16 results about "Autologous plasma" patented technology

The invention discloses an NK cell culture solution and an NK cell culture. The culture solution comprises a cell activation culture solution and a cell amplification culture solution, the cell activation culture solution comprises a basic culture medium, and the basic culture medium contains autologous plasma with the final concentration of 1-10%, IL-2 with the final concentration of 50-1000U / mL, IL-15 with the final concentration of 1-15ng / mL, OK432 with the final concentration of 0.1-1ug / mL and ginseng exosome with the final concentration of 1-10ug / mL; the cell amplification culture solution comprises a basic culture medium, and the basic culture medium contains IL-2 with the final concentration of 100-2000U / mL. The ginseng exosome component in the cell culture fluid can promote NK cell proliferation and remarkably improve the immune killing function of the NK cells, and the ginseng exosome serving as an activating factor can effectively improve the activity of the NK cells and enhance the immune effect of the NK cells.

A method for large-scale expansion of natural killer (NK) cells in a cell culture medium according to the invention, comprising the steps of: adding said cells to a culture system comprising a growth medium supplemented with serum selected from the group consisting of human serum and autologous plasma; cRX 527 with an effective concentration is added; the cells are expanded and heated within the system wherein the effective concentration of CRX 527 in the growth medium is in the range of 0.01 [mu] g / ml to 10 [mu] g / ml, preferably 100 ng / ml, and the ratio between the cell density and the total volume of the growth medium is in the range of 1.5 to 2.

The invention provides an NK cell in-vitro efficient culture system and application, the culture system comprises a K562 engineering cell, and six proteins of IL21, IL12, IL15, CD40L, CD155 and CD112 are expressed on a cell membrane of the K562 engineering cell in a transmembrane mode; the NK cell culture composition comprises the following components: a liquid serum-free cell culture medium, 1-5% (v / v) of autologous plasma, 100-250 [mu] g / ml of an astragalus extract and 200-500 [mu] g / ml of an angelica sinensis extract, and the dosage ratio of the astragalus extract to the angelica sinensis extract is 1: 2. The NK cell with high amplification multiple, high purity and high lethality can be obtained by a culture system established by taking the K562 engineering cell constructed by the invention as a trophoblast cell, taking an astragalus extract and an angelica sinensis extract as culture additives and taking PBMC as an original culture material, and the culture system has important significance for treating cancers by cell drugs.

The invention belongs to the field of biological medicine, and particularly discloses a culture method of domesticated mesenchymal stem cells (MSCs), which comprises the following steps: separating plasma and platelet lysis buffer from autologous peripheral blood, and obtaining CTL and Treg through magnetic bead sorting; the method comprises the following steps: sequentially co-culturing umbilical cord MSCs for three generations: co-culturing the P1 generation and CTL in a culture medium containing 30% of alpha MEM, 68% of DMEM / F12, 1% of autologous plasma and 1% of platelet lysis buffer, and adding 0.1-1 [mu] M of resveratrol and 10-50 [mu] M of spermidine; co-culturing P2 and Treg in a culture medium containing 50% of alpha MEM, 48% of DMEM / F12, 1% of autologous plasma and 1% of platelet lysis buffer; the P3 generation is cultured and harvested in 90% of alpha MEM, 5% of DMEM / F12 and 5% of PRP. The obtained MSCs surface marker is CD105 < + > / CD90 < + > / CD73 < + > / CD45 <-> / CD34 <-> / HLA-DR <->, the expression quantity of CD24 is low (CD24low), the expression quantity of CD47 is high (CD47high), the expression quantity of PD-L1 is high (PD-L1 high), the IDO activity is larger than or equal to 25 U / mg, the retention rate of the lung after vein transplantation is smaller than or equal to 12%, and the survival period is larger than or equal to 28 days. The cell is suitable for treating ARDS or hepatic fibrosis.

The present disclosure relates to a composition including non-autologous plasma or serum and a polymer; method of treating ophthalmic diseases (e.g., dry eye syndrome) or moisturizing and / or repairing non-keratinized surfaces including non-healing wounds with a composition including non-autologous plasma or serum and a polymer; and a therapeutic agent delivery device for delivery of the composition to the eye or a non-keratinized surface of a subject.

This invention relates to the fields of biomedical engineering and regenerative medicine, and particularly to a plasma matrix composite membrane activated by autologous keratinized epithelial tissue fragments, its preparation method, and its applications. The composite membrane is obtained by mixing autologous keratinized epithelial tissue fragments with a liquid autologous plasma matrix in a specific ratio, utilizing tissue factors on the surface of the tissue fragments to trigger platelet activation, spontaneously forming a gel, and then pressing it into a membrane. This invention is the first to utilize the biointerface of autologous keratinized epithelial tissue fragments to activate platelets, eliminating the need for exogenous thrombin and achieving completely autologous in-situ gelation. The composite membrane formed after pressing possesses excellent mechanical properties and barrier function, and can be prepared in a single operation in the operating room without the need for in vitro cell culture. The composite membrane of this invention is used for oral soft tissue augmentation, such as widening of keratinized gingiva and alveolar ridge soft tissue augmentation, and has advantages such as no donor site damage, high biosafety, and ease of operation.

The invention relates to a method for efficiently culturing and amplifying NK (Natural Killer) cells in vitro. The method comprises the following steps: extracting peripheral blood and separating upper-layer plasma and lower-layer blood; inactivating the upper-layer plasma, and centrifuging to prepare inactivated autologous plasma; performing density gradient centrifugation on the lower-layer blood to obtain mononuclear cells, washing the mononuclear cells, and freezing the mononuclear cells in liquid nitrogen; the culture bottle is coated by using a CD16 antibody, a CD244 antibody, a CD226 antibody and an NKG2D antibody; the method comprises the following steps: inoculating mononuclear cells recovered by human serum albumin and MG53 protein into a coated culture bottle, culturing under the conditions of 37 DEG C and 5% CO2 by adopting a serum-free culture medium containing inactivated autoplasma, IL-2, IL-12, IL-18 and MG53 protein, supplementing liquid on the third, fifth, seventh, ninth and eleventh days of culturing to adjust the cell density, and collecting the cells cultured to the fourteenth day to obtain NK cells. The method is easy to operate, high in safety, good in amplification effect and high in clinical application value.

The invention discloses an NK cell culture kit and application thereof. The kit contains a first cell induction culture solution, a second cell induction culture solution, a cell amplification culture solution, a first coating solution and a second coating solution, a basic culture medium is used as a basic solution, and the culture medium comprises IL-15, ALKS4230, N803, IL-27 and autologous plasma; the second cell induction culture solution takes the first cell induction culture solution as a basic solution and contains OK432; the cell amplification culture solution takes a basic culture medium as a basic solution and contains IL-2 and ascorbic acid; the first coating liquid comprises heparin sodium, a CD16 monoclonal antibody, an NKp46 monoclonal antibody and PBS (Phosphate Buffer Solution); the second coating solution comprises alendronumab, silizumab, fibronectin and PBS (Phosphate Buffer Solution). When the kit is used for culturing the NK cells, the combination of alendronumab and silizumab is beneficial to promoting selective amplification of the NK cells in PBMC (peripheral blood mononuclear cells), ALKS4230 can activate growth of the NK cells, and N803 can drive diffusion, proliferation and growth of the NK cells.

The invention belongs to the technical field of cell culture, and provides a preparation method of SNK cells, which comprises the following steps: step 1, centrifuging a blood sample, separating plasma and inactivating to obtain inactivated autologous plasma; then carrying out density gradient centrifugation to obtain blood sample mononuclear cells; washing and resuspending the mononuclear cells of the obtained blood sample; step 2, on the 0th day, re-suspending the mononuclear cells by using the No.1 liquid, inoculating in a culture bottle, adding inactivated autologous plasma, and standing and culturing; on the second day, supplementing the No.1 liquid and inactivated autologous plasma into each culture bottle; on the third day, adding No.3 liquid and inactivated autologous plasma into each culture bottle; on the fifth day, collecting the cells, counting, centrifuging, resuspending with a No.4 solution, adding inactivated autologous plasma, and continuing culturing; from the sixth day to the eleventh day, supplementing the liquid 4, and maintaining the cell density; on the 12th day to the 15th day, calculating the total number of cells and the variation trend of the motility rate, and finishing culture when the number of the cells reaches the predicted maximum quantity.

Provided are a tolerogenic DC cells culture medium and the use thereof in tolerogenic DC cells culturing. The culture medium comprises: a basic culture medium, a cytokine, berberine, and autologous plasma, wherein the concentration of berberine is 0.25-5 μM, and the cytokine comprises 100-1000 U / mL GM-CSF, 10-50 ng / mL IL -4, and 100-1000 U / mL TGF-β. The expression levels of costimulatory molecules CD80, CD83, CD86 and HLA-DR of DC cells cultured by means of the culture medium are significantly reduced.

The invention discloses a method for amplifying Treg cells and a Treg cell culture medium. The method for amplifying the Treg cells comprises the following steps: extracting PBMC (peripheral blood mononuclear cells) from autologous plasma; carrying out magnetic bead sorting on CD4 + T cells in the PBMC; the cell suspension is resuspended in a Treg cell culture medium, culture is conducted, cells are collected after 14 days, and a Treg amplification culture medium comprises 5% thermally inactivated autologous plasma, glutamine, HEPES, beta-ME, Rapamycin, Decitabine, IL-2 and alphaCD3 / CD28. According to the invention, CD4 + T cells can be efficiently induced to generate Treg cells, and the obtained autologous Treg cells can be applied to various autoimmune diseases and other clinical demand conditions for inducing peripheral immune tolerance.

The invention discloses a peripheral blood mononuclear cell cryopreservation protective agent and a cryopreservation method.The cryopreservation protective agent is prepared from, by volume, 5% of dimethyl sulfoxide (DMSO), 5% of dextran, 45% of autologous plasma and 45% of a serum-free culture medium. The method comprises the following steps: centrifuging a negative peripheral blood sample for infectious disease detection, counting mononuclear cells, preparing cryopreserved cell sap, carrying out programmed cooling, and transferring to low-temperature storage. The cryopreservation liquid has the advantages that the cryopreservation liquid can protect cells, and the effectiveness and the stability of the motility rate of the cryopreserved cells after recovery each time are ensured. The cell viability after recovery is high and can reach 95% or above, no heterologous animal component exists, and high-quality cryopreserved peripheral blood mononuclear cells are provided for recovery culture of immune cells later.

The invention discloses a cell culture method for in-vitro efficient induction culture of natural killer cells, and relates to the technical field of cell culture. The cell culture method comprises the following steps: (a) coating a culture vessel with a CD16 antibody; (b) separating PBMC (peripheral blood mononuclear cells) and autologous plasma from peripheral blood; (c) inoculating PBMC (peripheral blood mononuclear cells) into the coated culture vessel, and adding a culture medium for induced differentiation; the culture medium is prepared from a basic culture medium 581, 5%-20% of autologous inactivated plasma, 40 ng / mL to 60 ng / mL of IL-15 and 800 IU / mL to 1200 IU / mL of IL-2. Wherein 0.1-0.4 mL of glycyrrhizic acid monoammonium cysteine sodium chloride is added into every 10 mL of the culture medium, and 0.3-0.7 mL of 1 * 10 < 4 > pegylation recombinant human granulocyte stimulating factors are added into every 10 mL of the culture medium for initial culture; (d) supplementing liquid for the culture medium every 2-3 days in the whole culture process, wherein the volume ratio of liquid supplementation every time is 1: (0.9-1.1); (e) carrying out microorganism culture detection after the culture dosage is reached; and (f) harvesting the cells after the microbiological detection is qualified. According to the method, the motility rate and purity of the NK cells can be improved.

The invention discloses an efficient cord blood NK cell multiplication culture scheme separated from autologous plasma and application of the efficient cord blood NK cell multiplication culture scheme, by optimizing culture system design and regulating and controlling technological parameters, after 19 days of multiplication culture, the NK cell multiplication multiple can reach 2000 times or above, the cell purity is improved to 95% or above, and the tumor cell killing efficiency reaches 80% or above; the scheme successfully overcomes the core pain points of slow amplification rate, insufficient cell purity, low killing activity and the like caused by dependence of cord blood NK cells on autologous plasma in the prior art, avoids the problem of unstable culture effect of autologous plasma caused by dilution of an anticoagulant, and remarkably improves the preparation efficiency and application reliability of cord blood NK cells. And the method has extremely high clinical transformation value and wide industrial application prospect.

The present disclosure relates to a composition including non-autologous plasma or serum and a polymer; method of treating ophthalmic diseases (e.g., dry eye syndrome) or moisturizing and / or repairing non-keratinized surfaces including non-healing wounds with a composition including non-autologous plasma or serum and a polymer; and a therapeutic agent delivery device for delivery of the composition to the eye or a non-keratinized surface of a subject.