Method to produce epithelioid cells of light foetus of buffalo cow for cultivation of viruses

FIELD: biotechnologies.

SUBSTANCE: method is proposed to produce epithelioid cells of buffalo cow light foetus by means of long-term no-reseeding cultivation having high sensitivity to the virus of infectious rhinotracheitis of cattle, parainfluenza-3, viral diarrhea-disease of mucous membranes and adenoviral infection.

EFFECT: possibility to use in diagnostics of virus infections.

4 ex

 

The invention relates to biotechnology, in particular to a technology for cell cultures, and can be used in veterinary Virology in the diagnosis of viral infections of cattle.

Currently, there is a lot of material, indicating that the optimal cell systems upon receipt of viral preparations and for experimental purposes are the culture of diploid cells. Diploid cultures have several advantages over primary and permanent (stable) cultures, so long as they are able to proliferate in vitro with retention of the original properties, uniform cell structure and free from contaminants [2, 4].

Known diploid cell cultures derived from different tissues: kidney, skin, lung, heart, spleen, gimpa, segoviano gland and other organs of various animal species and humans [1-7].

A disadvantage of known methods is their complexity, which is the enzymatic and mechanical treatment of the source material that requires a lot of time, causes loss of cells, and the use of chemicals disrupts cell function. In addition, the culture obtained by prototypes enzymatic disaggregation of the tissue, were fibroblast-like Tina. It is known that a diploid cell culture fibroblast-like the IPA have a limited lifespan in culture in vitro and their sensitivity to viruses below, than to cells epitheliopathy type.

Closest to the proposed method to the technical essence and the achieved result is the method of cultivation of fibroblasts for substitution therapy, described in patent RU 2320720 [6]. In this way prompted characterized the culture of fibroblasts designed as a tool for regenerative therapy and cosmetology. The disadvantage of this method is "hard" processing of raw material (incubation with proteases of animal origin with constant stirring and pipetting), which is traumatic for the cells of the procedure, reducing the total number of viable cells in the resulting suspension dermal fibroblasts, as well as leading to violations of adhesion and receptor systems in part preserved cell viability.

The present invention represents a development in which taken Popelka get especiallyto from lung explants of fetal Buffalo, sensitive to respiratory viruses of cattle, with the use of technology for cell culture, which does not require enzymatic and mechanical treatment of the source material and culturing cells without reseeding.

The aim of the invention is to obtain epitheliopathy lung cells of the fetus Buffalo (LPB), case is valid for a wide range of respiratory viruses in cattle (cattle).

The technical result achieved by the proposed method is to simplify the process of cell epitheliopathy type with intensive reproduction and sensitivity to respiratory viruses of cattle.

To achieve the technical result in the production method of epitheliopathy cells, including the production of explants, incubation in a nutrient medium and the allocation of epitheliomata, exercise of lung tissue without enzymatic and mechanical treatment. For this lung tissue after getting in sterile conditions, cut into pieces 3...5 mm3and washed until full enlightenment supernatant. The resulting explants, washed with sterile Hanks solution with antibiotics, were placed in vials Carell, poured a nutrient medium consisting of equal volumes of 0.5%environment hydrolyzed lactalbumin (0.5% of GLA) with medium 199 with 10% serum of cattle, and incubated at 37°C. in 48-72 hours from the beginning of cultivation were changing environment. For 6-8 days of cultivation was mechanically removed explants and cells from lesions of the growth was removed with a glass of 0.02%solution of versene warmed to 35-37°C and perseval in the ratio of 1:1. Further cultured cells without reseeding for three weeks, and every 4-5 days spent changing environment by 30-50% with addition of 0.5%of the CSOs glutamine.

The DESCRIPTION of the PATENT

Epitheliopathy cells LPB was prepared as follows.

As the original fabric used is light fruit Buffalo 3...5th months of development, delivered no later than 2-3 h after slaughter of the animal. In a sterile room in the fetus removed easily and washed with sterile Hanks solution with antibiotics (200 units/ml penicillin and 200 μg/ml streptomycin). When processing authority pleura, trachea, bronchi, large blood vessels, cut the fabric into pieces 3...5 mm3and washed until full enlightenment supernatant.

Pieces of shredded tissue, rinsed with sterile Hanks solution with antibiotics, were placed in small culture flasks Carell, at a distance of 1-1 .5 cm from each other. Filled vials nutrient medium consisting of equal volumes of 0.5% of GLA with medium 199 with 10% serum of cattle, using a 10 ml pipette so that the explants were covered with medium. The vials were placed at 37°C for cultivation in thermostat.

The explants after 24 to 48 h was observed foci of cell growth, which are gradually increased. Colonies growing cells from explants were mixed (antilipogenic and fibroblast-like) type. The change of environment was performed 48-72 h from the beginning of cultivation. Later he entered the culture as follows.

After TRG is, as in vial appeared pockets of growth epitheliopathy cells (6-8 days), mechanical removal of explants and cells from lesions of the growth was removed with a glass of 0.02%solution of versene warmed to 35-37°C and perseval in the ratio of 1:1. The seeding cell concentration 300-350 thousand/ml On the 5th day of cultivation was obtained a homogeneous culture, composed mainly (with a predominance of 70-80%) of cells epitheliopathy type. This culture was used in further experiments on growing subcultures or epitheliopathy crops of the same type.

Further cultured cells without reseeding for three weeks, and every 4-5 days spent changing environment by 30-50% with the addition of 0.5%glutamine.

The result of the cultivation without reseeding observed a gradual withering away of remaining in the culture of fibroblast-like cells, and the vials were left cells epitheliopathy type, which are formed colonies of homogeneous cells. In the first passages of the multiplication of cells in the colonies was slow, and the accumulation of cells was sufficient for replanting in the same vial 1:1. Subsequent subcultures were made in the ratio 1:2, the concentration of cells was 200-250 cells/ml

The first 3-4 passage in monolayer culture were formed in 2-3 weeks. At this time every 4-5 days conducted a partial change of sredina 30-50%. Culture is stabilized at the 5th passage. Subsequent passages were performed as the formation of the monolayer, using a 0.25%solution of trypsin and 0.02%aqueous solution of versene, in the ratio of 1:9 basantapur way.

The obtained culture was subjected to subculturing many times and to date it is presented solely epitheliopathy cells. Culture was more passages 44.

Morphological characteristics. Culture has an intense growth. After 6-8 h of cultivation has been a steady growth with homogeneous cells that grew in a single layer without deposition. For 60-72 h is formed a monolayer. The coefficient reseeding is 1:2.

Ecomorphologically analysis of drugs at the level of 10, 20, 30 and 40 passages showed that culture LAB presented in monolayer cells epitheliopathy type. In stained preparations of cells had a polygonal shape, boundaries are firmly joined to each other. The nucleus of the cells is large, rounded, with 3-5 nucleoli. The structure of the nucleus had malosetti granular structure.

When karyological analysis of cells of strain modal class of chromosomes was 2n=50.

Information about contaminant. Bacteria, fungi and Mycoplasma bacteriological, radioautographic, cytological studies using fluorochrome-olivomitsina not found.

ultraline properties. Border cells are well expressed and culture good passerella. As the growth medium used: medium 199, 0.5% of GLA + 199 or 0.5% GLA + Needle in equal proportions with the addition of 10% cattle serum, inactivated at 56°C for 30 min and antibiotics (100 units/ml penicillin and 100 μg/ml streptomycin).

The monolayer of cells LAB was preserved in a thermostat at 36-37°C with medium without serum and 12-15 days, with no signs of degeneration.

Epitheliopathy cell culture LPB has found application in the work in the study of viral respiratory infections of cattle.

The described method allowed us to reliably obtain epitheliopathy culture of cells from the lung tissue of the fruit of Buffalo.

Example 1. Cell culture LAB were cultured in medium 199 with 0.5% GLA in the ratio of 1:1, with the addition of 10% cattle serum and antibiotics (100 u/ml penicillin and 100 μg/ml streptomycin). When reseeding the cells were removed from the glass 0.25%solution of trypsin and 0.02%solution of versene in the ratio (1:9), heated to 36°C. After counting the cells, the suspension was diluted to seed the concentration of nutrient medium 200-250 cells/ml

Cultural mattresses were placed in a thermostat at 37°C. the Monolayer was formed within 48-72 hours of Culture with increased monolayer used for infection with the virus of infectious bovine rhinotracheitis (RTI).

Example 2. Cell culture LAB cultivated on the environment And the La with 0.5% GLA in the ratio of 1:1, with the addition of 10% serum of cattle. After formation of the monolayer within 48-72 h of culture were used for infection with parainfluenza virus-3 (PG-3).

Example 3. Cell culture LAB were cultured in medium 199, with the addition of 10% serum of cattle. After formation of the monolayer after 48 to 72 h culture was used to infect virus viral diarrhea is a disease of the mucous membranes (BD-BS).

Example 4. Cell culture LAB were cultured in medium 199 with 0.5% GLA in the ratio of 1:1 with the addition of 10% serum of cattle. After formation of the monolayer after 48 to 72 h culture was used to infect virus adenovirus infection of cattle (ADI).

To identify the sensitivity of culture to the viruses were five serial passages of the virus. It is established that culture LAB sensitive to these viruses. Cytopathogenic action (JRC) of the virus RTIs occurred after 30 h after infection and was characterized by rounding of cells, loosening and destruction of the monolayer and subsequent rejection of the cells from the glass. In the infected culture was also mentioned in marginal chromatin in nuclei, then (after 25-30 h) is the formation of intranuclear inclusions typical of viruses of herpes group. JRS virus PG-3 occurred after 48-72 hours of culture Change, caused by a virus, characterized by the appearance of multinuclear cells (simpleton) and gradually the YM, after 72-96 h, the destruction of the monolayer. In the cytoplasm of cells was seen painted in pink cytoplasmic inclusions of different sizes. Sometimes they occupied almost the entire cytoplasm, in some cases in cell nuclei revealed a small pink inclusions, surrounded by a light area. The titer of the virus RTI reached 105,5-7,0TCD50/ml, virus NG 3 to 106,25-6,6TCD50/ml.

Cell culture LAB also susceptible to viruses VD-BS, ADVI with a characteristic expression of the JRS. For virus VD-BS is characterized by the appearance in the cells oraloader vacuoles, pikes nuclei and cells throughout infection - vacuolization of the cytoplasm. When the infection adenoviruses complete degeneration of the monolayer was accompanied by symplectomorphism, which was observed after 36-48 hours. The titer of virus VD-BS, ADVI in the culture has reached 104,75-5,4TCD50/ml and 10the 5.25-5,5TCD50/ml, respectively.

When examining one of outbreaks of respiratory infections of cattle, the maximum number of selected viral agents amounted to 35% on the primary-trypsinization culture kidneys calves, and culture LAB - 42% compared to the number of samples used for the study. Thus, culture is also suitable for the initial allocation of viruses from pathological material.

As can be seen from examples obtained epitheliopathy cell culture LAB Chu is stately to respiratory viruses of cattle.

In the dynamics of the cultivation was conducted cryogenical for long-term preservation of cells in createsite a medium consisting of 50% of media 199 with 0.5% GLA in the ratio of 1:1, 40% cattle serum and 10% DMSO. Spent freezing culture at 10, 20, 30 passages. After 15 and 30 days of snore in liquid nitrogen (-196°C) cell culture LPB held its defrost, rekultivirovanie. 48-72 h of rekultivirovanie formed a monolayer, represented by cells epitheliopathy type, which are not morphologically different from the original culture.

Literature

1. USSR author's certificate No. 1147748 from 30.03.1985.

2. Deacons L.N., Sitcov V.I. Animal cell culture (Methods and application in biotechnology). - M.: Satellite Company+. - 2000. - 400 C.

3. Look SO, Shitikova G.S., Ponomareva, NP and other fabrication and characterization of diploid lines of lung fibroblasts human embryo / Etiology and diagnosis of influenza and other acute respiratory diseases // Sat. scientific papers. - HP - H. - C.110-116.

4. Size J.F. ñ Obtaining cell cultures from animal tissues and their use in Virology. - M - 1975. - 103 C.

5. Pankov G., Sologub VK, Gololobova M.L., Rezovo TI Strain diploid lung cells of the embryo horses. Veterinary science. - 1978. No. 3. - P.42-43.

6. RF patent №2320720 from 27.03.2008. 7. Pozdnyakov A.A., Yurov C.P., MOTOZINE Z.P. and other Metadiscipline diploid strain cells testicular tissue foals and test its sensitivity to certain viruses. Bulletin of VIEW. - 1976. - VIP. - P.46-48.

The method of obtaining epitheliopathy lung cells of the fetus Buffalo for the cultivation of viruses in the implementation of which the Explant size 3-5 mm3obtained from the crushed tissue, cultured with the change of the nutrient medium within 48-72 hours from the beginning of cultivation, after 6-8 days, the explants were removed and subcultured cells grown in hotbeds of growth of explants remaining after their removal, removing them from the glass of 0.02%solution of versene warmed to 35-37°C, characterized in that the cells are cultivated without reseeding for three weeks with a change of medium every 4-5 days with addition of 0.5%glutamine.



 

Same patents:

FIELD: biotechnologies.

SUBSTANCE: method is proposed to cultivate human and animal cells on nutrient medium containing serum of buffalo cow foetus blood in the volume ratio of 5-7% serum and 93-95% medium, with the following incubation of cultures at 37°C for 3-5 days until formation of the monolayer.

EFFECT: invention may be used for cultivation of cells in a culture.

2 ex

FIELD: biotechnologies.

SUBSTANCE: method is proposed to treat experimental pulmonary tuberculosis in mice using transplantation of stem cells by introduction of a suspension of stem cells resistant to tuberculosis with "k" genotype in the area H-2E into a caudal vein once a week.

EFFECT: method improvement.

3 tbl

FIELD: biotechnology.

SUBSTANCE: means comprises at least one phenolic compound from the group of derivatives of cinnamic acid or a mixture of such compounds, and a nonionogenic surfactant or a mixture of such substances in an amount of not less than 75% by weight.

EFFECT: invention enables to obtain beauty products, biologically active additives and food products on the basis of the described means and to use them to stimulate the reparative processes in a cell and to reduce the side effects of aggressive cosmetic procedures.

30 cl, 7 dwg, 3 tbl, 38 ex

FIELD: biotechnologies.

SUBSTANCE: population of Tr1 cells is identified, being aimed against food antigen from regular ration of a human being and having phenotype CD4+CD25-FoxP3- in idle condition. The produced population of Tr1 cells in a combination with one or several pharmaceutically acceptable carriers, or in a combination with one or more pharmaceutical agents, used to treat inflammatory bowel conditions and selected from a group including anti-TNF, natalizumab, anti-IL1, anti-IL-6, anti-IL-12, anti-IL-17 and anti-IL-23, analogues of antagonist of a receptor IL-1.5 aminosalicylic acid and its analogues, corticoids, probiotics, methotrexate, azathioprine, 6-mercaptopurine, thalidomide, leflunomide, used within pharmaceutical compositions for treatment of inflammatory bowel conditions.

EFFECT: invention makes it possible to produce an efficient agent for treatment of inflammatory bowel conditions.

15 cl, 8 dwg, 1 tbl, 2 ex

FIELD: biotechnologies.

SUBSTANCE: permanent line of cells is produced from tissue of trout gonads by means of long-term passage in the medium of Igla MEM on Earl salts with 25 mM of HEPES from 10% embryonic serum. The line may be used as a test culture for extraction, accumulation, titration and study of a infectious pancreas necrosis virus (IPNV) of salmon fishers, virulent haemorrhagic septicemia virus (VHSV) of salmon fishes, infectious hemopoietic necrosis virus (IHNV) of salmon fish tissue, spring viraemia of carps virus (SVCV), koi herpes virus (KHV), sturgeon herpes virus (SbSHV).

EFFECT: permanent line of OMG cells will find wide application in laboratory research on virosology and biotechnology in production of virus vaccines and diagnosticums.

1 tbl

FIELD: medicine.

SUBSTANCE: patient's peripheral blood is simultaneously examined for the percentage of circulating stem hemopoietic cell CD34+ (SHC) and the volume fraction of lymphocytes (Lph, mln/ml); they are individually related as R by formula R=LPH/SHC, and an R (y axis) to SHC (x axis) law curve is drawn to be used as a calibration curve within the log-log grid, while the current recipient's state is assessed by examining the peripheral blood for LPH and SHC to be related as Rp and found in the calibration system of axis. If Rp is found either on the calibration curve, or above the same, the current recipient's state is considered to be favourable, otherwise the unfavourable state is shown by Rp found under the calibration curve.

EFFECT: using the declared method enables improving the accuracy of assessing the post-transplantation recipient's state.

4 tbl, 4 dwg, 3 ex

FIELD: biotechnologies.

SUBSTANCE: method is described to stimulate expansion of haematopoietic stem cells (HSC) in vitro or in vivo. A population of cells is incubated, which contains HSC, in presence of at least one HSC modulator, at the same time the HSC modulator is selected from the group that consists of PGE2, PGI2, linoleic acid, 13(s)- HODE, L Y 171883, MIDA acid, eicosatrienoic acid, epoxyeicosatrienoic acid, ONO-259, Cay 1039, an agonist of receptor PGE2, 16,16-dimethyl-PCE2, 19(R)-hydroxy-PCE2, n-(n-acetamide benzamide)phenyl esther 16,16-dimethyl-PCE2, 11-desoxy-16,16-dimethyl-PCE2, 9-desoxy-9-methylene-16,16-dimethyl-PCE2, 9-desoxy-9-methylene-POE2, butaprost, sulprostone, serynolamide PGE2, methyl ester PGE2, 16-phenyltetranor-POE2, 15(S)-15-MeTnn-PGE2 and 15(R)-15-MeTnn-PGE2. A method is proposed to strength HSC acceptance, and also a method to strengthen recovery of haematopoietic stem cells, which include HSC incubation in presence of at least one HSC modulator.

EFFECT: invention expands arsenal of methods for stimulation of growth of haematopoietic stem cells in a subject.

28 cl, 24 dwg, 11 tbl, 2 ex

FIELD: biotechnologies.

SUBSTANCE: by the recombinant method a line of cells CHO[V3D] is produced, which are transformed with plasmid pV3D, coding the human vessel endothelium growth factor, isoform A165, with C-terminal 3xDED-epitope (SEQ ID NO: 1), which is able to produce a biologically active recombinant protein VEGF-A165 of humans with C-terminal 3xDED-epitope in the amount of at least 0.2 mg 1 l of medium for 72 hours with 1 ml of growth medium per 5 cm2 of single-layer culture. The cell line is deposited in the All-Russian Collection of Industrial Microorganisms VKPM under the registration number VKPM H-123.

EFFECT: no impact at biological activity of protein and possibility to do protein treatment.

4 dwg, 7 ex

FIELD: biotechnology.

SUBSTANCE: method comprises adding to the part of the wells with lymphocytes of mitogens or test substances, incubation of the contents of the wells, selection of the contents of the wells, cytolysis and isolation of nucleic acids. The reaction of reverse transcription (RT) is carried out with primers for genes tpa and cdc2: primer for RT with mRNA of gene tpa 5'-CATCTTCATCTCACTCTTC-3', primer for RT with mRNA of gene cdc2 5'-CTGGAGTTGAGTAACGAG-3'. Real-time PCR is carried out with the obtained cDNA on the specific primers and with use of the labeled oligonucleotides for genes tpa and cdc2: for gene tpa the forward primer is 5'-TGTCGTGTCAGACCTTGAAGC-3', the reverse primer is 5'-CCTTGGATTTCTTGCTTGTGAC-3', the probe (FAM)-5'-TGTACCACTGTCTCAAGCCCTCCTGC-3'-(BHQ1), for cdc2 gene the forward primer is 5'-CTTCACTTGTTAAGAGTTATTTATAC-3', the reverse primer is 5'-CCAGAGTGTTACTACCTCATGTG-3', the probe (FAM)-5'-TGCCTTGCCAGAGC(FdT)TTTGGAATAC-3'-(BHQ1). The degree of proliferation of lymphocytes is expressed in proliferation index which is the number indicating how many times the amount of mRNA of gene tpa and/or cdc2 increases during culturing lymphocytes with/without addition of PHA or the test substance as compared with the amount of mRNA of the genes in lymphocytes freshly isolated from the blood of healthy donor.

EFFECT: invention enables to monitor directly the number of dividing cells, assessing the degree of expression of the genes involved and regulating the cell division.

4 dwg, 4 ex

FIELD: biotechnology.

SUBSTANCE: population of Tr1-cells directed against an antigen associated with disseminated sclerosis and having quiescent phenotype CD4+CD25-FoxP3- is isolated. The resulting population of Tr1-cells in combination with one or more pharmaceutically acceptable carriers, or in combination with one or more pharmaceutical agents used for treating disseminated sclerosis, selected from the group comprising interferon-beta, glatimer acetate, mitoxantrone, cyclophosphamide, methotrexate, asitropine, or natalizumab is used in pharmaceutical compositions for treating disseminated sclerosis.

EFFECT: invention enables to obtain an effective remedy for treating disseminated sclerosis.

11 cl, 2 dwg

FIELD: medicine.

SUBSTANCE: invention refers to medicine and veterinary science; it is used in transplantology, traumatology, surgery and oncology, and may be used to fill bone defects. What is described is a bioimplant which represents a donor bone deimmunised with chlorine-containing oxidants, a surface of which is covered with a multifunctional bioactive nanostructured coating (MBNC) of M-Ca-P-C-O-N or M-Ca-CON, wherein M is a metal specified in a group consisting of Si, Ti, Zr, Hf, Nb, Ta, and colonised by recipient's mesenchymal stem cells (MSC).

EFFECT: MBNC-coated bioimplant is in line with the anatomical and morphological characteristics of the replaced bone, provides cell adhesion, no transplant rejection, accelerated formation of connective tissue and callus.

8 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine and veterinary science, namely to reconstructive surgery it aims at the applications in transplantation, traumatology, surgery, and oncology. What is described is a method for producing bioengineered constructs for bone defect replacement, which is based on a bone anatomically compatible with the replaced one which is deimmunised in 5-10% solution prepared of a dry mixture of sodium chlorite, sodium perchlorate, sodium chloride in a ratio of 7:2:1 and distilled water; it is coated with heterogeneous implantable gel and colonised with multipotent mesenchymal stromal cells recovered from the recipient's bone marrow using immunomagnetic separation technique.

EFFECT: method provides sizeable bone defect replacement, high strength, fast fixation and repair of the construct in the implantation region, causes no reject phenomena.

1 tbl, 4 dwg

FIELD: medicine.

SUBSTANCE: what is described is a composition for fistula treatment in an individual, including stromal stem cells of fatty tissue where at least approximately 50 % of stromal stem cells of fatty tissue making a composition, express CD9, CD10, CD13, CD29, CD44, CD49A, CD51, CD54, CD55, CD58, CD59, CD90 and CD105 markers and where the contents of the stromal stem cells of fatty tissue in the composition makes at least approximately 3×106 cells/ml.

EFFECT: invention extends the range of products for fistula treatment.

8 cl, 7 dwg, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to chemistry of high-molecular compounds, namely to preparing film and spongy chitosan and collagen materials effective in human skin cell culture and wound grafting. A method for preparing the composite chitosan and collagen resorbable matrixes for human skin cell culture involves preparation of solutions polysaccharide chitosan and protein collagen, mixing them in preset proportions and formation of film and spongy matrixes of mixed polymer solutions. For this purpose, chitosan and collagen solutions in the concentration 1.0-4.0 wt % in a general solvent (aqueous 2 % acetic acid) are prepared, mixed in preset proportions, and the film and spongy matrix materials are formed from the prepared solutions. An amount of collagen in polymer mixtures makes 2.5-10 wt % (of chitosan). Further, films and sponges are heated to temperatures within 50-100°C for 1.0-5.0 h in an atmospheric environment.

EFFECT: use of the declared method allows preparing the film and spongy resorbable composite materials of natural polymers effective in human skin cell culture.

2 cl, 6 ex, 2 tbl

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to cardiovascular surgery. A biological material for cardiovascular surgery is made of a preserved carp swim bladder.

EFFECT: reduced calcification with maintained physical-mechanical properties.

1 ex

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to cardiovascular surgery. Described is biological material for prostheses, in which Glisson's capsule of horse is used. As base, Glisson's capsule of horse is used.

EFFECT: increase of elastic-strength characteristics of prostheses.

1 ex

FIELD: medicine.

SUBSTANCE: group of inventions relates to medicine, namely to dentistry, and deals with medications for restoration of periodontal tissues and methods of their obtaining. Biotransplant for treatment of periodontal diseases represents suspension which contains cultures of autological or allogenic fibroblasts, or fibroblast-like cells in 0.9% solution of sodium chloride in concentration 2.0-10.0x106 in 1 ml of biotransplant, integrated on pharmaceutically acceptable biocompatible biodegradable carrier, selected from the group: acellular matrix, hyaluronic acid preparation, acellular matrix and hyaluronic acid preparation, with specified volume ratio of cell culture suspension ands carrier, which makes it possible to obtain consistence convenient for injecting and, together with expressed clinical effect ensures low traumaticity and simplicity of injecting. Preferable, as carrier, used is crushed to size 100-200 mcm acellular matrix "Saimetra". Also claimed is method of treating periodontal diseases, which lies in the following: on mucous membrane of gum in region of defect one week before biotransplant introduction preliminarily made are cuts provoking an acute inflammatory process resulting in increase of tissue volume, which makes it possible to carry out lossless introduction of large volume of biotransplant. After that, into defect area by means of injection two times with interval 1-4 weeks introduced is claimed biotransplant, using 3.6-60×106 cells per treatment course, which ensures optimal time interval for cell engraftment and restoration of periodontal tissues.

EFFECT: increase of treatment efficiency.

7 cl, 3 ex, 2 dwg

FIELD: medicine.

SUBSTANCE: invention refers to medicine, and concerns a bioengineered structure for bony defect closure and osteogenesis which represents a hybrid implant comprising a porous polytetrafluorethylene membrane with a M-Ca-P-C-O-N or M-Ca-C-O-N doped multifunction, biocompatible, non-resorbed coating MBNC, where M is a metal chosen from a line including Ti, Zr, Hf, Nb, Ta, with autogenous or allogenic stromal cells recovered from fatty tissue or bone marrow passed on the surface thereof.

EFFECT: invention provides reliable degree of cell population attachment to the surface of the bioengineered structure with high cell culture adhesion and growth.

4 cl, 2 dwg

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to traumatology and orthopaedics, and can be used for correction of dystrophic and traumatic changes of cartilaginous tissue, and for early correction of growth processes in idiopathic scoliosis. The method is characterised by the fact that growth plates of newborn miniature pigs are placed in Hanks solution with cannomycin 1 g/l for 5 minutes, then growth plates are washed, crushed, transferred to test tubes with RPMI-1640 medium with 20% FBS and 1.5% collagenase of activity 240 U/ml added, and incubated; the suspension is centrifuged at 2000 rpm for 10 minutes; isolated chondroblasts are cultivated upon concentration 50-60 million; cell passage is enabled with mixed 0.25% trypsin and 0.02% EDTA; further, the cells are centrifuged at 2000 rpm for 10 minutes; then, a cell aggregate is moved to a well with nutrient medium RPMI-1640 with 10% FBS added, and cultivated for 4-6 weeks to prepare a chondral graft of 2×2×2±mm3.

EFFECT: invention prevents progressive spinal deformity at early stages, development of osteoarthrosis and osteochondrosis, reduced the postsurgical period, severely reduces number of postsurgical patient days and eliminates the staged expensive procedures on spine and joints.

1 ex, 5 dwg

FIELD: chemistry.

SUBSTANCE: porous matrix based on biocompatible polymer or polymer mix for tissue engineering is obtained by compression of polymer and sodium chloride particle mix with defined particle size, and further removal of sodium chloride by dissolution. Porosity grade of matrix lies within 93 to 98%, its pores fall into different sizes, with definite pore distribution by size within certain limits.

EFFECT: obtained matrices are free-shaped yet pertain stability and hardness characteristics required to withstand surgical implantation methods and counteract mechanical forces applied at the implantation point.

40 cl, 2 tbl, 8 ex

FIELD: medicine, transplantology, traumatology, orthopedics.

SUBSTANCE: the present innovation deals with the purpose to obtain osseous artificial block of vertebral bodies and trabecular bones of limbs at surgical treatment of traumatic lesions, degenerative-dystrophic osseous diseases, tumors, osteomyelitis and tuberculosis under conditions of systemic or local insufficiency of reparative osteogenesis. For local restoring the function of reparative osteogenesis in case of its different disorders, increased strength and rate of developing osseous block of vertebral bodies and maximal keeping achieved correction of vertebral deformation one should isolate stromal stem cells to cultivate and mobilize them upon fixing matrix out of porous titanium nickelide at 200 mln. cells/cu. cm, detect the implant's volume based upon measurements of pre-operational roentgenograms in standard projections by taking into account planned volume of operative interference, detect the quantity of stromal cells being necessary for developing local depot, calculate the quantity of desired medullary punctate, for the channel to apply the implant with mobilized stem cells.

EFFECT: higher efficiency.

Up!