V, A and 2D6 analyze in skin cells, consisting of normal and immortalized keratinocytes, using DNA-polymerase chain reaction at room temperature (mRNA expression). Profile CYP450, expressions is termed the keratinocytes, in particular, similar, even identical, with a profile of normal keratinocytes.

These cell lines respond to an inducing agent CYP450 consisting of benzo(a)pyrene, in the same way that you react naimportovane cells, even with the increased number of passages.

Markers of differentiation analyzed using specific antibodies against T-antigen (T-Ad), involucrin, filaggrin, loricrin, vimentin and keratins K4, K7, K8, K10/1, K13, K14, R17, K18 and C. Better ability to demonstrate the ability of differentiation could be shown for cell lines DK7-NR.

Glutathione-S-transferase (GST) analyzed using Western blot and Northern blot. All lines keratinocytes Express strongly the messenger RNA (mRNA) for gstπ. Profile exposure with GSTα, GSTμ and gstπ in these cell lines is similar to normal keratinocytes.

For analysis and comparison violations saturation and elongation added to the keratinocytes AGE immortalized keratinocytes treated with linoleic acid (LA, 15 µmol) and linolenic acid (LN, 15 mmol). For these experiments use the environment NR-2 (Biofluids Inc.), insufficient AEG. Cell culture process after reaching confluently and transferred from the environment to NR-2, having a high content of calcium (1.5 m is). Cells treated for 4 days of AGE (updated after 2 days). The analysis of these AGE performed through extraction and separation of phospholipids by using TLC (thin layer chromatography), and determine the amount of methyl esters of these fatty acids is carried out by means of GLC (gas liquid chromatography). It was possible to show the appearance of reducing the saturation and elongation of linoleic acid (20:4n-6 and 22:4n-6) and linolenic acid (20:5n-3, 22:5n-3 and 22:6n-3). The metabolic profile was consistent with the profile observed with normal keratinocytes.

All cell lines were hypodiploidy with the highest numbers of chromosomes in the range of diploid cells. In addition to the analyzed cells, no cells were not detected in these cultures. This result confirms the purity of the cell lines and the absence of any contamination originating from other sources.

Carcinogenesis immortalized keratinocytes determine subcutaneous injection (1-2×106keratinocytes) "Nude" mice. Lines tested keratinocytes and, in particular, the line DK-7-NR was not carcinogenic for "Nude" mouse.

Example 2: Getting epithelium

Prepare the environment NR-2, containing 750,000 cells cell line, in accordance with example 1, 0.5 ml of this medium is placed in the ear Falcon No.3180, they are placed in the wells of cups Falcon No.3043 already containing 2 ml of fresh cf the water NR-2, keratinocytes cultured for 2 days under conditions favorable for the growth of keratinocytes. On the third day the medium contained in the liner is removed and the cells left in open air. The environment contained in the wells are periodically replaced every 2 days medium NR-2, supplemented with EGF (5 ng/ml), vitamin C (38 µg/ml) and l2(1.5 mm). After 2-3 weeks in culture in the interphase air-liquid formed so epithelium harvested, fixed with picric acid and analyzing the morphology.

The results show that the cell line keratinocytes, in particular cell line DK7-NR, form a stratified and polarized epithelium, usually called stratum basale (main layer)containing cells having cuboidal morphology, identical morphology of normal cells. The Horny layer of the stratum corneum detects the morphology of ortho-keratinocytes, which do not contain cells that contain a nucleus. The formation of a cell layer ortho-keratinocytes has not been possible with other known termed cell lines so far. For example, under identical conditions line DK2-NR (EP 780496) forms a layer of the stratum corneum, which consists of a pair of keratinocytes, which is a layer organisa cells still containing cells with a nucleus. Only the morphology of ortho-keratinocytes of the stratum corneum reflects the normal situation of human skin. F. chicosci organise layer (stratum corneum), consisting of parakeratinized, characterized by an abnormal hypertrophy of the epithelium, leading to disorders such as psoriasis or neoplasia.

Example 3: Test for irritation

Cell line obtained in example 1, in particular, the line DK7-NR, cultivated in the medium NR-2. Induction "stress gene" TNFα (tumor necrosis factor alpha) after treatment skin irritant agent consisting of PMA (phorbol-12-myristate-13-acetate) and UV-B radiation UV-b)analyze the way Northern blotting and using biological tests. The results indicate that these cell lines and especially cell line DK7-NR, answer RMD and UV-B and Express the protein TNFαeven after an increased number of passages.

Example 4: Construction of artificial skin

The membrane liner Falcon No.3180 replace the layer of benzyl ester of hyaluronic acid (Hyaff 11). The bottom liner is seeded with primary human fibroblast (0,1×106cells in 0.2 ml medium), after 30 minutes the reaction, the liner is filled with DMEM containing 10% fetal calf serum, incubation was performed at 37°C in an atmosphere containing 5% carbon dioxide, in a few days, the liner drain and serve with 0.5 ml of fresh medium NR-2, containing 750,000 cells cell line DK7-NR, the liners are placed in the wells of cups Falcon No.3043 already containing 2 ml of fresh media is NR-2, and the cells cultured for 2 days under conditions favorable for the growth of keratinocytes. On the third day the medium contained in the liner is removed and the cells left on the air. The medium in the wells periodically replace every 2 days medium NR-2, supplemented with EGF (5 ng/ml), vitamin C (38 µg/ml) and CaCl2(1.5 mm). After 2-3 weeks of culture in the interphase air-liquid can observe the formation of artificial leather, exhibiting the characteristics of normal skin.

1. Cell line of human keratinocytes, immortalitya using pLXSHD+SV40(#328) and pLXSHD+E6/E7 used for immunological, pharmacological, photo - and chemical-Toxicological analysis of skin reactions and for the expression of heterologous genes, characterized in that it (1) is non-carcinogenic; (2) retains the ability to differentiate and to Express the proteins and enzymes expressed in normal differentiated keratinocytes, even after an increased number of passages in culture, and (3) forms a stratified and polarized epithelium with consisting of orthokeratosis Horny layer (stratum corneum), when cultivation in organotypic culture in serum-free environment and without a layer of feeder cells.

2. Cell line according to claim 1, used for artificial leather.

3. Cell line immortalized keratinocytes che is oweka DK7-NR (CNCM I-1996), used for immunological, pharmacological, photo - and chemical-Toxicological analysis of skin reactions and for the expression of heterologous genes.

4. Cell line according to claim 3, used for artificial leather.

5. The way the immortalization of human skin cells to obtain a cell line according to claim 1, comprising the following stages:

(i) the selection of the sample of human skin;

(ii) preparation of that sample skin for culture in vitro;

(iii) obtaining keratinocytes prepared from this sample of human skin and sowing serum-free medium for the cultivation of these keratinocytes in culture cups, top coating containing fibronectin, collagen type I and BSA, which facilitate the attachment and growth of cells;

(iv) the replacement of serum-free medium sufficient for optimal confluent growth of cells in culture by way of continuous maintenance coverage cups;

(v) the migration of keratinocytes in serum-free selective medium for the culture plates, pre-coated in the same way;

(vi) the infection of keratinocytes with the use of functional tumor genes of two different DNA viruses;

(vii) transferring the received immortalized keratinocytes in the environment for proliferation, suitable for proliferation of immortality the x keratinocytes, on cultural cups, covered in the same way; and

(viii) transferring the received prooperirovavshim keratinocytes in the environment for differentiation with a high content of calcium in the culture chambers, covered in the same manner, characterized in that the infection is two retroviral constructs representing vectors pLXSHD+SV40(#328) and pLXSHD+E6/E7.

6. The method according to claim 5, wherein the serum-free medium at stage (iii), (v) or (vii) is Wednesday NR-3.

7. The method according to claim 5, characterized in that environment for the differentiation stage (viii) is modified environment NR-2, having a calcium content of at least 1.5 mm.



 

Same patents:

The invention relates to biotechnology and can be used to obtain Malinov human IL-4 with an activating T-cell activity and reduced activating endothelial cells activity

The invention relates to biotechnology, in particular to the creation of transgenic plants with insecticidal properties

The invention relates to the field of genetic engineering and can be used in the biomedical industry

The invention relates to the field of genetic engineering and can be used in the biomedical industry for preparation of medicinal products for gene therapy

The invention relates to the medical industry and refers to polypeptides that are specific against CD19 and CD3, and their application

The invention relates to biotechnology and can be used to create a functioning tyrosine-specific chimeras proteinkinase

FIELD: biology, genetic engineering.

SUBSTANCE: invention relates to preparing immortalized cellular lines from health human skin tissues and can be used in immunological, pharmacological, photo- and chemical-toxicological analysis of cutaneous response, for expression of heterologous genes and for construction of artificial skin. Keratinocytes are immortalized by infection of keratinocytes of health human. The human skin sample is isolated and prepared its for culturing in vitro. Keratinocytes are prepared from this prepared human skin sample and plated in serum-free medium for growing keratinocytes in cultural plates with cover alleviating attachment and growth of cells. In the process for culturing keratinocytes the serum-free medium is replaced to provide preparing the optimal confluent growth of cells in culture with continuous maintenance of cup cover. Keratinocytes are transferred in selective serum-free medium in cultural cups with cover and infected with vectors pLXSHD + SV40(#328) and pLXSHD + E6/E7. Then prepared immortalized keratinocytes are transferred in cultural cups with cover to useful medium for proliferation. Then prepared proliferated keratinocytes are transferred in medium with high calcium content for differentiation in cultural chambers with cover. Invention provides preparing the human keratinocyte cellular line that has no oncogenic property and retains capacity for differentiation and expression of proteins and enzymes expressing by normal differentiated keratinocytes being even after increased number of passages in culture. Also, this cellular line forms lamellar and polarized epithelium with keratinized layer (stratum corneum) consisting of ortho-keratinocytes in the process for culturing in organotypical culture in serum-free medium and without layer of feeding cells.

EFFECT: improved immortalizing method, valuable biological properties of cellular line.

7 cl, 2 dwg, 4 ex

FIELD: genetic engineering, immunology, medicine.

SUBSTANCE: invention relates to new antibodies directed against antigenic complex CD3 and can be used in therapeutic aims. Antibody IgG elicits the affinity binding with respect to antigenic complex CD3 wherein heavy chain comprises skeleton of the human variable region in common with at least one CD3 taken among amino acid sequences SEQ ID NO 2, 4 and 6 and their corresponding conservatively modified variants. Light chain comprises skeleton of the rodent variable region in common with at least one CD3 taken among amino acid sequences SEQ ID NO 8, 10 and 12 and their corresponding conservatively modified variants. Antibody is prepared by culturing procaryotic or eucaryotic cell co-transformed with vector comprising recombinant nucleic acid that encodes antibody light chain and vector comprising recombinant nucleic acid that encodes antibody heavy chain. Antibody is administrated in the patient suffering with malignant tumor or needing in immunosuppression in the effective dose. Invention provides preparing chimeric antibodies against CD3 that are produced by expression systems of procaryotic and eucaryotic cells with the enhanced yield.

EFFECT: improved preparing methods, valuable medicinal properties of antibody.

33 cl, 5 dwg, 1 ex

FIELD: biotechnology, molecular biology.

SUBSTANCE: method involves transfection of cells HKB with vector pCIS25DTR comprising a selective marker and a sequence encoding protein eliciting procoagulating activity of factor VIII. Cells are selected using the selecting agent and clones with high level for expressing protein eliciting procoagulating activity of factor VIII are isolated. Invention provides preparing the protein eliciting activity of factor VIII with high yield, and strain of cells HKB with improved production under protein-free conditions also. Invention can be used for preparing the protein eliciting activity of factor VIII in industrial scale.

EFFECT: improved preparing and isolating methods.

8 cl,, 6 dwg, 1 tbl, 5 ex

FIELD: biology, genetic engineering, biotechnology, medicine.

SUBSTANCE: invention relates to preparing glycosylated polypeptide (glycoprotein) as a component of human erythropoietin by using the technology of recombinant DNAs. This polypeptide shows ability to increase production of reticulocytes and erythrocytes, to enhance the level of hemoglobin synthesis and consumption of iron by marrow cells and characterized by the higher molecular mass as compared erythropoietin isolated from human urine. Invention describes variants DNA sequences encoding this polypeptide that comprise vector constructions with these sequences, a method for preparing transformed mammalian cell lines producing the recombinant human erythropoietin, and a method for its preparing and purification. Also, invention proposes pharmaceutical compositions comprising glycosylated polypeptide (glycoprotein) of erythropoietin as an active component. Applying this invention provides scaling the process for preparing active human erythropoietin useful for its using in medicine.

EFFECT: improved preparing method, valuable properties of polypeptide.

10 cl, 4 dwg, 21 tbl, 12 ex

FIELD: biotechnology, in particular biosensors.

SUBSTANCE: claimed method includes production of sensitive cells producing in exited state signals being detectable by peripheral device. In one embodiment cells cultivated in cell cultures prepared form animal receptor cells are used as sensitive cells. In another embodiment as sensitive cells receptor cells which are functionally analogous to animal receptor cells cultivated in cell cultures prepared form animal stem cells are used. As peripheral device electrical signal receiver is used, wherein said electrical signal is generated by cell in exited state. Claimed invention is useful both in investigations and in industry.

EFFECT: biosensors with increased sensitivity, accuracy and integrity.

14 cl, 1 dwg, 4 ex

FIELD: biotechnology, immunology, molecular biology, medicine, pharmacy.

SUBSTANCE: invention describes the isolated human antibody or its antigen-binding fragment able to bind the human tumor necrosis factor (TNF-α). Amino acid sequence is given in the description. Invention discloses nucleic acid encoding heavy and light chain of isolated human antibody. Nucleotide sequences are given in the description. Invention describes recombinant vector expressing variable region of heavy and light chains of isolated human antibody, Chinese hamster ovary cells CHO dhfr- carrying vector. Invention discloses a method for synthesis of isolated human antibody. The isolated human antibody or its antigen-binding fragment can be used as an active component of pharmaceutical composition used in treatment of disturbances when activity of TNF-α is harmful. Using the invention allows neutralization of effect of TNF-α in case when its activity is harmful. Invention can be used in medicine.

EFFECT: valuable medicinal properties of antibody, improved method for synthesis.

17 cl, 11 dwg, 17 tbl, 4 ex

FIELD: genetic engineering, virology, pharmacy.

SUBSTANCE: invention proposes the recombinant modified virus OF VACCINE Ankara able to express structural antigens of hepatitis C virus. Virus comprises DNA sequences encoding structural antigens of hepatitis C virus or their functional regions or epitopes of hepatitis C virus structural antigens. Also, invention proposes a pharmaceutical composition comprising such virus, eucaryotic cell infected with such virus, a method for preparing such virus and a method for preparing hepatitis C virus structural polypeptides. Invention can be used in virology and medicine for preparing hepatitis C virus antigen.

EFFECT: valuable properties of virus.

20 cl, 14 dwg, 1 tbl

FIELD: genetic engineering, medicinal virology, medicine.

SUBSTANCE: invention proposes recombinant RNA molecules, methods for preparing recombinant eukaryotic cell, methods for preparing chimera RNA virus, vaccine preparations and immunogenic compositions. The recombinant matrix NDV-viral RNA with negative chain has been constructed that can be used in viral RNA-dependent RNA polymerase for expression of products of heterologous genes in corresponding host-cells. Proposed group of inventions can be used against pathogens and antigens of broad row.

EFFECT: valuable properties of expression systems.

145 cl, 11 dwg

FIELD: genetic engineering, virology, medicine.

SUBSTANCE: invention relates to method for production of modified Vaccinia virus Ankara (MVA). Claimed method includes contamination of mammalian continuous cell line with Vaccinia virus Ankara (MVA) of wild type, followed by viruses cultivation and collection. Further fresh cells of the same cell line are infected with newly formed viruses. Abovementioned steps optionally are repeated. Also disclosed are strains of modified Vaccinia virus Ankara (MVA) and utilization thereof. Said strains are capable to growth in continuous cell lines.

EFFECT: strains having decreased virulence in relates to mammalians.

20 cl, 5 tbl

FIELD: biotechnology, in particular production of modified swine factor VIII (POL1212).

SUBSTANCE: DNA molecule encoding of modified swine factor VIII is cloned in expression vector, having functionality in mammalian cells. Modified swine factor VIII protein is obtained by cultivation of mammalian cell line BHK CRL-1632 (ATCC), BHK 1632, or CHO-K1, transfected with vector. Therapeutic composition for treatment of subjects suffering from deficit of factor VIII, such as haemophilia, contains effective amount of swine factor VIII protein.

EFFECT: effective agent for treatment of factor VIII deficit.

13 cl, 8 dwg, 7 ex

Up!