Two-layer, flat, transparent hydrogel substrate for long-term cell cultivation
SUBSTANCE: two-layer, flat, transparent hydrogel substrate is proposed for long term cell cultivation and the method to get it. The substrate in the hydrated state has a thickness in the range of 25-500 microns, and the radius of menisci curvature of 10-150 microns. The hydrogel substrate includes a structure forming layer consisting of a three-dimensional collagen hydrogel and an upper protein layer. The three-dimensional hydrogel comprises collagen of type I, collagen of type III and proteins that promote cell adhesion and migration. The upper protein layer contains collagen of type IV, laminin and fibronectin. The method of substrate preparing comprises mixing of solutions of collagen of type I, collagen of type III and proteins that promote cell adhesion and migration and further gelation and vitrification, followed by proteins immobilization on the surface.
EFFECT: preservation of phenotype by endothelial cells and epithelial cells in their long-term cultivation.
13 cl, 1 dwg, 3 tbl, 8 ex
SUBSTANCE: nutrient medium comprises sodium chloride, potassium chloride, magnesium sulphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, calcium chloride, sodium bicarbonate, L-arginine, L-glutamine, L-tyrosine, L-tryptophan, calcium pantothenate, pyridoxal HCl, thiamine, meso-inositol, nicotinamide, riboflavin, folic acid, glucose, enzymatic hydrolyzate of muscle proteins, lactalbumin enzymatic hydrolyzate, sodium salt of benzylpenicillin with activity of 0.09×105-1.1×105, kanamycin sulphate with activity of 0.09×105-1.1×105, nystatin with activity of 6×104 to 6.5×104 IU/l, choline chloride, succinic acid and distilled water in a predetermined ratio of components.
EFFECT: invention enables to improve the quality of the product obtained.
1 tbl, 1 ex
SUBSTANCE: invention refers to biotechnology. What is described is using specific conjugate molecules, which are cargo molecule transporters for transporting a substance of interest into leukocytes. There are described a method for producing the above conjugate molecules, which are the cargo molecule transporters, a method for transporting the substance of interest into leukocytes, and the leukocytes containing the above conjugate molecules, which are the cargo molecule transporters. The invention can be used in medicine.
EFFECT: above conjugate molecules, which are the cargo molecule transporters, can be used for treating, preventing, relieving and/or reducing the intensity of a disease and/or a disorder involving leukocytes.
26 cl, 34 dwg, 5 tbl, 29 ex
SUBSTANCE: blood is separated into plasma and cellular fraction. Then the blood cellular fraction is subjected to successive two-phase treatment first by the buffer PBS solution containing 5 mM of EDTA with subsequent centrifugation and collection of supernatant. Cells are treated by equal volume of 0.15-0.35% trypsin solution in PBS with subsequent centrifugation and collection of supernatant. Plasma and extracted supernatants from cellular fraction are merged, cellular debris is removed by centrifugation at 15000-17000 g within 10-20 minutes. Foreign particles of non-exosome origin by filtration through filters with the diameter pores 0.1 mcm, and the total pool of exosomes is settled by ultracentrifugation at 100000-160000 g during 60-120 minutes.
EFFECT: invention allows to increase yield and purity of target product, reduce duration and labour input of the method.
3 cl, 2 dwg, 1 tbl, 3 ex
SUBSTANCE: strain of cells of the Chinese hamster ovaries CHO-Inflix 20/5 is produced, transfected by vectors, expressing light and heavy chains of chimeric antibody against tumour necrosis factor alpha (TNF-α) of human being is produced. The strain is deposited into the Russian collection of cellular cultures under No. RKKK(P)760D.
EFFECT: invention allows to produce chimeric antibody with the specific efficiency no less than 33,5 picograms per cell a day.
4 dwg, 2 tbl, 4 ex
SUBSTANCE: invention relates to field of biotechnology. Claimed is method of cultivating epithelial stem cells or separated tissue fragments, containing said epithelial stem cells, including provision of extracellular matrix, incubation of epithelial stem cell or separated tissue fragment, containing said epithelial stem cells, with extracellular matrix, cultivation of epithelial stem cell or separated tissue fragment in presence of cell culture medium, which contains basal medium for animal or human cells, into which inhibitor of bone morphogenetic protein (BMP) and 5-500 ng/ml of mitogenic growth factor are added, as well as cell culture medium, its application, method of three-dimensional organoid cultivation, method of its obtaining, three-dimensional organoid and its application.
EFFECT: method of cultivating epithelial stem cells is claimed.
32 cl, 10 ex, 34 dwg
SUBSTANCE: invention relates to biotechnology. Method includes adhesion of cell population to microcarrier in medium, containing Pho-kinase inhibitor. After that, cultivation of cells, separation of cells from microcarrier and adhesion of obtained cells to second microcarrier in medium containing inhibitor of Rho-kinase. Invention can be used in medicine.
EFFECT: method of growing human embryonic stem cells is disclosed.
7 cl, 48 dwg, 3 tbl, 11 ex
SUBSTANCE: invention refers to biotechnology and medicine. What is presented is a method for differentiation of mesenchymal stem cells of fibrotic lungs into fibroblastic cells, characterised by recovering the cells self-maintaining in vitro for 60 days and having mesenchymal stem cell phenotype (CD44+, CD73+, CD90+, CD106+, CD31-, CD34-, CD45-) from the right lobe of fibrotic lungs and producing the fibroblastic cells when adding fibroblast growth factor 2 mcg/ml to the mesenchymal stem cell culture.
EFFECT: method enables producing the donor cells from the patients suffering idiopathic fibrosis from fibrotic pulmonary tissues.
3 dwg, 1 ex
SUBSTANCE: invention relates to the field of biotechnology. ENA-21/13-13-finite monolayer-suspension cell subline of kidney of new-born Syrian hamster is deposited in the Russian Collection of Cell Cultures (RCC) in the specialized collection of finite somatic cell cultures of agricultural and game animals at the All-Russian Scientific Research Institute of Experimental Veterinary Medicine n.a. Y.R. Kovalenko (Russian Academy of Agricultural Sciences of farm animals) under No. 89 and intended for reproduction of foot-and-mouth disease virus type A, O, C, Asia-1, CAT-1, CAT-2, CAT-3, and the rabies virus strain "Schyolkovo-51", used for production of antiviral vaccines against foot-and-mouth disease and rabies.
EFFECT: enhanced immunogenicity, infectivity and concentration of cells in industrial suspension cultivation in bioreactors.
SUBSTANCE: claimed invention deals with method of obtaining population of cells, expressing pluripotency markers and markers, characteristic of formed endoderm line. Claimed method includes the following stages (a) obtaining population of cells, expressing markers HNF-3β, GATA-4, Mixl1, CXCR4, and SOX-17, characteristic of formed endoderm line, and (b) cultivation of population of cells under conditions of hypoxia on tissue culture substrate, which is not subjected to preliminary treatment with protein or extracellular matrix, in medium with addition of activin A and ligand Wnt and IGF-1 or insulin, transferrin and selenium, where cultivated cells express markers of formed endoderm line and pluripotency markers.
EFFECT: characterised invention makes it possible to obtain cells, capable of cultivation under conditions of hypoxia, which do not require lines of feeding cells, coatings of complex matrix proteins and preserve differentiation potential.
16 cl, 39 dwg, 9 tbl, 32 ex
SUBSTANCE: group of inventions refers to medicine, namely to immunology, and can be used in laboratory diagnostics, as a test system and a method for measuring interferon alpha (IFN-α) antiviral activity in human blood serum. The test system for measuring IFN-α activity in the human blood serum comprises diploid cells, a virus-containing fluid and standard human interferon-α (IFN-α). As the diploid cells, the test system comprises cells of the characterised line of the diploid cells - human M-20 fibroblasts at 20-33 passages cultured in the medium with added 10% fibrinolytically active plasma (FAP). As the virus, the system contains the A vesicular stomatitis virus (VSV) adapted to M-20 cells, the Indiana strain; the test system additionally contains a viral dye based on two fluorochromes - acriflavine and rhodamine C. The group of inventions also involves a method for measuring IFN-α activity in the human blood serum with the use of the developed system.
EFFECT: using the given inventions enables the quantitative good-reproducible measurement of IFN-α activity in the examined blood serum sampled by the luminescent microscopy of the vital preparations coloured in a fluorochrome-based vital dye.
3 cl, 4 dwg, 1 tbl, 4 ex
SUBSTANCE: group of inventions relates to field of biomedicine. Set and method are claimed for preparation of multi-layered agarose blocks on the surface of mini-slides. Set consists of first component, second and third components. First component represents plane-parallel plate, on which separating blocks, higher than thickness of mini-slides, are installed with equal spaced between them. Mini-slides are installed between blocks on 50-100 mcm. Second component represents plane-parallel plate, one side of which represents polished surface, on sides of which two lateral legs in form of 50±5 mcm thick blocks are welded. Third component represents plane-parallel plate, one side of which represents polished surface, on sides of which two lateral legs in form of 100±5 mcm thick blocks are welded. Method of preparing multi-layered agarose blocks on the surface of mini-slides is realised with application of claimed set.
EFFECT: inventions provide preparation of series of slides with agarose blocks of fixed volume with standard thickness of agarose layers.
2 cl, 4 dwg, 3 ex
SUBSTANCE: biohybrid composite material for sorption and degradation of crude oil and petroleum products is proposed. The material is a thermoplastic polymer with fibre-forming properties - acrylonitrile copolymer with methyl acrylate. It comprises incorporated phosphorus-containing cationites and/or nitrogen-containing anionites, the cell walls of aquatic plants of the family Lemnaceae (Lemnaceae) and immobilized cells of bacteria-oil destructors.
EFFECT: composite material has high adsorption capacity and a higher degree of biodegradation of petroleum hydrocarbons.
SUBSTANCE: invention proposes a preparation method of an enzymatic agent based on immobilised butyrylcholin esterase and an enzymatic agent for determination of carbamates and organic phosphorus compounds. Starch or gelatine gel is prepared in a phosphate buffer pH 7.9-8.0. The gel is cooled down to 31-35°C. Then, it is mixed with the buffer solution of butyrylcholin esterase and a buffer solution of an indicator of thiolic groups of 5,5'-dithio-bis(2-nitrobenzoic acid) with a concentration of 0.56 M. The obtained mixture is dosed to a hydrophobic substrate and dried at the temperature of 8°C. The inventions allow simplifying an enzyme immobilisation procedure, increasing storage time of an enzymatic agent without any loss of its activity during more than one year.
EFFECT: agent has high sensitivity to action of organic phosphorus compounds and carbamates.
2 cl, 7 dwg, 7 ex
SUBSTANCE: invention relates to biotechnology. Method of obtaining complex of organic solvents, which includes acetone, butanol and ethanol, from renewable natural vegetable cellulose-containing raw material includes milling to particles with size 20-80 mcm. Preliminary saccharification of milled raw material is performed for 1-5 h at 45-55°C. The following simultaneous saccharification of carbohydrates and fermentation of obtained hydrolysates are carried out at 28-34°C for 45-50 h under impact of biocatalyst. Said biocatalyst represents cells of bacteria acetobutylicumB-4786, immobilised in cryogel of polyvinyl alcohol in form of cylindrical granules, which have diameter and height 3-5 mm.
EFFECT: method makes it possible to increase productivity of process to 0,464 g/l/h and the total concentration of organic solvents to 23,3 g/l.
4 cl, 9 ex
SUBSTANCE: immobilised biocatalyst for microbial biotransformation of steroid compounds comprises cells of a microorganism having 1,2-dehydrogenase activity, included in the polyvinyl alcohol cryogel matrix. As cell having 1,2-dehydrogenase activity the immobilised biocatalyst comprises biomass of actinobacteria Pimelobacter simplex. The ratio of components in the biocatalyst is (wt %): actinobacteria cells Pimelobacter simplex - 1-5 (in dry substance), polyvinyl alcohol - 7-20, the aqueous phase - up to 100.
EFFECT: implementation of stereo, regioselective biotransformation of respective steroid substrates at their elevated initial concentration, obtaining the desired product with high yield, the ability to perform multiple, at least 30 cycles, use of the biocatalyst without loss of activity.
1 dwg, 1 tbl, 1 ex
SUBSTANCE: method provides for suspension of a biomass of cells till concentration is 15-30 wt % in the solution of polyvinyl alcohol with concentration of 6-9 wt %, which is prepared on a feed medium specific for growth of a certain phototrophic microorganism, freezing and storage of aliquots of the obtained suspension with the volume of 0.1-0.5 cm3 at -80÷-70°C.
EFFECT: method allows simplifying a cryopreservation process of cells of phototrophic microorganisms, increasing survival capacity of cells, providing this level of survival capacity at storage of cells during the period of up to a year and a half and using unfrozen specimens of cells as a concentrated seed grain.
SUBSTANCE: invention proposes a method for obtaining immobilised biocatalyst for synthesis of water solutions of amides, including acrylamide and nicotinamide from nitriles of carboxylic acids. Chitosan microgranules with diameter of 1-2 mm are obtained as a result of forcing-through of its 2-4% solution in 2% acetic acid by means of an extrusion machine to 1M of potassium hydroxide solution. Then, chitosan is activated with 0.01-0.5% solution of benzoquinone in the ratio of 1:1. Then, ferment preparation of nitrile hydrase is immobilised at 20 minutes of incubation at the temperature of 22-25°C.
EFFECT: immobilised nitrile hydrase at conversion of nitriles keeps its activity at least in fifty cycles of reactions.
5 dwg, 5 ex
FIELD: process engineering.
SUBSTANCE: invention relates to biotechnology and may be used for wasteless cleaning of water and solid surfaces from oil and oil product outflow. Proposed material comprises outer layer of polyester fibers and mid layer of polypropylene fibers comprising incorporated phosphorus-containing cationites and/or nitrogen-containing anionites, cell walls of hydrophytes and immobilised cells of oil destruction bacteria. Proposed biohybrid material features sorption capacity of, at least, 25 g/g of sorbent and degree of bacteria cell load of, at least, 150 mg/m2 and may be used may times: at least, 5 regeneration cycles with extraction of, at least, 90% of sorbate in first cycle.
EFFECT: wasteless cleaning of water and solid surfaces from oil and oil product outflow.
SUBSTANCE: what is presented is a composition for producing a polymer film for microorganism immobilisation in biosensor analysers. The composition consists of polyvinyl alcohol and N-vinylpyrrolidone copolymer. The initial ingredients are taken in the molar ratio of PVA-VP 239:(9.0-56.2).
EFFECT: composition provides high biosensor sensitivity in stable operation up to 40 days.
1 dwg, 1 tbl, 1 ex
SUBSTANCE: invention relates to a collagenic film and a method of making said film. The film contains at least one collagenic layer. The surface of the collagenic layer is formed by a plurality of domains with predominant orientation of collagenic fibres in each domain and continuous variation of orientation of fibres from one domain to another. There are pores on the domain boundary.
EFFECT: possibility of stable formation of structures similar to natural collagen.
22 cl, 21 dwg, 13 tbl
SUBSTANCE: bioprinter comprises the unit of cell reprogramming and the unit of output of the cells on the substrate, as well as the successively connected unit of loading and storing the colony of somatic cells, the unit of cell reprogramming with a device of inclusion of one or more reprogramming factors and a device of inclusion of methyltransferase inhibitor GSK126, mounted on it, having a reprogramming compartment, a unit of culturing the pluripotent stem cells with a device of insertion of the agent modifying the epigenetic status of the cell, the device of insertion of protein Bax, the device of insertion of protein Bak, the device of insertion of nitrogen monoxide, the device of insertion of proliferation inhibitor, and the device of insertion of apoptosis inhibitor, having a compartment of cell culturing, and a mechanism of changing the nutrient medium for the cells, a unit of cell differentiation with a device of insertion of one or more growth factors, mounted on it, having a compartment of cell differentiation, a unit of storage of differentiated cells, having a compartment of storage of differentiated cells, a unit of output of the cells on the substrate with a device of hydrogel insertion on the substrate, mounted on it, made with the ability to operate according to the principle of three-dimensional ink jet printer, and a control system.
EFFECT: invention provides the ability to print organs and tissues without signs of carcinogenesis, without the necessity in use of additional equipment.
3 cl, 2 dwg