Method for dielectric microcontainer administration into a mammal cell using femto-picosecond laser pulses
SUBSTANCE: optical trap for microcontainer is created using a focused laser beam with a wavelength of 830 nm. The microcontainer is brought into contact with the cell membrane by exposing it to laser pulses trains with a wavelength of 780 nm, with subsequent cutting of cell membrane by focused laser pulses and controlled introduction of the microcontainer into the cell using an optical trap.
EFFECT: invention allows to deliver the microcontainer with genetic material to the given mammal cell coordinate with high precision.
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: invention relates to field of medicine, genetic engineering and biotechnology. Claimed is method of providing tumour-free tissue-substituting therapy on the basis of obtained from adult somatic cells induced pluripotent stem cells (iPSC), where the latter are genetically modified by means of artificial chromosome (AC), carrying bicistronic cassette with suicide-gene and gene of sensitivity to antibiotic under control of regulatory element, specific for pluripotent stem cells, with modified iPSC being selected in presence of respective antibiotic, absence of AC integration into iPSC genome is confirmed, are introduced into recipient organism directly, without preliminarily differentiation in vitro, after 1-14 days patients are given a 5-10-day course of therapy with inductor of toxicity of suicide-gene product.
EFFECT: invention makes it possible to reduce to zero risk of cancer transformation of transplanted cells and development of teratomas, increase clinical efficiency of recovery of cell mass of injured organ or tissue, reduce waiting time for potential recipients.
6 cl, 1 tbl, 1 dwg
SUBSTANCE: as the present invention the method for production of polyfunctional magnetic nanoparticles based on bacterial magnetosomes and the hybrid protein MGG is provided, which enables to obtain magnetosomes binding immunoglobulins of the IgG class on the fragment Fc. The result is achieved in that in the lipid membrane of bacterial magnetosomes by ultrasonic treatment the hybrid protein MGG is integrated, which amino acid sequence comprises the transmembrane domain and the binding area of immunoglobulins.
EFFECT: obtaining polyfunctional magnetic sorbent bearing on the surface of magnetic nanoparticles of ligand, that enable to connect to the particles the immunoglobulins of IgG class.
SUBSTANCE: carrier is proposed for targeted delivery of nucleic acids to cells expressing the receptor CXCR4, which consists of a sequence-ligand to the receptor CXCR4 with the amino acid sequence KPVSLSYRSPSRFFESH, the linker part of two molecules of ε-aminohexanoic acid linking the sequence-ligand to the sequence for compaction of nucleic acids, the sequence providing compaction of nucleic acids and the complex output from endosomes CHRRRRRRHC.
EFFECT: invention can be used for targeted delivery of genetic structures into cells with the receptor CXCR4 on the surfaces, such as malignant tumour and stem cells, in order to correct genetic defects, influence on processes of implementation of the genetic information and prevention of diseases.
3 cl, 7 dwg, 4 ex
SUBSTANCE: invention refers to biotechnology, more specifically to a method for introducing an siRNA molecule into the cytosol of a cell, and can be used in medicine. The method involves contacting said cell with an siRNA molecule, a carrier and a photosensiting agent, and irradiating the cell with light of a wavelength effective to activate the photosensitising agent. The carrier comprises a cationic polyamine such as a lipopolyamine in a non-liposomal formulation, branched polyethyleneimine (PEI), a betacyclodextrin amine polymer, a PAMAM dendrimer molecule, and a cationic peptide such as polyarginine or L- or D-arginine copolymers. The method is used to inhibit the target gene expression, to obtain a cell or a cell population containing the siRNA molecules, as well as to treat or to prevent a disease where the inhibition of one or more genes may be effective, including to treat a malignant growth.
EFFECT: invention enables the PCI-mediated site-specific siRNA delivery into the cytosol of a cell.
27 cl, 15 dwg, 1 tbl, 13 ex
SUBSTANCE: immunostimulatory complex is described, which includes an RNA in the form of a complex with one or more oligonucleotides. Besides, the oligonucleotide has properties of peptide penetrating into a cell (CPP), contains from 8 to 15 amino acid remains and is characterised by the following general formula: (Arg)1(Lys)m(His)n(Orn)o(Xaa)x. At the same time the main number of remains is selected from Arg, Lys, His, Orn.
EFFECT: invention may be used for transfection of a cell, tissue or organism or for modulation, preferably induction or increasing immune response.
16 cl, 22 dwg, 11 ex
SUBSTANCE: method includes processing of plant cells with intact cellular barriers by complex of carrier - carried material consisting of at least one carried polypeptide and/or polynucleotide component connected with the carrier - internalised positively charged peptide (IPCP), and designed for obtaining the transduced cells and propagation of plant cells for cultivation of plant. And these plant cells are somatic cells preliminary treated with agent permeabilising cells, or gametophytes.
EFFECT: invention enables to obtain effectively genetically engineered plants under conditions of intense cellular uptake of the complex of carrier-carried material.
13 cl, 11 dwg, 5 tbl, 12 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention relates to field of molecular biology and biotechnology and can be used in medicine and in pharmaceutical industry. RNA molecule, capable of target-specific RNA interference, represents double-stranded RNA molecule, 23 nucleotides long, which has 3'-overhang from 1-5 nucleotides. It is obtained by joining two RNA strands and used for obtaining pharmaceutical composition. When introduced into multicellular eukaryotic organism or in a cell of multicellular eukaryotic organism, said RNA molecule ensures promotion of target-specific RNA interference and leads to reduction of target-gene expression level or to target-gene knockout. Method of promoting target-specific RNA interference by means of said RNA molecule is applied for determination or modulation of gene function. Cell, containing endogenic target nucleic acid, RNA molecule, capable of target-specific RNA interference, and exogenic target nucleic acid, is used in analytical procedures.
EFFECT: application of invention ensures target-gene silencing, mediated by target-specific RNA interference.
40 cl, 23 dwg, 3 ex
SUBSTANCE: method provides contact of said cell with a peptide nucleic acid (PNA) molecule and a photosensitising agent and cell exposure to light at wave length effective to activate the photosensitising agent where said PNA molecule is conjugated with positive peptide. Also, there are described compositions containing such conjugated PNA molecules, the cells produced with the use of the method, and application of the method.
EFFECT: effective cell capture of peptide nucleic acid molecules conjugated with positive peptide.
37 cl, 13 dwg, 9 ex
SUBSTANCE: invention refers to biotechnology and genetic engineering, namely to a genetically engineered construct pGoatcasGCSF for human granulocyte colony-stimulating factor (GCSF) expression . The offered invention can be used for producing transgenic animals that are human granulocyte colony-stimulating factor producers. It involves creating the genetically engineered construct pGoatcasGCSF of the size 6386 bps, and a specified nucleotide sequence shown in SEQ ID 1. The genetically engineered construct pGoatcasGCSF includes a 5'-regulatory sequence of the goat CSN1S1 asi-casein gene of the size 3387 bps connected with the full-size human GCSF gene of the size 1485 bps, and a 3'-flanking region of the cow gene CSN1S1 of the size 1514 bps.
EFFECT: stable effective level of human GCSF expression in milk of the transgenic animals, eliminated possibility of ectopic transgenic expression.
9 dwg, 2 tbl, 6 ex
SUBSTANCE: invention relates to carbosilane dendrimers and synthesis method and use thereof. Disclosed are novel dendrimers, the ends of branches of which include primary, secondary, tertiary and quaternary amino groups, synthesis method thereof, composition based on said dendrimers and versions of using said dendrimers. The disclosed dendrimers may be used to transport anionic molecules in blood, such as nucleic acid molecules, including ODN and RNAi molecules and other anionic medicinal agents with which they can interact, thus protecting them from interaction with proteins in the plasma and/or enhancing their degree of penetration into target cells. The dendrimers can be used to bind anionic molecules with surfaces, and can also be administered as active components for preventing or treating diseases caused by viruses such as HIV or hepatitis C, or prions whose life cycle can be disrupted by dendrimers.
EFFECT: obtaining novel carbosilane dendrimers, having a wide range of application in medicine and which are processable and biocompatible.
172 cl, 35 dwg, 19 tbl, 57 ex
SUBSTANCE: invention relates to biotechnology. The method of obtaining a bifidogenic factor includes the separation of deoxyribonucleic acid from a biomass of bifidobacteria by triple processing with ultrasound with a frequency of 40 kHz for 30 minutes with the further chromatography of combined supernatants on sepharose Sepharose CL-4B. The spectrophotometric characteristic of the purity of separated DNA constitutes 1.88.
EFFECT: method provides obtaining a highly pure DNA sample from the biomass of bifidobacteria.
2 dwg, 2 tbl, 2 ex