682results about "Cell dissociation methods" patented technology

The invention discloses a preparation method and application of a sea cucumber exosome with an immune cell repairing function, and belongs to the technical field of biomedicine. The method comprises the following steps: feeding sea cucumbers by using a combined method of composite stress and an echinacea extract, extracting sea cucumber cells, and obtaining sea cucumber exosomes through ultracentrifugation; wherein the condition of composite stress is set as follows: the day and night temperature difference is not lower than 3 DEG C, and meanwhile, the dissolved oxygen content of the water body is adjusted to 75-90% of the dissolved oxygen content of the conventional feeding water body. The obtained sea cucumber exosome has a remarkable immune cell repairing function, and can effectively promote proliferation and activity recovery of immune cells, inhibit expression of inflammatory factors and improve the level of anti-inflammatory factors. The sea cucumber exosome prepared by the preparation method disclosed by the invention has an important application prospect in the research and development of immune repair drugs, and can provide a technical support for a novel immune regulation preparation in the field of biomedicine.

The invention discloses a cord blood-derived regulatory T cell amplification culture medium and a use method thereof, and relates to the field of animal cell culture and immune cell therapy, the culture medium is a serum-free culture medium, the culture medium is composed of a basic culture medium, a serum substitute combination, a combination of four cell factors, an immunosuppressor, an apparent stabilizer and an antioxidant, and all the components play a role synergistically; according to the use method, cell amplification is realized through a collaborative process of staged environmental regulation and targeted activation. According to the method, the problems of exogenous pollution and batch difference caused by dependence of a serum-containing system on cord blood-derived regulatory T cell amplification in the prior art are solved, the defect that a serum-free scheme is difficult to consider the amplification efficiency and the function stability at the same time is overcome, the cell amplification quality and the clinical application safety are guaranteed, and the method is suitable for large-scale clinical transformation requirements.

Provided are a fusion protein (for example, in the form of a bispecific antibody) binding to CD235a and CD3, a preparation method therefor, and a related use thereof.

The invention discloses a preparation method of fresh taxus chinensis stem and leaf protoplasts. The preparation method comprises a preparation process and a purification process. The preparation method of the taxus chinensis stem and leaf protoplast is simple and easy to operate, raw materials are easy to obtain and low in price, reagent components have good biocompatibility, and the taxus chinensis stem and leaf protoplast is free of harmful components, safe and environmentally friendly. The invention provides a simple and rapid taxus chinensis stem and leaf protoplast enzymolysis preparation method, which adopts vacuum filtration, accelerates the permeation of enzymatic hydrolysate and improves the enzymolysis efficiency, so that the protoplast can be rapidly obtained. Meanwhile, high-purity separation of the protoplast is carried out in combination with density gradient sedimentation of the iodixanol solution, so that the protoplast with uniform size and complete form is obtained, and convenience is provided for scientific researches such as subsequent conversion and single cell transcriptome sequencing.

Provided are a method and system for enriching natural killer (NK) cells in a blood sample.

The invention relates to injectable temperature-sensitive hydrogel loaded with traditional Chinese medicine active exosomes, and belongs to the technical field of clinical treatment of diabetic foot refractory wounds, and the injectable temperature-sensitive hydrogel is obtained by poloxamer 407 hydrogel and a hyaluronic acid loaded composite carrier; the composite carrier body is formed by coating a traditional Chinese medicine active exosome with a coating solution; the traditional Chinese medicine active exosome is obtained by inducing adipose-derived mesenchymal stem cells with a modified astragalus extract. The injectable temperature-sensitive hydrogel is obtained by loading a composite carrier with poloxamer 407 hydrogel and hyaluronic acid, the poloxamer 407 and the hyaluronic acid quickly form a physical barrier on a wound surface after being injected, a moist healing environment is provided, and continuous and slow release of exosome and active ingredients is realized by means of the temperature-sensitive characteristic and the network structure of the poloxamer 407 and the hyaluronic acid, so that the wound healing effect is improved. And over-high local concentration or premature loss caused by sudden release of the medicine is avoided.

The invention provides a marker of spinal cord functional neural precursor cells, a detection method and a sorting method, and belongs to the technical field of biological medicine. The invention provides application of a marker detection reagent in preparation of a kit for detecting spinal cord functional neural precursor cells. The invention further provides a sorting-based method for separating the SFNPCs cells, and the cells are sorted through the Marker of the specific SFNPCs, so that the cell purity of the SFNPCs is remarkably improved, the difference between batches is reduced, and a high-purity and homogeneous SFNPCs cell population is obtained. The survival rate of the SFNPCs cells separated and subcultured to the P9 generation by the method disclosed by the invention is more than 85%, and the SFNPCs cells have a good treatment effect on a spinal cord injury model.

The invention belongs to the field of biotechnology and aquaculture, and particularly relates to a method for improving hypoxia resistance of pelteobagrus vachelli based on hypoxia induction of liver extracellular vesicles of the pelteobagrus vachelli. The method comprises the following steps: extracting high-purity extracellular vesicles EVs in liver tissues of pelteobagrus vachelli subjected to low-oxygen treatment by adopting a method of combining differential centrifugation and high-precision iodixanol density gradient centrifugation; diluting the extracted EVs to 2mu g / mu L by using PBS (Phosphate Buffer Solution) to prepare an EVs suspension; an intraperitoneal injection mode is adopted, the EVs suspension is injected into the body of the pelteobagrus vachelli, and after injection is conducted for 24 hours, the hypoxia resistance of the pelteobagrus vachelli is evaluated through a suffocation point detection test. The invention opens up a brand new path for improving the hypoxia resistance of the pelteobagrus vachelli, provides a new idea and a new method for efficiently enhancing the hypoxia resistance of economic fishes in the field of aquaculture, is expected to play an important role in actual aquaculture production, and assists the sustainable development of the aquaculture industry.

The invention relates to the technical field of rhinitis spray preparation, in particular to a method for preparing allergic rhinitis spray from umbilical cord mesenchymal stem cell exosomes, which comprises the following steps: 1, umbilical cord treatment; 2, culturing umbilical cord mesenchymal stem cells; 3, preparing an umbilical cord mesenchymal stem cell exosome; and 4, preparing the rhinitis spray. The rhinitis spray prepared by the method disclosed by the invention is simpler and more convenient to use. The exosome, the platelet growth factor and other substances in the rhinitis spray well solve the core problems of single curative effect, large side effect, low compliance and the like in the traditional rhinitis treatment, and through the induction and repair functions of the stem cell exosome and the bactericidal ability of the antibacterial peptide, the nasal cavity environment is improved, the anaphylactic reaction is reduced, the nasal mucosa is repaired, and the rhinitis treatment effect is improved. The allergic rhinitis is expected to be fundamentally treated.

The invention discloses application of tea extracellular vesicles in preparation of a product for preventing and / or treating UVB-induced skin photoaging, and belongs to the technical field of application of the tea extracellular vesicles. The tea extracellular vesicles are derived from tea leaves and / or stem leaves and are prepared by a method of combining enzymolysis, multi-stage filtration and ultracentrifugation, and the particle size of the tea extracellular vesicles is mainly distributed in a range of 60-100nm. Experiments show that the tea extracellular vesicles can effectively reduce active oxygen accumulation and lipid peroxidation caused by UVB radiation, repair mitochondrial membrane potential and inhibit expression of matrix metalloproteinase and inflammatory factors, so that photoaging symptoms such as skin wrinkles, erythema and barrier function damage are relieved. The tea extracellular vesicle provided by the invention has the advantages of natural raw materials, good biocompatibility, small side effect and the like, can be used for preparing a plurality of external dosage forms such as gel, cream, spray and the like, and provides a safe and effective new natural product choice for prevention and treatment of skin photoaging.

The invention discloses an application of exosome nano-vesicles derived from artemisia annua cells and peroxidase A0A2U1N9S9 contained in the exosome nano-vesicles in preparation of anti-colorectal cancer drugs. According to the present invention, the anti-CRC activity analysis results show that the artemisia annua cell-derived exosome nano-vesicles can inhibit the growth of DLD-1 and HCT116 cell strains, and the active substance base of ACDENVs for inhibiting the growth of DLD-1 and HCT116 cell strains, namely peroxidase A0A2U1N9S9, is innovatively revealed for the first time based on the difference analysis of the proteomics technology; and a new research thought and a theoretical basis are provided for the research of anti-colorectal cancer drugs.

The invention discloses a sleep-promoting nasal spray based on mesenchymal stem cell-derived exosomes and preparation and application thereof.The preparation method comprises the steps that 1, mesenchymal stem cells separated from umbilical cord tissue are cultured to P3-P5 generations, cell culture supernate is collected, cells are digested, and then cell debris and macromolecular impure protein are removed through pretreatment; (2) filtering and concentrating the pretreated cell culture supernatant and replacing a part of solution; (3) carrying out degerming filtration on the solution obtained in the step (2) through a 0.22 [mu] m filter membrane to obtain an MSC-Exos solution; and (4) adding a stabilizer with a proper concentration into the MSC-Exos solution to obtain a compound MSC-Exos solution, namely the sleep-promoting nasal spray. The product is a cell-derived natural active substance, can significantly improve sleep, and does not involve any pharmaceutical ingredient.

The invention discloses a method suitable for extracting extracellular small vesicles in fresh water pearl shell tissue waste liquid, which comprises the following operation steps: (1) solid-liquid separation: slaughtering pearl shells, and filling slaughtering waste liquid into a waste liquid crude extraction device; (2) waste liquid crude extraction: performing multi-stage gradient filtration and gravity settling on the slaughtering waste liquid by using a waste liquid crude extraction device to obtain clarified waste liquid; (3) removing impurities: removing impurities for the first time and removing impurities for the second time; (4) concentration of the extracellular vesicles: concentrating the extracellular vesicles by using an ultra-speed centrifuge to obtain a high-concentration extracellular vesicle solution; and (5) preservation of extracellular vesicles: according to the final volume of the high-concentration extracellular vesicle solution obtained in the step (4), carrying out sample standardization through protein concentration, sub-packaging the sample, and carrying out long-term preservation in a low-temperature environment. The particle sizes of the extracted vesicles are concentrated, high-purity and complete extracellular small vesicles can be obtained, and the method is suitable for biomedical research and product development.

The invention discloses application of a 3D hypoxia mesenchymal stem cell derived exosome preparation in preparation of an immune cell proliferation inhibitor or a medicine for treating acute myocardial infarction, belongs to the technical field of biology, and solves the problems of low cell survival rate and immunological rejection in application of a cell treatment method in the prior art. The 3D hypoxia mesenchymal stem cell derived exosome is used as a medium for treating acute myocardial infarction, and the problem of immunological rejection can be solved.

The invention belongs to the technical field of biological medicine, and particularly relates to an extracellular vesicle drug delivery system, a preparation method thereof and application of the extracellular vesicle drug delivery system in treatment of Alzheimer's disease. According to the invention, one or more of stilbene glucoside, emodin, kaempferol, resveratrol and quercetin are used for regulating and controlling the expression of miRNA (micro Ribonucleic Acid) related to the disease progress in the extracellular vesicles, and the result shows that one or more of miR-let-7c-5p, miR-486-5p, miR-132-3p, miR-160b and miR-129-5p is remarkably up-regulated, and one or more of miR-342-3p, miR-16-5p and miR-125b-5p is remarkably down-regulated. The regulated extracellular vesicles can be used for treating Alzheimer's disease, relieving Tau protein abnormal phosphorylation, reducing amyloid protein beta deposition, inhibiting microglial cell activation, protecting neuronal cells and relieving memory deficits.

To provide a method for separating and purifying extracellular vesicles.SOLUTION: Wherein the hollow fiber membrane has an inner diameter of 0.2 mm to 1.4 mm and a molecular weight cut-off of 100,000 to 1,000,000, and the filtration method comprises a first filtration step of pressurizing and filtering a culture supernatant of mesenchymal stem cells through a first opening on one end side of the hollow fiber membrane to separate the culture supernatant into a permeated liquid and a first concentrated liquid, and a second filtration step of pressurizing and filtering the first concentrated liquid through a second opening on the other end side of the hollow fiber membrane; A method for separating and purifying extracellular vesicles, the method comprising a second filtration step of separating the extracellular vesicles into a permeate and a second concentrate, wherein a concentrate having an increased concentration of the extracellular vesicles is obtained by alternate tangential flow filtration in which the first filtration step and the second filtration step are alternately performed a plurality of times, and a membrane surface speed in the first filtration step and the second filtration step is 0.3 to 2m / sec.SELECTED DRAWING: None

The embodiment of the invention discloses a method for constructing a cartilage micro-tissue by compounding an extracellular matrix with a vascular matrix component and application. The method comprises the following steps: preparing a porcine decellularized cartilage extracellular matrix; generating a cartilage extracellular matrix suspension; carrying out screening treatment on the cartilage extracellular matrix suspension; preparing a porcine acellular cartilage extracellular matrix microcarrier; preparing a target suspension of a common system from the porcine acellular cartilage extracellular matrix microcarrier and vascular matrix component cells, and transferring the target suspension to a bionic floating microenvironment of a shaker bioreactor for dynamic culture; carrying out culture treatment on the porcine acellular cartilage extracellular matrix microcarrier; and taking out the cell-loaded porcine decellularized cartilage extracellular matrix microcarrier from the cultured and treated porcine decellularized cartilage extracellular matrix microcarrier as the constructed cartilage micro-tissue. According to the embodiment, the condition of secondary injury to a user can be improved and reduced, the repairing effect is improved, and the adaptability of the constructed cartilage micro-tissue is improved.

The invention discloses a PDRN-loaded umbilical cord mesenchymal stem cell source nano-vesicle and a preparation method thereof, and belongs to the technical field of biological medicines. The preparation method provided by the invention comprises the following steps: mixing an umbilical cord mesenchymal stem cell suspension with a PDRN solution, and carrying out freeze thawing treatment; incubating the mixed solution subjected to freeze thawing treatment; extruding the incubated mixed solution for multiple times by using a filter membrane; and collecting the extruded product, and separating and purifying to obtain the PDRN-loaded umbilical cord mesenchymal stem cell source nano-vesicle. According to the preparation method disclosed by the invention, the encapsulation efficiency of PDRN is remarkably improved, meanwhile, secondary damage of a traditional post-loading technology to a preformed vesicle structure is avoided, the membrane integrity and biological activity of a final product are better maintained, the yield is high, and the preparation process is rapid; the introduction of exogenous impurities is avoided from the source, and the high purity and biological safety of the obtained nano-vesicle component are ensured.

The invention relates to the technical field of aquatic animal cell culture, in particular to a rapid enrichment and separation method for gonad somatic cells of plectropomus leopardus. Comprising the following steps: selecting healthy plectropomus leopardus for anesthesia; the method comprises the following steps: taking gonad tissues, performing sterile treatment, cutting, centrifugal washing and filtering, inoculating the gonad tissues into a culture medium for primary culture to obtain gonad cells emigrated from tissue blocks, performing trypsin-EDTA digestion and centrifugal washing, and treating the gonad cells with 1 * PBS containing polysorbate; after closing, antibody incubation and washing, the marking effect is verified through a fluorescence microscope, and specific enrichment of gonad somatic cells is achieved; and filtering and purifying the enriched fluorescence-labeled cells, placing the cells under a fluorescence microscope, accurately picking fluorescence-positive cells under a fluorescence visual field, transferring the fluorescence-positive cells into a culture dish containing a complete medium, inoculating the separated fluorescence-positive cells into the optimized complete medium, and carrying out purification culture, so that the cells grow in a wall-attached manner and stable passage is realized.

The invention relates to the technical field of bioengineering, in particular to a preparation method of a probiotic and exosome preparation applied to immunoregulation. The probiotics and the umbilical cord stem cell exosome are combined for use, the immune regulation effect is enhanced from multiple targets and multiple ways, the immune function of the body is improved, the exosome carries a specific molecular marker and acts on immune cells in a targeted mode, precise immune regulation is achieved, and the preparation is in an oral form and convenient to use; the exosome is extracted from umbilical cord stem cells, has no pathogenicity and immunogenicity, and is low in adverse reaction risk after being used for a long time, and the prepared combined preparation can be used for treating and preventing various immune-related diseases, and also can be used for daily conditioning of people with low immunity and improving the body resistance; meanwhile, the combined preparation adopts micro-capsule embedding, so that the probiotics can be effectively protected from gastric acid and bile erosion, the survival rate and colonization efficiency of the probiotics in intestinal tracts are improved, the immunoregulation effect is further enhanced, and the combined preparation is convenient to store and transport and has a good application prospect.

The application discloses a kind of neutrophil membrane fusion liposome biomimetic nanomedicine carrier and its preparation method and application, belong to medical field.The carrier includes liposome and the neutrophil membrane fused to liposome;Liposome raw materials include DOPE, DPPC and cholesterol, and mass ratio is 6:3:1 in turn;Liposome and membrane protein mass ratio is 1:1.The application establishes quantitative analysis strategy based on NanoFCM, can accurately evaluate the fusion efficiency and fusion uniformity of NM@lipos at single particle level.Based on DOPE, the hybridization efficiency of NM@lipos (D) reaches 92.0%, which is significantly higher than that of NM@lipos (L) based on lecithin (70.1%).In in vivo experiment, the tumor enrichment capacity of NM@lipos (D) is increased by 5.1 times compared with Lipos (D) in 24 hours, and shows significantly prolonged circulation time.

Methods, systems, and compositions are provided for extracting bone marrow cells from bone obtained from deceased donors, for preparing the bone marrow for cryopreservation, and for obtaining desired cells from cryopreserved and fresh bone marrow

The invention relates to the technical field of bioengineering, in particular to an exosome separation and concentration method with marine animal source tissue as a raw material. Comprising the following steps: freeze-drying and crushing a marine animal source tissue sample; performing sample enzymolysis; and removing impurities and concentrating. According to the method disclosed by the invention, the exosome in the cells is fully dissociated through freeze-dried powder crushing and composite enzymolysis processes, and the filtering speed of the sample is accelerated by virtue of ultrasonic oscillation, so that the rapid and high-purity separation and concentration of the exosome in the marine animal source tissue are realized.

The invention discloses a method for extracting and preparing angelica keiskei exosomes. The method comprises the following steps: S1, selecting an angelica keiskei sample and selecting chitosan; s2, crushing and filtering the angelica keiskei sample to obtain an extracting solution; s3, dissolving chitosan through acetic acid, forming a film through a rotary coater, and carrying out drying and heat treatment; s4, mixing the extracting solution with the dried chitosan membrane, and adsorbing the exosome through the charge effect; s5, putting the chitosan membrane into a tangential flow filtration system, collecting effluent after tangential flow filtration, and purifying the effluent; and S6, pre-freezing the purified solution, and carrying out drying treatment. The invention also discloses application of the method. The method has the beneficial effects that the angelica keiskei sample is crushed and filtered to obtain the extracting solution, then the extracting solution and the dried chitosan film are mixed at room temperature, the exosome is adsorbed through the charge effect, and the exosome is purified after being filtered by the tangential flow filtering system, so that a large amount of high-purity exosome is obtained, and the method is simple, convenient and efficient.

The invention discloses a tissue dissociation liquid, a tissue dissociation method and application thereof, and relates to the technical field of cell biology. In a DMEM / F12 culture medium, the tissue dissociation liquid only contains the following raw materials in concentration: 0.5 to 3 mg / mL of type II collagenase, 0.5 to 2 mg / mL of type IV collagenase, 0.5 to 2 mg / mL of hyaluronidase, 10 to 18 [mu] M of Y27632 and 80 to 150 [mu] g / mL of primocin. The tissue dissociation liquid provided by the invention can dissociate normal tissues or tumor tissues into single cells or cell clusters, the single cells obtained by the digestion method are high in proliferation rate and easy to successfully culture, and the tissue dissociation liquid has the technical advantages that the cell yield and survival rate are maintained at a relatively high level. Therefore, the tissue dissociation liquid provided by the invention has good application prospects in tumor tissue dissociation, cell line construction and organoid model construction.

Use of a bitter melon exosome and an oral medicament prepared therefrom. The bitter melon exosome obtained from the bitter melon juice can be used for preparing a pharmaceutical formulation embedding a protein, a polypeptide, a small molecule peptide, a nucleic acid, or a small molecule compound. The compositions of the oral medicament of the present invention comprise the bitter melon exosome and an active pharmaceutical ingredient encapsulated in the bitter melon exosome. The bitter melon exosome is suitable for preparing a drug that is easily damaged by the gastrointestinal tract and is mostly administered by means of an injection route into an oral agent, and the oral agent can produce a similar therapeutic effect to that of the injection administration route by oral administration, and is convenient to use. The extraction method for the bitter melon exosome is simple and has a low cost. Moreover, the bitter melon exosome has no immunogenicity, has relatively high biocompatibility and biosafety, and has relatively strong tolerance to gastric acid and digestive tract proteolytic enzymes, and intestinal barrier permeability.

The invention discloses application of a tripterygium wilfordii exosome in preparation of an anti-cervical cancer medicine and a tumor angiogenesis inhibiting medicine, and relates to the field of biological medicine. The medicinal plant exosome derived from tripterygium wilfordii is successfully separated, and it is proved that the medicinal plant exosome can regulate related pathways through delivery of functional nucleic acid molecules to inhibit proliferation and migration of cervical cancer cells and promote active oxygen related apoptosis so as to be used for tumor treatment. The tripterygium wilfordii exosome can further act on vascular endothelial cells in a tumor microenvironment, the migration function of Hela cells is inhibited, and then the effect of resisting cell proliferation in the tumor microenvironment is achieved. The tripterygium wilfordii exosome has the advantages of being simple in preparation method, remarkable in tumor growth resistance and tumor cell migration inhibition effect, good in biocompatibility, high in delivery efficiency and the like, and can be used as a novel nano-drug for tumor treatment.

The invention relates to the technical field of cell manipulation and analysis, and relates to an automatic single cell continuous capturing and screening system and a use method thereof. Comprising a bias liquid storage module, a cell injection module, a micro-fluidic chip, a cell capturing module, a cell screening module and an imaging system, the bias liquid storage module, the cell injection module, the cell screening module and the cell capturing module are connected with the micro-fluidic chip; the bias liquid storage module is used for generating bias flow; the cell injection module is used for providing cells; the micro-fluidic chip is used for capturing and screening cells; the cell capturing module is used for capturing cells; the cell screening module is used for screening cells; and the imaging system is used for acquiring captured cell images and performing visual analysis. Micro-fluidic pipeline double-bias-flow stable cell flow sample introduction is designed, a high-speed electromagnetic valve is matched to realize rapid control of a gas path on cell capturing and screening channel fluid, and a high-speed camera is combined to analyze cell positions in different areas in real time, so that automatic single cell continuous capturing and screening are realized.

The present invention relates to a method for preparing Gram-negative bacterial outer membrane-derived nanovesicles (OMNVs) and uses thereof. Additionally, the present invention provides a method for treating or diagnosing cancer using OMNVs. Furthermore, the present invention provides a method for delivering therapeutic or diagnostic substances to target cells or tissues by loading them into OMNVs. Also, the present invention provides a method for preventing and treating various diseases, including cancer and bacterial / viral infections, by co-administering OMNVs with antigens or administering OMNVs expressing antigens.

The invention discloses nucleus pulposus cells as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The method comprises the steps that exosomes are collected from immortalized spherical nucleus pulposus cells and used for culturing the nucleus pulposus cells, the cultured nucleus pulposus cells have higher multiplication capacity, and large-scale in-vitro amplification of the nucleus pulposus cells is achieved; meanwhile, the cultured nucleus pulposus cells have higher extracellular matrix secretion capacity. The cultured nucleus pulposus cells are used for treating the intervertebral disc degeneration disease, the clinical use requirement can be met, and an effective treatment scheme is provided for treating the intervertebral disc degeneration disease.