1935results about "Cell dissociation methods" patented technology

The invention discloses an extraction and purification method and application of plant-derived exosome-like nanoparticles, and relates to the technical field of plant exosome preparation. The extraction and purification method comprises the following steps: washing fresh ginseng, airing for later use, adding a proper amount of buffer solution, mixing, breaking walls, filtering, centrifuging filtrate to obtain supernate, carrying out ultracentrifugation on the supernate to obtain ginseng exosome nano-particle precipitates, resuspending the precipitates, respectively adding pure water sucrose solutions with different concentrations, and carrying out ultracentrifugation to obtain ginseng exosome nano-particles; and re-suspending the target strip, and carrying out ultracentrifugation to remove redundant cane sugar so as to obtain the purified ginseng exosome-like nano-particles. The ginseng exosome-like nano-particles disclosed by the invention have a remarkable effect when being applied to cosmetics or medicines with skin photoaging resistance, skin oxidation resistance and wrinkle resistance. In an anti-UVB (Ultraviolet B) induced cell injury experiment, the ginseng exosome-like nano-particles have an excellent anti-UVB induced cell injury effect.

The invention provides a preparation method of mitochondria-targeted antioxidant hybrid vesicles. The preparation method comprises the following steps: S1, preparing and separating adipose-derived stem cell nano-vesicles; s2, preparing a liposome loaded with dihydromyricetin and distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-triphenylphosphine targeting peptide by an ethanol injection method; and S3, preparing the hybrid nano vesicles loaded with the dihydromyricetin and the distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-triphenylphosphine targeting peptide. The invention also provides the hybrid vesicles prepared by the method and application of the hybrid vesicles in preparation of refractory diabetes wound treatment drugs. The lipidosome and the adipose-derived stem cell nano-vesicles are hybridized to prepare the hybridized vesicles capable of targeting mitochondria and resisting oxidation, the hybridized vesicles are applied to wound treatment for the first time, the problem of oxidative stress of diabetic wounds is solved, wound healing is accelerated, and a new strategy is provided for optimizing mitochondrial function defects and oxidative stress of the diabetic wounds.

The invention relates to the technical field of biological medicines, and particularly discloses an anti-human CD3 rabbit recombinant monoclonal antibody as well as a preparation method and application thereof. The antibody obtains anti-CD3 immune response by immunizing a rabbit, is obtained by combining single B cell sorting, gene cloning and a mammalian cell expression system, and has high affinity and good specificity. The antibody can effectively recognize CD3 positive T cells in human peripheral blood and can be applied to the fields of flow cytometry, immunohistochemistry, T cell function research and the like. Experimental results show that the antibody has the advantages of high titer, strong fluorescence signal, low background and the like, and is superior to the traditional murine anti-CD3 antibody. Amino acid sequences of light chain and heavy chain variable regions of the antibody are further obtained, and controllability and repeatability of antibody expression are achieved. The antibody has important scientific research value and industrial application prospect, and is suitable for various scenes such as T cell marker detection, immune state evaluation, immunotherapy auxiliary research and the like.

The invention discloses a porcine fibroblast line capable of being passaged for a long time and application of the porcine fibroblast line. According to the invention, pig fibroblast tissues are taken as a source, pig fibroblasts at different parts are obtained through a tissue block separation method, in-vitro long-term passage is carried out, the cells are triggered to break through the Harike limit, monoclonal screening is further carried out through a limited dilution method, and a pig fibroblast line with a single source is obtained. After screening, porcine fibroblasts with two parts can be subjected to long-term passage in vitro, namely ear edge fibroblasts and rear leg fibroblasts, have collagen secretion capability and are preserved in China Center for Type Culture Collection with preservation numbers of CCTCC NO: C202511 and CCTCC NO: C202512, the cell line breaks through the Harike limit, is already transferred to 42 generations at present, and has the advantages of being capable of secreting collagen, capable of secreting collagen, capable of secreting collagen and capable of secreting collagen. The cells are in a healthy and full long fusiform state, the proliferation level is not influenced after long-term passage, and the method can be applied to preparation of cell culture meat.

The embodiment of the invention discloses a method for extracting extracellular vesicles from a traditional Chinese medicine dry product. The method comprises the following steps: taking a dry traditional Chinese medicine product as a raw material, and sequentially performing crushing, soaking, first-order enzymolysis, wall breaking, differential centrifugation, second-order enzymolysis, high-speed centrifugation, ultracentrifugation and ultrafiltration enrichment to obtain the extracellular vesicles. A two-step enzymolysis method is adopted in the enzymolysis process, impurities are removed to the maximum extent in a sequence difference enzymolysis mode, and the structure and function stability of the exosome are protected. According to the invention, the differential centrifugation and the enzymolysis method are alternately used, so that impurities with different sizes and different properties can be removed more thoroughly. According to the method, the cost is strictly controlled, the exosome purification efficiency is relatively high, the concentration of the obtained dry exosome is relatively high, and the operability is extremely high.

The invention relates to the technical field of biological medicines, in particular to an umbilical cord mesenchymal stem cell exosome and application of the umbilical cord mesenchymal stem cell exosome in preparation of a medicine for treating male infertility caused by hidden testis. The prepared high-concentration umbilical cord mesenchymal stem cell exosome suspension preparation is directly used for treating cultured spermatogonial cells and spermatoblasts in vitro, is injected into the testis of a mouse, and is used for improving the seminiferous tubule and spermatogenic microenvironment of testis tissue and promoting division and proliferation of the spermatogonial cells and the spermatoblasts. The method can effectively reverse oligospermia and azoospermia caused by the hidden testis, and compared with a model without intervention of treatment means, the method can effectively promote increase and even regeneration of sperms in the testis.

The invention discloses a preparation method and application of plant-derived extruded nano-vesicles, and relates to the technical field of biological medicines. The preparation method of the plant-derived extruded nano-vesicles comprises the following steps: juicing cleaned plant roots, stems, leaves, flowers or fruits to obtain plant juice, and filtering with gauze to obtain first filtrate; centrifuging the first filtrate at a low speed of 300-6000 * g to obtain a second filtrate; performing 8,000-20,000 * g centrifugation on the second filtrate for 0.5-3 hours at the temperature of 0-5 DEG C, so as to obtain a first precipitate; adding the first precipitate into a buffer solution, carrying out ultrasonic treatment, diluting, and sequentially passing through a filter membrane with the pore diameter of 0.4-10 microns and a filter membrane with the pore diameter of 0.1-0.4 microns, so as to obtain a third filtrate; and centrifuging the third filtrate, taking the precipitate, and resuspending to obtain the extruded nano-vesicles. Experimental detection shows that the extruded vesicles are similar to natural vesicles in characteristics, but the yield is far higher than that of the natural vesicles, and the extruded vesicles can serve as nano-carriers to be applied to a foundation or clinic.

The invention provides human placenta-derived vasogenic stem cells (hPASCs) and application thereof. The human placenta-derived angiogenic stem cells (hPASCs) disclosed by the invention are preserved in China Center for Type Culture Collection, Wuhan University, Wuhan, China; the preservation date is April 2, 2025; the preservation number of the strain is CCTCC (China Center For Type Culture Collection) NO: The hPASCs disclosed by the invention contain angiogenic cell subgroups, have stem cell stemness, have an angiogenesis effect in vivo and in vitro, and can be used for treatment of diseases such as cardiovascular diseases and vascularization construction of artificial organs and tissues.

The invention provides application of an enhanced and optimized mesenchymal stem cell exosome in knee osteoarthritis and muscle cartilage injury, and belongs to the technical field of biological medicine. The treatment activity of the umbilical cord mesenchymal stem cell exosome is remarkably improved by combining a composite pretreatment technology with a 3D culture system. Through the synergistic effect of the total flavonoids of rhizoma drynariae and tanshinone IIA, the pathological process of knee osteoarthritis can be regulated and controlled in multiple dimensions, including inflammatory reaction inhibition, cartilage repair promotion and joint function improvement, and the functional limitation of single-component pretreatment is broken through. The invention provides a safe and efficient novel treatment technical strategy for relieving knee osteoarthritis and muscle cartilage injury, and has a wide clinical application prospect.

The invention relates to a method for preparing a Chinese wolfberry exosome, the Chinese wolfberry exosome prepared by the method and application of the Chinese wolfberry exosome. Specifically, the preparation method of the lycium barbarum plant exosome disclosed by the invention comprises a layer-by-layer filtration step, and the layer-by-layer filtration step sequentially comprises deep filtration and optional membrane filtration.

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 fruit and vegetable exosome extraction method based on ligand fishing. The fruit and vegetable exosome extraction method comprises the following steps: suspending amino-functionalized Fe3O4 (at) SiO2 nanoparticles in a glutaraldehyde solution, and adding activated agglutinin to obtain Fe3O4 (at) SiO2-agglutinin nanoparticles; adding a glutaraldehyde solution into agarose gel to obtain a size exclusion chromatographic column filler; the method comprises the following steps: dispersing Fe3O4 (at) SiO2-lectin nanoparticles in a PBS buffer solution to obtain a uniform suspension, adding the uniform suspension into a size exclusion chromatographic column filler to obtain a uniform slurry, filling the size exclusion chromatographic column with the uniform slurry, and flushing with the buffer solution to form a magnetic bead stationary phase; and loading fruit and vegetable supernatant into a size exclusion chromatographic column, passing through the magnetic bead stationary phase, cleaning residual impurities combined on the exosome-magnetic bead compound by using a buffer solution, and dissociating the exosome from the magnetic beads by using a competitive eluent of the exosome to obtain the high-purity fruit and vegetable exosome. The problem that an existing exosome extraction method is difficult to meet the requirements of high efficiency, simplicity, high purity and high activity at the same time is solved.

A brain organoid containing optic vesicles generated based on H9 induction and an eye-brain fusion culture method thereof are provided. H9 embryonic stem cell induction is used to generate a brain organoid containing optic vesicles with primitive visual field. Based on an established optic vesicle brain organoid culture system, microscopic imaging is combined with specific marker antibodies related to early retinal development and photoreceptor cell maturation to structurally and functionally identify the brain organoid containing optic vesicles.

The invention relates to a medicine, a medicine composition and application of the medicine and the medicine composition in treatment of liver fibrosis. The medicine and the medicine composition comprise an umbilical cord mesenchymal stem cell membrane; the umbilical cord mesenchymal stem cell membrane is prepared by the following steps: coating the surface of a temperature-sensitive culture dish with a matrix; adding the cell suspension of the umbilical cord mesenchymal stem cells into a temperature-sensitive culture dish for culturing; the temperature is reduced, the umbilical cord mesenchymal stem cells and the extracellular matrix secreted by the umbilical cord mesenchymal stem cells are separated in a lamellar mode, and the umbilical cord mesenchymal stem cell membrane is obtained. The medicine and the medicine composition provided by the invention can effectively relieve liver fibrosis.

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.

The invention relates to a cell collection method capable of simultaneously collecting three cells in a co-culture model, and belongs to the technical field of biology. The invention provides a cell collection method capable of simultaneously collecting three cells in a co-culture model, and the cell collection method comprises the following steps: after a three-cell co-culture model is constructed, taking out a Transwell chamber, retaining cells in the lower chamber, and collecting the cells in the lower chamber; respectively digesting the cells on the two sides of the Transwell cell membrane by using a trypsin solution with the concentration of 0.5 g / 100mL so as to respectively collect the cells on the two sides of the Transwell cell membrane. According to the cell collection method disclosed by the invention, the three cells in the three-cell co-culture model are simultaneously collected in a manner of digesting the cells on the two sides of the Transwell membrane step by step by using pancreatin, so that not only is the cell and consumable cost saved, but also the experimental synchronism of the three cells is ensured, and convenience is provided for optimizing the experimental process.

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 stem cell exosome based on targeted fusion polypeptide modification, a preparation method and application of the stem cell exosome in treating male erectile dysfunction. According to the exosome, a specific targeting fusion polypeptide (sequence: EPLQLKM-GGGS-DPPV) is anchored on the surface through a chemical coupling technology, so that the targeting property of the exosome on cavernosum smooth muscle cells is enhanced; meanwhile, an anti-TrkA monoclonal antibody (TrkA-2E7-HL) is loaded through an electroporation method, and a fibrosis pathway mediated by a nerve growth factor (NGF) is inhibited. The exosome induces secretion of the umbilical cord mesenchymal stem cells by adopting an anti-oxidation culture medium, so that the anti-oxidation activity of the exosome is remarkably improved. Experiments show that in a diabetes ED rat model, the erectile function (ICP / MAP ratio) of the modified exosome is improved to 0.79 + / -0.05, the fibrosis area is reduced to 9.2 + / -1.6%, and the NO secretion level is improved by 3.5 times compared with that of a common exosome. Through triple mechanisms of polypeptide targeting, antibody synergy and oxidation resistance, effective reversion of the ED pathological process is realized, and the polypeptide has remarkable novelty and clinical transformation potential.

The invention relates to the technical field of stem cell injections, in particular to a human umbilical cord mesenchymal stem cell injection and a preparation method thereof. The cell cryoprotectant comprises the following components: a cell cryoprotectant and P6-generation human umbilical cord mesenchymal stem cells. By selecting P6-generation human umbilical cord mesenchymal stem cells, the capacity of secreting hepatocyte growth factors (HGF) can be improved; the cell cryoprotectant contains 6-9wt% of dimethyl sulfoxide and a certain amount of human serum albumin, can better protect cells and maintain the motility rate and biological functions of the cells, and is more beneficial to preparation of cryopreserved human umbilical cord mesenchymal stem cell injection. The P6-generation human umbilical cord mesenchymal stem cells are obtained by culturing a culture medium containing fetal calf serum, and the ability of the injection to repair liver injury can be improved.

The invention relates to the technical field of freeze-dried powder preparation, and discloses a white tomato exosome freeze-dried powder preparation method, which comprises: 1, manually picking white tomatoes 3-5 days after a color changing period, carrying out double-frequency ultrasonic cleaning, and pre-cooling in a 4 DEG C phosphate buffer solution containing 0.1% of vitamin C; according to the method, by virtue of an ultrasonic-cellulase synergistic crushing process, cells are mildly and efficiently crushed, so that the damage to an exosome structure is reduced, and the release efficiency and activity of the exosome are improved; the zebra fish embryo melanogenesis can be obviously inhibited, and the whitening and freckle-removing effects are relatively good.

The invention relates to the technical field of biological medicines, in particular to rapamycin-induced extracellular vesicles and application thereof in preparation of a medicine for promoting wound repair. The rapamycin-induced extracellular vesicles can enhance the migration and tube forming ability of endothelial cells, so that angiogenesis is promoted; meanwhile, the proliferation and migration capabilities of macrophages can be inhibited, and the proportion of M1 type macrophages in a wound is reduced, so that the inflammatory response mediated by the macrophages is relieved; and the composition shows extraordinary effects in the aspect of accelerating wound repair. The biological activity of the extracellular vesicles induced by the rapamycin depends on the activation of a PI3K / Akt signal channel.

The invention provides an exosome enrichment method based on a modified cellulose material, which comprises the following steps: carrying out TEMPO oxidation on plant residue cellulose to prepare a cellulose material with a carboxyl structure; the cellulose structure is further refined by adopting mechanical shearing and high-pressure homogenizing means, so that the specific surface area and the dispersion stability are remarkably improved; through polyethyleneimine PEI grafting, a large number of amino functional groups are introduced, the surface electropositivity of the material is enhanced, and efficient adsorption and enrichment of exosomes with negative charges are achieved. The invention also provides the exosome prepared by the method. The modified cellulose material disclosed by the invention is wide in source, simple and convenient to prepare and mild in enrichment process, the separation purity and the enrichment efficiency of the exosome can be remarkably improved, and the modified cellulose material has good biocompatibility and environmental friendliness; compared with existing ultracentrifugation, density gradient centrifugation and immunoaffinity separation methods, the method has the advantages that the operation process is simplified, the equipment dependence and reagent cost are reduced, and the potential of large-scale production and clinical application is achieved.

The invention discloses a preparation method of polygonatum-sibiricum-sourced exosome-like nano-vesicles and application of the nano-vesicles in antitumor drugs, and relates to the technical field of biological medicines. The preparation method comprises the following steps: crushing fresh rhizoma polygonati slices, filtering and collecting to obtain a first filtrate; carrying out gradient centrifugation treatment on the first filtrate to obtain supernate; carrying out first membrane filtration treatment on the supernate to obtain second filtrate with the particle size of less than 450 nm; centrifuging the second filtrate to obtain a precipitate, and resuspending the precipitate with a PBS buffer solution to obtain a rhizoma polygonati exosome crude extract; and performing secondary membrane filtration treatment on the rhizoma polygonati exosome crude extract to obtain a third filtrate with the particle size of less than 220nm, thereby obtaining the rhizoma polygonati-derived exosome-like nano-vesicles. The polygonatum-sibiricum-sourced exosome-like nano-vesicle has remarkable inhibitory activity on tumors, provides a new strategy and means for tumor treatment, and has important scientific value and clinical application potential.

The invention relates to the technical field of biological products, and discloses a mesenchymal stem cell hair growth liquid and a preparation method thereof, and the mesenchymal stem cell hair growth liquid comprises the following components: hair follicle mesenchymal stem cells; a hair follicle mesenchymal stem cell exosome; growth factors: a vascular endothelial growth factor (VEGF), a fibroblast growth factor (FGFs), a keratinocyte growth factor (KGF-7) and interleukin-10 (IL-10); vitamins: vitamin E, vitamin B5, vitamin B2, vitamin B6, vitamin C and biotin; the plant extracts comprise a cacumen biotae extract, a polygonum multiflorum extract, a ginseng extract, a peppermint extract, a purslane extract and a rhodiola rosea extract; other active ingredients: tripeptide and digoxin; the auxiliary material is a hyaluronic acid-collagen peptide graft copolymer; and the balance of a phosphate buffer solution.

The invention discloses a mesenchymal stem cell membrane bionic nano-enzyme for targeted therapy of Parkinson's disease as well as a preparation method and application of the mesenchymal stem cell membrane bionic nano-enzyme. The mesenchymal stem cell membrane bionic nano enzyme comprises an inner core and a mesenchymal stem cell membrane, wherein the inner core is composed of mesoporous polydopamine nanoparticles, and the mesenchymal stem cell membrane coats the inner core. The medicine contains the mesenchymal stem cell membrane bionic nano enzyme. The mesenchymal stem cell membrane bionic nano-enzyme disclosed by the invention shows good biocompatibility, and can be homed to a Parkinson disease region in the brain under the mediation of CXCR4 and VLA-4 specific membrane proteins. In addition, through the CD47 membrane protein expressed on the surface, the removal of the immune system can be avoided, so that the action time in the body is prolonged. The nano-enzyme is helpful for eliminating reactive oxygen free radicals, reducing oxidative stress level and reducing damage of neuroinflammation to dopaminergic neurons, so that safe and efficient treatment of Parkinson's disease is realized.

The invention relates to the technical field of plant extraction, in particular to a preparation method of a plant cell exosome, the plant cell exosome and application of the plant cell exosome. The preparation method comprises the following steps: after the surface of a plant leaf is scratched, inoculating the plant leaf into a first culture medium for induction culture, then transferring the plant leaf into a second culture medium for subculture to obtain a callus, and then adding the callus into a buffer solution for grinding to obtain a filtrate; centrifuging the filtrate again to obtain supernate; and adding an exosome complexing agent into the supernate, incubating overnight, centrifuging, and freeze-drying to obtain the exosome. The first culture medium comprises an MS culture medium and the following components added into the MS culture medium: pullulan, 2, 4-D, agar and NAA; the second culture medium comprises an MS culture medium and the following components added into the MS culture medium: pullulan, 2, 4-D, agar, NAA and sodium phytate. The preparation method can increase the protein content in the exosome.

The invention discloses bacillus subtilis for secretory expression of collagenase and application of the bacillus subtilis. The invention provides bacillus subtilis for secretory expression of collagenase and application of the bacillus subtilis in fat cell extraction, and after optimization of an expression element promoter and a signal peptide and large-scale fermentation of a fermentation tank, the highest enzyme activity can reach 669U. ML <-1 >. After purification, adipose-derived cells are successfully extracted and separated from mouse adipose cells, and after passage, the adipose-derived cells present the form of adipose cells. The method shows that the method has a good application prospect in the aspect of fat cell extraction.

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.

A method for preparing exosomes is provided. The method includes preparing a human platelet, treating the human platelet with a buffer to obtain a platelet solution, centrifuging the platelet solution, and collecting supernatant of the centrifuged platelet solution to obtain an exosome solution. The buffer is calcium ion buffer, phosphate buffered saline (PBS), tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) buffer, or 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) buffer.

The invention relates to the technical field of biological medicines, in particular to rhizoma drynariae extracellular vesicles for treating periodontitis by targeting macrophage ETS2 genes as well as a preparation method and application of the rhizoma drynariae extracellular vesicles. The extracellular vesicles are creatively extracted from the traditional Chinese medicine rhizoma drynariae by combining a differential ultracentrifugation method with a sucrose density gradient centrifugation method, the functions of the extracellular vesicles are studied, and it is found that the extracellular vesicles can effectively slow down alveolar bone resorption in a periodontitis state and reduce periodontal soft tissue inflammation and have a good treatment effect on periodontitis. It is found for the first time that the rhizoma drynariae extracellular vesicles can reduce the inflammatory ability of macrophages in an inflammatory state and can reduce expression of proinflammatory factors TNF-alpha and IL-6. The invention has important scientific research value and clinical significance for further development and application of various traditional Chinese medicine plant extracellular vesicles including rhizoma drynariae extracellular vesicles.