18 results about "Large cell" patented technology

The invention relates to the technical field of neutrophile granulocyte separation, in particular to a mouse neutrophile granulocyte separation method which comprises the following steps: (1) anesthetizing and disinfecting a mouse; (2) tissue sampling; (3) tissue digestion; (4) preparing a cell suspension; (5) purifying cells; (6) cell culture; according to the method disclosed by the invention, isoflurane is adopted in the anesthesia disinfection step, so that central nervous activity of mice can be rapidly inhibited, tissue damage caused by stress reaction is avoided, and chemical stimulation can be reduced while body surface microorganisms are cleared through combination of ethanol disinfection and sterile normal saline flushing, and aortic tissues with intact activity are provided for subsequent material taking. In the compound enzyme digestive juice, collagenase I can specifically degrade aortic wall collagenous fibers, trypsin dissociates adhesion connection between cells, neutral protease assists in decomposing elastic fibers, and DNA enzyme I is supplemented to inhibit cell aggregation, so that the contradiction between incomplete digestion and large cell damage of the traditional enzyme juice is effectively solved.

The invention discloses double-mesoporous silica nanoparticles for modifying ionizable amino and a preparation method and application of the double-mesoporous silica nanoparticles. The core technical process of the double-mesoporous silica nanoparticles comprises the following steps: firstly, synthesizing mesoporous silica through a double-template method of a block copolymer and a surfactant; then grafting ionizable amino to the surface of the mesoporous silica through a silane coupling agent; and finally, RNA is loaded on the modified mesoporous silica by electrostatic adsorption, and efficient tumor treatment is finally realized. The invention also discloses an application of the silicon dioxide nanoparticles in RNA delivery. The silicon dioxide nanoparticles prepared according to the method disclosed by the invention have high RNA loading efficiency and cell uptake efficiency. The double-mesoporous silica nanoparticles modified with ionizable amino groups have the advantages of large drug loading capacity, high cell uptake rate, high biocompatibility, simplicity in synthesis, mild reaction conditions, convenience in cutting and the like, and have a good application prospect.

The invention discloses a stem cell-based microvesicle as well as a preparation method and application thereof, belongs to the technical field of biological tissue engineering, and particularly relates to a preparation method of the stem cell-based microvesicle, which comprises the following steps: culturing stem cells until the cell fusion degree is 80-90%, performing hypoxia treatment, and cleaning for 2-3 times; standing and culturing the cleaned stem cells, then adding a calcium ion carrier, and carrying out ultrasonic treatment; the serum-free basic culture medium containing the calcium ion carrier is immediately absorbed and removed, the cells are cleaned through PBS, culture is conducted for 24 h, and cell supernatant is collected and merged respectively; performing first centrifugation to remove cell debris; performing second centrifugation to remove dead cells and large cell debris; carrying out third centrifugation to remove organelles; fourth centrifugation is carried out, large vesicles are removed, and a final supernatant is obtained; and filtering and centrifuging to obtain a microvesicle precipitate. The preparation method of the microvesicles aims at systematically improving the yield and biological activity of the stem cell microvesicles through low-oxygen pre-stimulation, ultrasonic-calcium ion carrier synergistic induction and differential centrifugation.

According to the invention, a novel immune checkpoint molecule SIDT1 is found. The SIDT1 is a powerful T cell inhibition factor mainly expressed on CD8 < + > T cells. A series of SIDT1 inhibitors are identified in the invention. The SIDT1 inhibitor can enhance the anti-tumor CD8T cell reaction and limit tumor progression, so that a new thought is provided for treatment and prevention of related tumor patients with PD-1 treatment resistance.

The invention relates to methods for extracting, purifying and concentrating mesenchymal stromal cell exosomes enriched with micro-RNA, proteins and lipids, and can be used in cosmetology and pharmacology for rehabilitation and regeneration. The technical result of the invention is a more efficient process for producing exosomes. This technical result is achieved in that the present method for extracting, purifying and concentrating exosomes from mesenchymal stromal cells includes cultivating cells, collecting the conditioned culture medium, depleting high-molecular-weight proteins, extracting, purifying and concentrating exosomes, and freezing and lyophilizing the exosome concentrate; in order to enrich the culture medium with exosomes, multipotent mesenchymal stem cells (MSCs) are grown in a medium having a lowered glucose concentration and containing platelet lysate and 5 mM of L-alanyl-L-glutamine, wherein, in the case of static cell cultivation, the growth medium is placed in a culture vessel and once a level of not less than 80% monolayer confluence is reached, the culture medium is harvested and replaced once every 24 hours, and in the case of flow-through cell cultivation, cells are placed on cell carriers in a bioreactor and 72-96 hours after the start of cultivation, the culture medium is harvested and replaced once every 24 hours, the exosome-enriched culture medium is subjected to concentration, which includes successive stages of centrifugation, tangential flow filtration and isopycnic centrifugation: in the first stage, the culture medium is centrifuged for 10 mins at 2000 g to remove large cell particles and other inclusions; in the tangential flow filtration stage, the supernatant is concentrated using hollow fibre filters having a molecular weight cutoff of 500 kDa, after which the concentrate is subjected to fivefold diafiltration with an equal volume of DPBS phosphate buffer solution using the same hollow fibre cartridge; and in the isopycnic centrifugation stage, the concentrated supernatant is mixed with a saccharose solution and deuterium oxide and subjected to isopycnic centrifugation at 100000 g and 4˚С for 75 mins, after which the concentrate is subjected to further fivefold diafiltration with a buffer solution, then the concentrate is filtered through filters with a pore diameter of 0.22 µm and 0.1 µm, the exosome concentrate is subjected to initial freezing to -75˚C in a freezing chamber, and drying is carried out in a lyophilization chamber, wherein in a first drying stage, the concentrate is cooled to -40˚С and subsequently held for 100 mins at an absolute pressure in the chamber of not more than 10 Pa, then cooled to -30˚С and subsequently held for 610 mins at an absolute pressure in the chamber of not more than 10 Pa, then cooled to -10˚С and subsequently held for 315 mins at an absolute pressure in the chamber of not more than 10 Pa, after which it is cooled to 0˚С and subsequently held for 130 mins at an absolute pressure in the chamber of not more than 10 Pa, then heated to +10˚С and subsequently held for 130 mins at an absolute pressure in the chamber of not more than 10 Pa; in a second drying stage, drying is carried out at +22˚С for not less than 130 minutes at an absolute pressure in the chamber of not more than 10 Pa.

This disclosure provides medicaments and methods for treating envenomation using small molecule inhibitors of secretory phospholipase A2 (sPLA2). The sPLA2 inhibitor AZD2716 and related compounds can be used as monotherapy. Alternatively, the sPLA2 inhibitor varespladib and / or methyl varespladib can be used in combination with a statin. When a subject has been envenomed (for example, by a snake bite or bee sting), the medicaments can be administered to prevent lethal effects or tissues damage cause by the venom: for example, acute kidney injury, mast cell degranulation, or cerebral edema. The medicaments may be contained in an automatic injection device or an injection pen to deliver an effective dose as soon as possible following envenomation.

The present application relates to a molecular marker combination and detection reagent for identifying human blood mast cell precursor cells and application, and belongs to the technical field of cell detection. The molecular marker combination can realize efficient identification of human blood mast cell precursor cells, provide a new means for diagnosis of allergic diseases, and provide a new target for intervention of allergic diseases. The present application provides a molecular marker combination for identifying human blood mast cell precursor cells, wherein the molecular marker comprises a human cell surface molecule and a human intracellular molecule, the human cell surface molecule comprises CD34 and Fc epsilon R1 alpha, and the human intracellular molecule comprises CPA3, TPSAB1 and GATA2.

The present application belongs to the technical field of nucleic acid delivery and gene editing, and particularly relates to a method for knocking out a target gene of a cell in vitro by using an LNP delivery system. In view of the technical problems of unstable editing efficiency, large difference in editing efficiency between different individuals, low transfection efficiency, large cell damage, unstable knockout effect and the like when a T cell is transfected by using an existing LNP for gene editing, the present application provides a method for efficiently knocking out a target gene of a T cell in the form of LNP by optimizing the LNP and LNP transfection operation parameters in a systematic manner. The method can effectively solve the above technical bottlenecks, realize efficient and stable knockout of a target gene of a T cell, and provide reliable technical support for T cell function research and immunotherapy application.

According to the present invention, a pharmaceutical composition for preventing and treating lung adenocarcinoma, comprising K-ras-specific activated T cells, is used as an antigen composition designed such that a K-ras mutant (G12D, G12V, and G13D) recombinant overlapping peptide is sequentially divided into a total of 12 epitopes (n=1 to 12, and the last epitope (n=12) consists of 23 amino acids) in units of 30 amino acids in the amino acid sequence of K-ras, and 15 amino acid sequences overlap between the epitopes. Thus, the pharmaceutical composition is more effective in recognizing and killing lung adenocarcinoma in which K-ras, K-ras mutant G12V, K-ras mutant G12D, or K-ras mutant G13D is detected. Therefore, according to the present invention, the pharmaceutical composition for preventing and treating lung adenocarcinoma, comprising K-ras-specific activated T cells, can be used to effectively prevent and treat lung adenocarcinoma, particularly lung large cell adenocarcinoma, in which K-ras as well as K-ras mutants are detected.

Disclosed are a delivery system based on sensitized mast cells, a preparation method therefor, and an application thereof, as well as a pharmaceutical composition and a drug administration device involving the delivery system of sensitized mast cells. The delivery system utilizes the specific recognition of tumor antigens by sensitized mast cells to targetedly deliver drugs to tumor cells, while protecting the drugs from the clearance of the circulating immune system. Sensitized mast cells rapidly degranulate under the activation of tumor antigens to release drugs, thereby enhancing infection, exerting an oncolytic effect, lysing tumor cells and releasing antigens, and activating an anti-tumor immune response. The delivery system can be used as a promising platform technology for personalized therapy.

The invention belongs to the technical field of exosome application, and discloses application of a sarcodon aspratus exosome in preparation of a medicine for treating interstitial cystitis. The prepared sarcodon aspratus exosome is perfused into the bladder of a rat with interstitial cystitis, and it is found that the sarcodon aspratus exosome can significantly improve urination disorder of the model rat and relieve symptoms of interstitial cystitis. In addition, the invention also finds that (1) the sarcodon aspratus exosome can obviously reduce the expression of IL-1beta, IL-6, MCP-1, CXCL-1, ICAM-1 and BAX in the bladder tissue; (2) the sarcodon aspratus exosome can significantly reduce the activation number of mast cells and inhibit degranulation of the mast cells; and (3) the sarcodon aspratus exosome can significantly improve epithelial cell apoptosis. The research shows that the sarcodon aspratus exosome can be used for treating interstitial cystitis. The research provides a new idea for the treatment of interstitial cystitis.

A method for obtaining a genetically engineered leukocyte composition is provided. The method includes: (a) enriching a population of large cells from a biological sample obtained from a subject, wherein the biological sample comprises leukocytes without performing density gradient centrifugation; (b) contacting the population of large cells with an activating agent; and (c) transducing the population of large cells with a viral vector comprising a polynucleotide. The enriching comprises an array-based separation comprising a microfluidic device configured for deterministic lateral displacement comprising an array of obstacles, and the obstacles are elongated so that their length perpendicular to bulk fluid flow (P1) is longer than their width parallel to bulk fluid flow (P2) by at least 10%.

The application discloses a high-purity NKT cell preparation and a preparation method and a cryopreservation regulation method, and belongs to the fields of cell biology technology and immunotherapy. The method systematically solves multiple challenges of low purity, insufficient amplification efficiency, poor cell viability, high cost and difficulty in large-scale production of NKT cells in the prior art through a unique secondary cryopreservation process, innovative application of platelet lysate and specific induction of alpha-galactosylceramide (alpha-GalCer). The prepared NKT cell preparation has the advantages of high purity, large amplification fold, strong cell function activity and good safety, and provides a high-quality and standardized cell source for clinical applications such as tumor immunotherapy, and has great industrialization prospects.

Devices and methods for preparing adipose tissue for transplantation from leaflet fat extracted, for example by fat aspiration, the fat consisting of a fluid component comprising an oil component, a blood component and / or a disinfecting solution, and a solid component comprising an oil component, a blood component and / or a sterilizing solution, the invention relates to a device for washing and separating solid components comprising cell debris, cells and one or more large cell masses of different sizes, in which the device comprises at least one washing and separating container (1) having a washing chamber (101), said container (1) having an inlet (102) and an outlet (103), the invention relates to a washing chamber (101) for letting liposuction material into the washing chamber (101) through an inlet (102) and for letting at least a portion of said material out of said chamber (101) through said outlet (103), said washing chamber (101) comprising stirring means for forming an emulsion of a fluid component.

A drug delivery system based on sensitized mast cells, its preparation method, and its application are disclosed, along with a pharmaceutical composition and delivery device involving the sensitized mast cell delivery system. This delivery system utilizes the specific recognition of tumor antigens by sensitized mast cells to target and deliver drugs to tumor cells while protecting them from clearance by the circulating immune system. Upon activation by tumor antigens, sensitized mast cells rapidly degranulate to release the drug, thereby enhancing infection response, exerting an oncolytic effect, lysing tumor cells and releasing antigens, and activating an anti-tumor immune response. This delivery system can serve as a promising platform technology for personalized therapy.

The invention discloses a synthesis method and application of tumor microenvironment GSH / pH dual response type copper-based self-assembly nanoparticles, and belongs to the technical field of nano material preparation. The preparation method comprises the following steps: self-assembling copper ions, 3, 3 '-dithio-bis-propionyl hydrazide and a copper chaperonin inhibitor DCAC50, and carrying out surface modification by using F-127, so as to obtain the copper-based self-assembled nanoparticles. The material can respond to GSH / acidic pH of TME, released Cu < 2 + > is reduced into Cu under the action of high-concentration GSH, a large amount of active oxygen is generated through a Fenton-like reaction while copper death is triggered, and oxidative stress in cells is amplified; the released DCAC50 inhibitor can block Cu efflux mediated by copper chaperonin Atox1, and copper ion outflow is inhibited. Through double regulation and control of copper metabolism kinetics, tumor cell copper death is synergistically induced, oxidative stress is enhanced, and the efficient anti-tumor effect is achieved.

Inhibiting mast cell activation by binding sialic acid-binding immunoglobulin-like lectin-9 (Siglec-9) is described. Inhibiting mast cell activation by binding Siglec-9 can be used to treat mast-cell associated inflammatory disorders, such as allergic diseases, rheumatoid arthritis, and mastocytosis.