3491results 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 relates to a stem cell bioactive nano-vesicle with efficient inflammation regulation and tissue repair effects, the average particle size of the nano-vesicle is 100-350nm, the polydispersity index (PDI) is 0.05-0.40, and the expression rate of phosphatidylserine (PS) on the surface of the nano-vesicle is greater than 40%. According to the preparation method, stem cells are subjected to apoptosis induction, and then a programmed serial extrusion process is adopted to prepare the nano-vesicles. The nano vesicles are moderate in particle size, uniform in distribution and high in production efficiency, the preparation process is easy to operate, the quality is stable and controllable, and the nano vesicles have excellent technical effects in the aspects of repair and regeneration of inflammation injury type tissues and organs.

The embodiment of the invention discloses a preparation method of exosome-loaded hydrogel, the exosome-loaded hydrogel and application of the exosome-loaded hydrogel. A specific embodiment of the method comprises the following steps: carrying out oxidation treatment on hyaluronic acid; carrying out mixed reaction on oxidized hyaluronic acid and methacrylic anhydride under a preset environmental condition to obtain a first mixed solution; performing dialysis and freeze-drying treatment on the first mixed solution to obtain target oxidized hyaluronic acid; adding the target oxidized hyaluronic acid into the exosome solution to obtain an exosome-loaded oxidized hyaluronic acid solution; the exosome-loaded oxidized hyaluronic acid solution and polylysine are subjected to a mixed reaction, the exosome-loaded hydrogel is obtained, and the mass ratio of target oxidized hyaluronic acid to polylysine in the exosome-loaded hydrogel ranges from 1 to 2. According to the embodiment, the mechanical property and controllable degradability of the exosome-loaded hydrogel can be improved, so that the applicability of the exosome-loaded hydrogel and the retention capacity and activity of the exosome are improved.

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.

There is provided herein methods generating a population of primed mesenchymal stem cell-derived exosomes. A method in accordance with the disclosure may comprise expanding a population of mesenchymal stem cells (MSCs) in culture, administering one or more priming agents in the culture to prime the population of MSCs and obtain a population of primed MSCs: growing the population of die primed MCSs in culture to produce a primed-MSC-derived conditioned medium; collecting the primed MSC-conditioned medium; and purifying a population of exosomes from the primed MSC-conditioned medium. The one or more priming p agents may comprise a conditioned media derived from a population of stem cells different from the population of MSCs, a Nrf2 activator, or a combination thereof. There is also provided herein methods of treating tissues such as cornea and liver using the population of exosomes from the primed MSC-conditioned medium.

The invention relates to a universal nano biological vesicle preparation method. The preparation method comprises the following steps: carrying out crude extraction on cells from bacteria, fungi, plants or animals to obtain a crude extracting solution; the crude extracting solution is placed in a low-temperature and high-pressure homogenizer to be subjected to low-temperature and high-pressure homogenizing treatment, then the nano vesicles are obtained, the low-temperature and high-pressure homogenizer comprises a high-pressure cavity, a homogenizing valve, a working cavity, a conveying system and a temperature control system, a slit is formed in the homogenizing valve, and the crude extracting solution located in the high-pressure cavity enters the working cavity through the slit of the homogenizing valve, the pressure intensity of the high-pressure cavity is 300-3000 bar, and the working temperature of the high-pressure homogenizer is 0-10 DEG C. The activation energy is reduced by controlling high-pressure and low-temperature environments, so that the original cell membrane structure is broken, the cell wall structure is destroyed, and phospholipid bimolecules are driven to aggregate and self-assemble to form the vesicles. For biological membranes derived from bacteria, fungi, plants and animals, the vesicles can be prepared by the preparation method disclosed by the invention, and the method has universality.

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 medical biology, and provides a dopaminergic neuron precursor cell surface marker F3 and / or SSTR2, and the dopaminergic neuron precursor cell surface marker F3 and / or SSTR2 can be used for screening dopaminergic neuron precursor cells to obtain high-purity dopaminergic neuron precursor cells. The dopaminergic neuron precursor cell has relatively high efficiency of differentiating to the dopaminergic neuron.

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 of NK (Natural Killer) cells. The method comprises the following steps: (1) separating and purifying the NK cells; (2) primarily amplifying the NK cells in a low-oxygen environment; (3) co-culturing the NK cells; and (4) continuously amplifying and harvesting the NK cells. The NK cell prepared by the method shows relatively high cell purity and activity, and shows a remarkable anti-tumor effect in a malignant tumor treatment mouse model. The method has the advantages of high efficiency and reliability, and provides good reference for research and clinical application of the NK cells.

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 present invention relates to methods of preparing a therapeutic exosome using a protein newly-identified to be enriched on the surface of exosomes. Specifically, the present invention provides methods of using the proteins for affinity purification of exosomes. It also provides methods of localizing a therapeutic peptide on exosomes, and targeting exosomes to a specific organ, tissue or cell by using the proteins. The methods involve generation of surface-engineered exosomes that include one or more of the exosome proteins at higher density, or a variant or a fragment of the exosome protein.

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 relates to the field of cell biology, and particularly discloses an exosome as well as a preparation method and application thereof. The method for preparing the exosome comprises the following steps: (1) introducing nucleotide sequences for coding a signal peptide TPA and an epidermal growth factor into an AAVS1 region of a host cell, such as a sequence shown in SEQ ID No.9 and a sequence shown in SEQ ID No.10, so as to obtain a modified host cell; and (2) collecting the exosome secreted by the modified host cell. The expression level of EGF mRNA in the exosome can be remarkably improved, so that the obtained exosome has a better effect in skin wound treatment.

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

The invention discloses a culture medium for culturing a liver cancer micro-tumor model and a culture method thereof. The invention relates to the technical field of biology, and provides a culture medium for culturing a liver cancer micro-tumor model, and the culture medium is composed of an antibacterial and antifungal agent tri-antibody, HEPES, GlutaMax, a human recombinant protein EGF, FGF-10, HGF, IL-2, IL-15, R-spondin 1, calomel, gastrin, A83-01, N-acetyl-L-cysteine, nicotine, an N2 additive, B27 and a basal culture medium. The liver cancer micro-tumor model obtained by the method can accurately reflect various characteristics of the original focus of a patient, and is a good scientific research experiment model and a preclinical experiment model in the field of precise tumor diagnosis and treatment. It can be foreseen that the culture method has wide application prospects in the fields of liver cancer research and clinical diagnosis and treatment.

The invention discloses a method for obtaining pigment cells through in-vitro culture of plectropomus leopardus skin tissues. The method comprises the following steps: step A, preparing a primary cell culture solution; step B, pretreating the plectropomus leopardus skin tissues to obtain pretreated fish skin tissues; step C-1, carrying out a digestion reaction on the pretreated fish skin tissue by an enzymolysis method to obtain an explant; and step C-2, carrying out in-vitro culture on the explant through an explant method to obtain the plectropomus leopardus skin tissue primary pigment cells. The plectropomus leopardus skin tissue is successfully separated and cultured into a plectropomus leopardus skin tissue primary pigmented cell group by combining an enzymolysis method and an explant method, so that coexistence culture of several kinds of pigmented cells is realized. Raw materials are provided for exploring the self function and gene regulation and control mechanism of the pigmented cells, and preconditions of in-vitro verification experiments are provided for further exploring the influence of the pigmented cells on the body color. Meanwhile, a foundation is laid for breeding of improved varieties of the plectropomus leopardus.

The invention relates to application of sea cucumber-derived nano-vesicles in preparation of a medicine for promoting wound repair. The nano-vesicles (SC-EVs) extracted and prepared from sea cucumbers can effectively promote cell migration, scratch repair and tube forming capabilities of human skin epithelial cells (HACAT) and human skin microvascular endothelial cells (HM DEC) and inhibit cell apoptosis, so that the capability of repairing cells is achieved, and particularly the effect of repairing wound surfaces in a high glucose state or diabetes mellitus is achieved. The invention provides a new direction and thought for the application of the sea cucumber-sourced nano vesicles (SC-EVs), provides a brand new drug research and development and clinical treatment direction for wound repair, especially for solving the problem of difficult healing of wounds of patients with high glucose or diabetes, and has extremely high social value and market application prospect.

The invention relates to application of a tea exosome in preparation of a medicine for improving skin pigmentation, the tea exosome is creatively extracted and purified, and the tea exosome can effectively inhibit proliferation of melanoma cells and inhibit expression of melanin generation related protein by scavenging free radicals and inhibiting tyrosinase activity, so that the effect of improving skin pigmentation is achieved. The intracellular melanin removal is promoted. In a mouse in-vivo experiment, after the tea exosome is applied, it is observed that the skin epidermis thickness of a mouse is reduced, the dermis thickness is increased, collagen arrangement is tighter and more ordered, the content is increased, and meanwhile generation of inflammatory factors is reduced. The tea exosome can significantly increase the content of a melanin autophagy key factor LC3B and reduce the content of P62 to promote melanin autophagy, and has good percutaneous infiltration capacity, thereby playing a role in improving skin pigmentation.

The invention provides a breast milk source exosome as well as a preparation method and application thereof. The preparation method of the breast milk source exosome comprises the following steps: pre-treating breast milk, and then carrying out primary purification concentration and fine purification on the breast milk source exosome after the pre-treatment; wherein the pretreatment comprises the step of mixing degreased breast milk with a sodium phosphate solution; the primary purification concentration comprises tangential flow ultrafiltration treatment; the fine purification comprises purification treatment by adopting a multi-mode chromatography. The invention provides a large-scale purification process of the breast milk exosome, which can be used for treating a large number of samples, improving the purity and yield of the breast milk exosome and ensuring the morphological integrity of the breast milk exosome.

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 provides a drug-loaded callicarpa nudiflora-derived extracellular vesicle as well as a preparation method and application thereof, and relates to the technical field of pharmaceutical preparations. The drug-loaded callicarpa nudiflora-derived extracellular vesicle has relatively high encapsulation efficiency and drug loading rate and good gastrointestinal tract stability and biocompatibility, can target a colitis disease part to reduce adverse reaction of a drug and improve the curative effect, solves the problem that the drug is insoluble in water, and can be used for preparing the drug-loaded callicarpa nudiflora-derived extracellular vesicle. The invention provides a novel drug delivery strategy for treating the ulcerative colitis, and is expected to become an effective strategy for clinically treating the ulcerative colitis, thereby achieving the effects of synergistically treating the ulcerative colitis and improving the effectiveness and safety of drug delivery.

The invention discloses a construction method and application of a mouse airway organoid inflammation model, and relates to the technical field of biology. The construction method comprises the following steps: (1) taking a mouse airway tissue, digesting with digestive juice, and centrifuging to obtain a cell suspension; (2) centrifuging the cell suspension, discarding the supernatant, adding matrigel, uniformly mixing, incubating, and culturing by using an airway organoid complete medium; (3) adding a cell recovery solution into the cells cultured in the step (2), incubating, degumming, centrifuging, discarding supernatant, adding matrigel, uniformly mixing, incubating, and adding an airway organ complete culture medium for continuous culture; and (4) adding 1-20 [mu] g / ml of LPS into the airway organoid complete culture medium, and incubating. The AO constructed by the invention covers four main cell types of cilia cells, goblet cells, basal cells and rod-like cells, causes epithelial injury in the AO and expression of AO inflammatory factors, and can provide reference for development of CRD new drugs.

The invention belongs to the technical field of biological medicines, and particularly relates to a sperm injury repair model and an establishment method thereof. Comprising the following steps: step 1, establishing a sperm lead poisoning model; and 2, establishing a lead poisoning sperm repair model. The method for establishing the lead poisoning sperm repair model in the step 2 comprises the following steps: (1) preparing a preparation for optimizing sperm functions in an extracellular vesicle way; (2) repairing the damaged sperms by using the preparation for optimizing the sperm function through the extracellular vesicles; and (3) detecting the activity of the repaired sperms. In the invention, by establishing a sperm injury repair model and adopting an extracellular vesicle modification technology, the key functional protein is introduced into the extracellular vesicle, and the method has important significance on auxiliary researches such as development and research of related drugs for improving sperm injury and improving sperm motility of asthenospermia patients in vitro.

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 plant exosome extraction method and application thereof, and belongs to the technical field of exosome extraction. The extraction method of the plant exosome comprises the following steps: S1, mixing a plant sample with a buffer solution, performing juicing treatment, and filtering to obtain a crude extract; s2, carrying out continuous flow centrifugal treatment on the crude extract to obtain a refined extract; s3, carrying out concentration treatment on the refined extract to obtain a concentrated solution; and S4, carrying out microfluidic purification treatment on the concentrated solution to obtain the plant exosome. According to the method, the exosome of the plant sample is extracted by adopting a treatment method combining juicing treatment, continuous flow centrifugal treatment, concentration treatment and microfluidic purification treatment, so that large-volume, high-throughput, high-efficiency and low-cost exosome extraction and separation of the plant sample are realized; in addition, it is found for the first time that the panax notoginseng source exosome not only can effectively inhibit expression of inflammatory factors, but also has a good effect on psoriasis treatment.