Method for making medical preparations

FIELD: medicine.

SUBSTANCE: diploid cell cultures are reduced and collected by propagation in a monolayer in an Eagle's growth medium. The conditioned growth medium is separated from the formed cell layer by sterile bottling. The cell layer is removed, washed by centrifugation. The prepared cell suspension is reduced to the required and bottled in the sterile environment. The conditioned growth medium bottles and cell suspension bottles are consistently single frozen to temperature -20°C and higher and thawed at temperature +25°C and lower, and bottled in the sterile environment. The conditioned growth medium and cell suspension are frozen in open bottles at temperature minus 50°C and lower, kept in the frozen environment for at least 48 hours and then lyophilised in two stages. The first stage involves desorption at temperature minus 50°C to 0°C for at least 20 hours; and the second stage requires sublimation for at least 20 hours at temperature 0°C to 30°C; the finished lyophilised bottles are closed in the sterile environment. The invention allows preparing the lyophilised preparations effective in treatment of deep vast burning wounds, frostbites, degrees III (a, b) and IV subdermal burns, treatment of oral and nasopharyngeal mucosa, are storage-stable at temperature +4°C for at least 12 months.

EFFECT: higher efficacy of the preparations for treatment of diseases.

1 dwg, 3 tbl, 2 cl

 

The invention relates to biology and medicine and can be used for restoration and correction functions of the tissues damaged by their treatment in the treatment process liofilizirovannami biologically active substances diploid cells.

The use of cell cultures of alloparental (FBA) is a basis of a method of treatment of lesions of the skin and mucous membranes of the skin and injuries. Currently, cell cultures are widely used in several countries, mainly for the treatment of superficial lesions of the skin and mucous membranes, in particular severe burns, trophic ulcers and other surface lesions of skin and mucous coats. Biotechnology cell system is the Central element for cellular therapeutic technologies that are created for use in cardiology, neurology, Hematology, Hepatology, combustology, dentistry, endocrinology, and for the purpose of revitalization.

There is a method of treatment of burn wounds by transplantation of living cells fibroblasts grown on hydrophilic base, covered with collagen and subjected to multiple passirovanny (RF patent No. 1699047, IPC AC 35/48, 1989).

There is a method allows to shorten the healing of wounds in 5-7 days and provide epithelization throughout the affected surface. However, the very cell TRANS who Lancet suitable for use in a short shelf life, which makes transportation and correspondingly wide use.

Also known to treat skin diseases and injuries from external causes, the method of preparation of the drug, which is a cellular transplant of fetal tissues. The drug is produced by the selection and accumulation of cells suspended last selected slurries with a high content of regional stem cells with defined characteristics, cultivated add partially differentiated cells, specialized cells and biologically active substances in fetal tissues. Cell transplant may contain additions, including specific proteins and/or antioxidants and/or growth factors and the like (patent RF № 2160112, IPC AC 35/48, 2000).

The resulting product can only be stored in liquid nitrogen, which is difficult for ordinary transportation. Despite the fact that known drug allows lyophilic drying, the latter is held against sludge or supernatant to a concentration of the material does not allow to obtain the product a long shelf life in the absence of liquid nitrogen. In addition, raw materials for obtaining of the drug is limited, and the process of its reception requires considerable time, which also makes it difficult wide is using.

Also known cell culture for substitution therapy, which uses a strain of diploid cells LEC-4 (81) (Patent RF № 2213775 IPC C12N 5/08; A61R 35/12; C12N 5/08 C12R 1:91, 2003).

The use of cultured fibroblasts showed high efficiency in replacement therapy, does not require expensive nutrient media, growth stimulants, which reduces the cost and allows to considerably reduce the time for obtaining grafts, ready to use in clinics. However, the drug has a short shelf life and suitable for storage only in liquid nitrogen requires individual preparation of the sample and its immediate use, which makes it difficult transportation and wide application.

A method of obtaining strain of diploid cells, which can be used in substitution therapy for the treatment of skin lesions. (Application for the invention of the Russian Federation No. 2007104923, IPC C12N 5/08, 2008). The method includes the use of pre-shredded embryonic tissue, which was washed with enzyme solution at a temperature of 25-37°C, followed by trypsinization not less than three but not more than five times by processing the pieces of fabric with a solution of trypsin proteolytic activity within 65-80 Ed under stirring, plum separated cells treated with nourishing the environment and centrifuged, the precipitate resuspended and cultured in monolayer in the growth medium on the basis of eagle medium, incubated with subsequent passirovannym at least five times, the target product is treated with an enzyme solution with a view to its removal from the surface of the mattress, the obtained cell suspension is centrifuged, and then the target product is subjected to cryopreservation in liquid nitrogen.

A known method can improve the efficiency of the process of obtaining strains of diploid mammalian cells, which is characterized by increased stability and is effective in the treatment of skin lesions. However, the obtained product is not suitable for long term storage in the absence of liquid nitrogen and is not subject to transport in the absence of special equipment that makes its widespread use.

There is also known a method of obtaining a dry product by separating the target substance - alpha-fetoprotein serum, followed by fractionation of proteins, purification by double affinity chromatography on sepharose, sterilizarea filtration and lyophilization (RF patent No. 2283131, IPC AC 38/17, OR 37/02, 2006).

Receive the drug should be stored at +4°C, possesses immunosuppressive properties, is used to reduce reactions transplantation of immunotherapy transplantation of organs and tissues. However, the process of obtaining the drug is time-consuming, requires significant amounts of reactants, whereas the yield of the target product is 75%, and, in addition, the target product lacks a strong effect in the treatment of skin diseases.

Closest to the present invention is a method of obtaining a lyophilized insulin by sterile extract of the pancreas of embryos, they crumble into fragments, cultivation in vitro in a nutrient medium Needle with the addition of 10% human serum in a rotational setting, centrifugation, removal of toxins, Department of supernatant followed by freezing, thawing, by repeated centrifugation with subsequent lyophilization of frozen supernatants (inventor's certificate SU # 1806751, IPC AL 37/26, 35/39, 1993).

Get the drug does not have its own strong effect in the treatment of lesions of skin and mucous coats.

The present invention is to obtain a highly effective drug for the treatment of lesions of skin and mucous coats, with long shelf life at a temperature of +4°C with simultaneous provision of waste received.

The problem is solved in that a method of obtaining cell drugs for the treatment of porage the s skin and mucous membranes of the skin by lyophilization cell cultures, where used as raw material recovered after cryopreservation diploid cell culture, in this case, before lyophilization cell cultures, surgery recovery and accumulation of cell culture by means of its propagation in the monolayer in the growth medium based on the medium Needle, air-conditioned growth medium is separated from the resulting cell formation by its sterile drain into vials, and the cell layer is removed, washed by centrifugation, adjusted to the desired concentration and the resulting cell suspension sterile poured into vials vials with air-conditioned growth medium and the flasks with cell suspension sequentially subjected to a single freeze to a temperature not lower than -20°C and thawing at a temperature not above +25°C With subsequent aseptic filling into vials, air-conditioned growth medium and the cell suspension is subjected to freezing in open vials at a temperature not higher than minus 50°C, kept in a frozen state not less than 48 hours, followed by lyophilization in two stages, the first stage for at least 20 hours lead desorption at a temperature of minus 50°C÷0°C, and the second stage are sublimation for at least 20 hours at a temperature of 0°C÷30°C, the vials with cell culture sterile closed.

As a source of raw materials used is neoconservative diploid cell culture according to regulatory documents (Selection, cultivation and control strains of diploid cells." Methodical instructions. The Ministry of health of the USSR, M., 1979, p.16-22). As a source of raw materials can be used in cell cultures derived from embryonic tissues of mammals, mainly tissues of embryos pigs, sheep. For carrying out the claimed method is the most preferred are the embryonic tissue of the first half of pregnancy (4-6 weeks). Cells derived from embryonic tissues such an early age, less differentiated, which positively affects the quality and quantity of the final product. As a source of embryonic tissue can be used embryos of the second half of pregnancy (10-12 weeks), but in this case the quality of the cells is impaired, and time efficient cultivation significantly reduced.

Preparation of cell culture includes the use of pre-shredded embryonic tissue of a mammal, washed enzyme solution at a temperature of 25-37°C, followed by trypsinization tissue by repeated processing of the pieces of fabric under stirring with a solution of trypsin, plum separated cells treated with culture medium and centrifuged, the precipitate resuspended and cultured in monolayer in the growth environment on the basis of the environment of the Needle, incubated with subsequent passirovannym for a period of not less than five times, the target product is treated with an enzyme solution with the purpose of removing from the surface of the mattress, the obtained suspension is centrifuged and the resulting cell mass is used to produce the claimed drug after its cryopreservation.

The inventive method can be implemented to retrieve the target drugs with any other permitted use of cell culture.

In the present method for cultivation in the monolayer used growth medium based on medium Needle, which can be used medium consisting preferably of equal amounts of the growth medium Needle MEM and 0.5% solution of hydrolyzed lactalbumin (GLA) with 10% fetal serum of calves (ETS) or a mixture of medium growth Needle MEM and environment 199 or other known growth medium containing the growth medium Needle. The use of these environments ensures the preservation of the viability of cell cultures and their maximum output.

Eat cells produce a mixture of solutions of 0.25% trypsin and 0.02% of Versene then adding eagle medium or GLA. The mode of removal of cells provides the maximum quantity and quality removable cell suspension and subsequent centrifugation - quality deposition of cell mass.

According to the claimed method is similar cell culture used after its cryopreservation. Getting dry preparation is carried out by lyophilization pre-treated cell cultures. Processing a cell culture is conducted as follows. Restored a cell culture is grown in a monolayer in the above growth medium based on the medium Needle, preferably, within 4-5 days, then eat produce cell culture and its separation from air-conditioned growth medium. The last pick in the bottles and removed the cell layer washed by centrifugation, the obtained cell suspension was adjusted to the desired concentration and pour into bottles. Samples for sterility taken from the vials with air-conditioned growth medium from the flasks with a cell suspension. Vials with air-conditioned growth medium and the vial containing the cell suspension is subjected first to freezing to temperatures below -20°C, and then thawing at a temperature of +20°C ÷ +25°C. the Frozen vials of cell suspension and air-conditioned growth medium is stored up for bacteriological analyses, but not more than 3 weeks. After thawing the contents of the vials sterile poured into bottles for medicines, then freeze in open vials in a sterile tape for freeze-drying at a temperature not higher than minus 50°C, kept in a frozen state at least 48 is aces followed by lyophilization in two stages, in the first stage lead desorption for at least 20 hours at a temperature of minus 50°C÷0°C, and the second stage are sublimation for at least 20 hours at a temperature of 0°C÷30°C at a pressure not higher than 15 PA, vials with dry target product sterile closed.

The target product is a:

- dried product on the basis of the air-conditioned growth medium - (LS);

- dried product on the basis of cell culture - cells (LC).

The resulting preparations can be stored at a temperature of +4°C ÷ +6°C is not less than 12 months, which makes them easy to transport and use.

Conducted research, it was found that the obtained lyophilized drugs have shown efficacy in the treatment of extensive deep burn wounds, frostbite, when subdermal burns III (a, b) and IV degree early after admission of the patient, in the treatment of the mucous membranes of the oral cavity and the cavity of the nasopharynx.

The inventive method provides a waste-free technology for the production of drugs for the treatment of skin lesions from cell cultures, the yield of the target product exceeds 90%.

Comparison of the proposed method with the known leads to the conclusion conforms to the criterion "novelty", as to obtain a dry product used cryopreserved the cell culture, and its lyophilization is accompanied by a special pre-processing, in which the target product is obtained from the cell culture and conditioned growth medium in which the cell culture was replicated.

In science and technology known methods of freeze drying various substances, including methods of drying tissue cell cultures. There are also known methods of cultivation of cell cultures in monolayer in the growth environment based on the environment the Needle. In the inventive method, the set of techniques used allowed us to obtain new technical result, which is expressed in maintaining the quality of cell cultures, the possibility of using air-conditioned growth medium to obtain the desired product (PA), which led to the creation of non-waste production of the maximum yield of target product, suitable for long-term storage and large-scale use. The foregoing leads to the conclusion about conformity of the proposed method the criterion of "inventive step".

The claimed invention can be used in medicine for the treatment of lesions of the skin and mucous membranes of the skin by known methods, target product capable of industrial production, for its preparation are known in medicine drugs. Of the above, the its allows to make a conclusion on the conformity of the proposed solutions to the criterion of "industrial applicability".

The inventive method is illustrated by the scheme shown in the drawing.

For implementing the inventive method were prepared for cell culture according to the following method. As a source of raw materials use of cryopreserved cell culture, which is obtained by trypsinization homogeneous tissue from a mammal animal donor, 4-6-week-old embryos of the pig. Selected fabric washed three times with sterile Hanks solution or 0.25%solution of trypsin or 0.02%solution himopsina with gentamicin at a temperature of 25-37°C, collected in a sterile flask, washed up probably full of bleeding and crushed with a scalpel into pieces the size of 2-3 mm3. The washed pieces of fabric are placed in a sterile flask for carrying out the process of trypsinization on a magnetic stirrer. Trypsinization lead by adding to the flask with 0.25% trypsin solution, preferably proteolytic activity within 65-80 Units at a temperature of 32-37°C. the Cycle trypsinization repeated at least 3 times. To ease digestive trypsin activity in the drained portion of the suspended cells to1/2volume add 0.5% solution of hydrolyzed lactalbumin (GLA). After each cycle of trypsinization a suspension of cells is placed in pre-cooled to a temperature not more than +6°C sterile centrifuge Cup the us and poured a medium Needle or a mixture of mediums Needle and 0.5% GLA. Centrifugation selected the suspension of cells is carried out at 1200-1500 rpm for 10 minutes in a refrigerated centrifuge at a temperature of +4 to+6°C. the precipitate resuspended 0.5% solution, the GLA and the cell suspension thoroughly pipeinput. Select a sample and hold control and counting of cells in the cell Goryaeva. Pietrobono cell suspension is prepared at a concentration of 300 thousand cells /ml in medium Needle MEM with the addition of 10% fetal bovine serum (ASCRS) and poured into sterile disposable plastic mattresses. Incubation is carried out in thermostat at 37°Scheres 1 day are changing the growth medium to remove detritus. The monolayer of cells is formed on the 5th day. Reseeding performed by standard methods in the ratio 1:2. On the second and third passages of the cells subcultured in 5 days regardless of the density of the monolayer. From the 4th passage is formed a dense monolayer with oriented growth zones unimorph fibroblast-like cells with clear boundaries. Removal of cell formation while passirovannye lead 0.25% solution of trypsin or 0.02% solution of versene or their mixture at a ratio of 1:1 at a temperature of about 37°C for 0,5-1,0, minutes with the addition of eagle medium MEM or GLA, followed by centrifugation at 1000-1200 rpm for 10-15 minutes.

Quantitative karyological analysis of the obtained cell culture p is Casali, the relative number of cells with chromosome set, the corresponding chromosomes of the animal donor, is 95-97%. The formation of culture is in the process 1-5 passages, accumulation obtained diploid strains cells are from 6 to 20 passages. The growth of established cultures of diploid strains of cells of adequate quality is not less than 45 of the passage.

Cryopreservation established cell culture carried out by a standard procedure in the growth medium with addition of 10% glycerol in software freezer according to the standard procedure followed by placement in a low-temperature refrigerator (not less than 86°C) or in liquid nitrogen (-196°C). Control and recovery of the resulting cells showed that the viability of the recovered culture is 85-95%.

Samples established cell culture that is not affected by cryopreservation, continue to passivate prior to the manifestation in the monolayer signs of nonspecific degeneration or significant changes in morphological parameters.

Upon receipt of fibroblast cells of embryos of the pig from the lung of pig embryos (LES) as described above, the average output of the cells with 1 g of tissue is 30-40 million cells.

For preparation of the inventive method for the treatment of lesions of the skin and mucous coats take the restored settlement is E. cryopreservation of cell cultures, obtained from the embryonic tissue of mammals and prepared mainly by the above described method.

Control of bacteriological and virological sterility is done according to approved methods of TORMENT 1/4.2.588-96 method of direct seeding; method of staining DNA fluorochromes for RD 42-28-10-89 or by electron microscopy in accordance with TH the USSR Ministry of health, M., 1979; reaction haemadsorption with Guinea pig erythrocytes in the THROES of 1/4.2.588-96. Preparation drugs to determine karyological parameters of culture carried out in accordance with the requirements of RD 42-28-10-89, control karyological parameters are known methods (see, e.g., "isolation, cultivation and control strains of diploid cells." Methodical instructions. The Ministry of health of the USSR, M., 1979, p.16-22).

Cryopreserved cell culture restored by known methods within 14-18 days. Restored cryopreserved cell cultures cultured in monolayer in the growth environment based on the environment the Needle, which is used, wt.%: nutrient medium Needle MEM - 45, 0.5%solution of hydrolyzed lactalbumin - 45 (GLA) and fetal serum of calves - 10 (ETS). Cultivation of conduct for 4-5 days, then eat produce cell formation and separation from air-conditioned growth medium by the Liwa. Eat cell layer of lead with the aforementioned method. Air-conditioned growth medium selected vials for blood substitutes a volume of 250 ml or 500 ml from each vial take samples to determine the bacteriological and virological sterility. Shot of the cell layer washed by centrifugation for 10 minutes at 1500 rpm the Resulting cell suspension adjusted to a concentration of approximately 200 thousand cells/ml and dispensed into vials. Samples for bacteriological and virological sterility taken from the vials with air-conditioned growth medium from the flasks with cell culture. Vials with air-conditioned growth medium and the flasks with cell culture is subjected to first freeze to a temperature not lower than minus 20°C, preferably -16°C ÷ -18°C, and then spend the thawing temperature +20°C ÷ +25°C. the Frozen vials with cell culture and the spent growth medium stores to conduct bacteriological tests, but not more than 3 weeks. Thawing vials are for obtaining data bacteriological studies. After thawing the contents of the vials poured into sterile 5 ml vials for pharmaceuticals by volume of 10 ml, then freeze in open vials in a sterile tape for freeze-drying at a temperature not higher than minus 50°C, the optimal m is the Nusa 70°C, kept in a frozen state not less than 48 hours. Freeze-dried preparations is carried out in the apparatus LZ-9.2 at a pressure not higher than 15 PA in two stages, first conduct a stage of desorption in the temperature range of minus 50°C÷0°C for at least 20 hours, and then the stage of sublimation at the temperature 0°C ÷ +30°C. the Rate of temperature rise of 5°C per hour. The total duration of the drying cycle is 40-42 hours. After drying the obtained drugs and LK sealed, sterile closing plugs and rolling aluminum caps.

Preparations obtained as described above, were examined in preserving the original properties and tested for the treatment of burns. The obtained data are presented in tables.

To determine the healing of burn wounds II, III (A. B) extent were used lyophilized preparations obtained from cryopreserved cells diploid strain obtained in the above way. Clinical studies of the effects of drugs were carried out on groups of patients aged 6 months to 15 years with burns wounds II, III (a, B) class (group 1 - control, receiving conservative treatment methods using ointments, group 2 receiving drug treatment, group 3, receiving treatment LK). Claimed the drugs were prepared by the dilution in 5 ml of water for injection sludge is in a physiologically sterile solution. Treatment of skin lesions led by spraying or applying a bandage soaked in diluted drug. The timing of complete epithelialization, formation of the scab and the type of epithelialization was assessed visually; pain was assessed by the survey, by the reaction in the process; exudation was determined morphologically. The data obtained are presented in table 1, table 2 and table 3.

As can be seen from the presented data, the use of drugs as air-conditioned growth medium (PM)and cell culture (LK)obtained by the claimed method allows to increase the effectiveness of the treatment of skin lesions and its quality, reducing pain and preventing the occurrence of purulent processes and the formation of scars.

From the presented data also shows that the effectiveness of the LC is higher than the efficiency of drugs. The use of the drug LK is indicated for the treatment of patients with severe skin lesions (e.g., extensive deep burns III and IV), whereas the treatment of less severe skin lesions (e.g., burns II and III), and treatment of the mucous membranes of the skin can be effectively carried out using a drug drug.

Table 1
The healing of burn wounds II, III (a, B degree under the influence of the processing liofilizirovannam medication
OptionsThe treatment
Control (conservative method)Lyophilized growth medium (BOS)Liofilizirovannaya cell culture (LK)
Age8 months. 15 years6 months. 14 years1 year - 14 years
The timing of complete epithelialization (days)14,3±1,99,3±1,28,5±1,1
Formation of the scab (%)10010-117-9
Suppurative complications (%)5nono
Type epithelializationwith the formation of a scarbezrucovabezrucova

Table No. 2
Tolerability of the patient and burns III (A, B) extent
Patient groupPainThe duration of painful reactionReaction
group 1 (control)++++-
group 2 (PA)++-
group 3 (LK)++-
where: + - measurable; ++ - denominated.

Table No. 3
The effect of the drug on the wound healing process for burns III (a, B) extent
Patient groupThe exudationEpithelialization (days)Thermal responseLeukocytosis n×109
group 1 (control)+++13,6±/-1,6N 10,3±/-1,2
group 2 (PA)++12,1±/-1,3N10,9±/-1,7
group 3 (LK)++10,5±/1,7N10,4±/1,4
where: +++ - expressed strongly; ++ - expressed weakly.

Monitor the suitability of drugs within 12 months showed that during storage of drugs and LK in terms of +4°C +6°C preparations retain their original properties. Obtaining the claimed preparations which are suitable for use for at least 12 months and do not require special temperature conditions for storage, you can extend their use for the treatment of skin lesions.

1. The method of obtaining drugs for the treatment of lesions of skin and mucous membranes of the skin by lyophilization cell cultures, which is used as raw material recovered after cryopreservation diploid cell culture, in this case, before lyophilization cell cultures, surgery recovery and accumulation of cell culture by means of its propagation in the monolayer in the growth medium based on the medium Needle, air-conditioned growth medium is separated from education is bassegoda cell formation by its sterile drain vials and the cell layer removed, washed by centrifugation, the obtained cell suspension was adjusted to the desired concentration and sterile poured into vials vials with air-conditioned growth medium and the flasks with cell suspension sequentially subjected to a single freeze to a temperature not lower than -20°C and thawing at a temperature not above 25°C With subsequent aseptic filling into vials, air-conditioned growth medium and the cell suspension is subjected to freezing in open vials at a temperature of not higher than -50°C, kept in a frozen condition for at least 48 h followed by lyophilization in two stages, the first stage for a period of not less than 20 h lead desorption at a temperature of -50 to 0°C, and the second stage are sublimation for at least 20 hours at a temperature of 0 to 30°C, the vials are ready liofilizirovannam drug sterile closed.

2. The method according to claim 1, characterized in that as the growth medium on the basis of eagle medium use medium consisting, wt.%: nutrient medium Needle MEM - 45, 0.5%solution of hydrolyzed lactalbumin - 45 and fetal serum of calves - 10.



 

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13 cl, 7 ex, 8 dwg

FIELD: chemistry; biochemistry.

SUBSTANCE: invention discloses a strain of hybrid animal cells Mus musculus L.4 A2, which is deposited in the Collection of cell cultures of the State Research Center of Virology and Biotechnology VECTOR, which is a producer of monoclonal antibodies which are specific to the matrix protein VP40 of the Ebola virus, Zaire subtype (Mainga strain), and a strain of hybrid animal cells Mus musculus L. 1C1 which is deposited in the Collection of cell cultures of the State Research Center of Virology and Biotechnology VECTOR, which is a producer of monoclonal antibodies which are specific to the matrix protein VP40 of the Ebola virus, Zaire subtype (Mainga strain). The invention is also aimed at obtaining monoclonal antibodies 4A2 which are produced by the 4A2 hybridome, (subclass of immunoglobulins IgGl which have a heavy 55 kDa and a light 25 kDa chain) and are used as binding antigens in the "sandwich" format immunoenzymometric system for exposing the matrix protein VP40 of the Ebola virus, Zaire subtype (Mainga strain), and monoclonal antibodies 1C1 produced by the 1C1 hybridome (subclass of immunoglobulins IgGl which have a heavy 55 kDa and a light 25 kDa chain), used as biotin labelled indicators in the "sandwich" format immunoenzymometric system for exposing the matrix protein VP40 of the Ebola virus, Zaire subtype (Mainga strain). The disclosed antibodies are used together in a "sandwich" format immunoenzymometric system for exposing the matrix protein VP40 of the Ebola virus, Zaire subtype (Mainga strain).

EFFECT: invention enables to obtain monoclonal antibodies which are specific and do not compete with each other for antigen epitopes and which, when used together in a "sandwich" format immunoenzymometric system, ensure high reliability of results for exposing the matrix protein VP40 of the Ebola virus.

5 cl, 3 dwg, 1 tbl, 6 ex

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: organic chemistry, natural compounds, medicine, oncology.

SUBSTANCE: invention represents new saponin mixtures used for inhibition of initiation and activation of mammalian epithelial cell in pre-malignant or malignant state, for stimulation of apoptosis of mammalian malignant cell, prophylaxis of anomalous proliferation of mammalian epithelial cell, for treatment of inflammatory and regulation of angiogenesis in mammal. These mixtures are isolated form plants of species Acacia victoriae. Also, invention relates to methods for their applying. These compounds can comprise triterpene component, such as acacic or oleanolic acid to which oligosaccharides and monoterpenoid components are joined. Mixtures and compounds elicit properties associated with regulation of apoptosis and cytotoxicity of cells and strong anti-tumor effect with respect to different tumor cells.

EFFECT: valuable medicinal properties of compositions.

43 cl, 53 tbl, 50 dwg, 44 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, medicine, genetic engineering, pharmacy.

SUBSTANCE: the hemopoietic protein comprises the amino acid sequence of the formula: R1-L1-R1, R2-L1-R1, R1-R2 or R2-R1 wherein R1 represents the modified ligand flt-3; R2 represents the modified human IL-3, the modified or unmodified colony-stimulating factor. Modification of R1 is carried out by addition of N-end with C-end directly or through linker (L2) that is able to join N-end with C-end to form new C- and N-ends. The modified human IL-3 is prepared by replacing amino acids at positions 17-123. The human G-CSF is modified by exchange of amino acids. The hemopoietic protein is prepared by culturing cells transformed with vector comprising DNA that encodes the hemopoietic protein. The hemopoietic protein stimulates producing hemopoietic cells and this protein is used as a component of pharmaceutical composition used in treatment of humans suffering with tumor, infectious or autoimmune disease. Invention provides preparing multifunctional hemopoietic proteins eliciting the enhanced activity with respect to stimulation of hemopoietic cells and eliciting the improved physical indices. Invention can be used for preparing chimeric multifunctional hemopoietic proteins.

EFFECT: improved preparing and producing method, valuable medicinal properties of protein.

22 cl, 19 dwg, 18 tbl, 117 ex

FIELD: cellular biology, medicine.

SUBSTANCE: invention relates to isolating and cryopreserving precursor-cells. Methods involve treatment of human liver tissue for preparing the essentially monocellular suspension containing precursor-cells and cells that are not precursor-cells, a single or more lines of cellular differentiation presenting in the human liver. Invention describes methods involving stage for separating cellular population resulting to reducing amount of cells that are not precursor-cells and providing preparing the separated suspension enriched with precursor-cells expressing one or more markers and associated with a single or more lines of the cellular differentiation. Also, invention describes a method for selection cells from the separated suspension wherein these cells or their progeny, or their more matured forms express one or more markers associated with lines of the cellular differentiation. These markers involve: CD14, CD34, CD38, CD45 and ICAM. Hepatic precursor-cells have diameter size 6-16 mc, they are diploid and show indices: glycoforin A-, CD45-, AFP+++, ALB+, ICAM+ and they comprise subpopulations varying with respect to expression of CD14+, CD34++, CD38++ and CD117++. These cells are useful for carrying out cellular and genetic therapy in liver treatment and for preparing artificial organs also.

EFFECT: valuable biological and medicinal properties of cells.

41 cl, 7 tbl, 13 dwg, 15 ex

FIELD: medicine, surgery, transplantology.

SUBSTANCE: embryonic spleen should be sampled, washed in nutritive medium № 199 to be placed into fresh medium № 199 to obtain homogenate in teflon homogenizer followed by centrifuging; then one should isolate the upper, medium and inferior layers, suck off medium layer and the upper part of inferior layer; the cell mixture obtained should be diluted in nutritive medium № 199 to be then introduced by injections into mesentery of small intestine or rectus muscle of abdomen. The present innovation favors the activation of immune system in patients undergone splenectomic operation and in those in case of surgical immunodefficient state due to high functional and regenerating activity of transferred embryonic splenic cells.

EFFECT: higher efficiency of prophylaxis.

6 dwg, 2 tbl

FIELD: medicine, genetic engineering.

SUBSTANCE: invention relates to applying genetic engineering approaches for treatment of autoimmune diseases, in particular, for treatment of cerebrospinal sclerosis. This is achieved by incorporation of one or some recombinant genes encoding autoantigens that represent a target for autoimmune response. In particular, invention claims a method for designation of gene encoding encephalitogenous epitope of proteolipid protein and expression of gene product in vivo by using the recombinant retroviral vector. Expression and secretion of encephalitogenous epitope improves histopathological and clinical indices in experimental autoimmune encephalomyelitis in mice that is used as a model of cerebrospinal sclerosis. The advantage of invention involves the development of a method for recovery the tolerance in treatment of cerebrospinal sclerosis being without suppression of immune system.

EFFECT: improved and valuable method for treatment.

6 cl, 13 dwg, 3 ex

FIELD: biotechnology and pharmaceutical industry.

SUBSTANCE: title operations are accomplished by following way. Three-dimensional culture of stromal cells is cultured in piston flow bioreactor, in particular being introduced in fibrous matrix incorporated into substrate, which is placed in container constituting a part of bioreactor piston. Stromal cells are grown until density 5 x 106 cell/cm3 substrate is attained, whereupon non-differentiated hemopoietic cells are either sowed directly into piston flow bioreactor or cultured in conditioned medium of stromal cells obtained by gathering medium from indicated flow bioreactor. Non-differentiated hemopoietic cells obtained by cultivation in presence of three-dimensional culture of stromal cells or their conditioned medium are transplanted to into recipient.

EFFECT: enabled growth of large amounts of stromal cells within a relatively small volume to provide longer maintenance of vital activity and reproduction of non-differentiated hemopoietic stem cells or precursor cells.

77 cl, 9 dwg, 3 tbl

FIELD: biotechnology and pharmaceutical industry.

SUBSTANCE: title operations are accomplished by following way. Three-dimensional culture of stromal cells is cultured in piston flow bioreactor, in particular being introduced in fibrous matrix incorporated into substrate, which is placed in container constituting a part of bioreactor piston. Stromal cells are grown until density 5 x 106 cell/cm3 substrate is attained, whereupon non-differentiated hemopoietic cells are either sowed directly into piston flow bioreactor or cultured in conditioned medium of stromal cells obtained by gathering medium from indicated flow bioreactor. Non-differentiated hemopoietic cells obtained by cultivation in presence of three-dimensional culture of stromal cells or their conditioned medium are transplanted to into recipient.

EFFECT: enabled growth of large amounts of stromal cells within a relatively small volume to provide longer maintenance of vital activity and reproduction of non-differentiated hemopoietic stem cells or precursor cells.

77 cl, 9 dwg, 3 tbl

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

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