50 results about "Dispase" patented technology

This invention relates to pork liver cell immortalities and its producing method. Procedures are showed: high live ratio fresh original pork liver cell is got by dispase-collagenase perfusion method; it is cultured for 24 hours. Then cell upper heat removing of recombination retronituse that contains SV40 big T antigen is used to infect the pork cell under condition of polybrene which concentration is 8ug / ml. Then it is medicine pressure filtrated by 500ug / ml G418 after one week, single clone cell is picked for large culture when the cell clone appears and grows to 1.0-2.0cm to get pork cell that can passage. Reinfection is done under condition of 8ug / ml polybrene. Then it is medicine pressure filtrated by 2ug / ml puromycin after one week. Single clone cell is picked for large culture when the cell clone appears and grows to 1.0-2.0cm to get immortality pork cell.

The present invention relates to a biological implantation material and method of preparing the same, which comprises the steps of:(i) treating a tissue derived from animal or human with alcohol;(ii) contacting the said tissue with an enzyme selected from the group consisting of dispase, DNAse, RNAse and pepsin in a solvent;(iii) treating the tissue obtained in step (ii) with alkaline solution; and(iv) treating the tissue obtained in step (iii) with acid solution.

The invention discloses a sea cucumber egg nutriment and a preparation method thereof. The sea cucumber egg nutriment is white or light yellow hydroscopic powder free of odor and taste and highly soluble to water and ethanol. The preparation method comprises sea cucumber egg and intestine breaking and enzymolysis, metal ion removal through ion exchange, and freeze drying, wherein the enzymolysis employs conjugated enzymes of exogenous enzymes and endogenous enzymes or single exogenous enzymes; single exogenous enzyme enzymolysis refers to the enzymolysis using single pepsinase or using the mixture of trypsinase, dispase and alkaline proteinase. The invention not only makes wastes in sea cucumber processing with traditional methods into an invigorant rich in nutrition but also reduces pollution of the wastes to the environment. The processing method is simple and reasonable and can be industrialized easily.

Production of low-molecular weight chitoglycan or chitooligose is carried out by fixing dispase on N-succinchitoglycan aquogel ball from cross-linking method, degrading chitoglycan from fixed enzyme, controlling enzymolysis time, regulating degraded product pH to 8-9 from sodium hydroxide, depositing by alcohol, washing and vacuum drying to obtain low-molecular weight chitoglycan or chitooligose with free amidogen. It achieves much yield, less molecular weight distribution and continuous industrial production. It can be used for medicine and food.

The invention discloses an in-vitro separation and cultivation method for tooth-sourced mesenchymal stem cells. The in-vitro separation and cultivation method comprises the following steps: (A), gingiva on a tooth surface and surrounding tissue are scraped off, and the tooth is stored in tooth preserving fluid; (B), a culture solution is added to a centrifuge tube, dental pulp is collected to the centrifuge tube and cut into pieces by scissors; (C), collagenase type I and dispase are added to digest dental pulp tissue; (D), single-cell suspension is prepared; (E), the obtained single cell is subjected to primary culture, and the planting density of primary cells is (1-5)*10<4> / cm<2>; and (F), the cells are subjected to subculturing. The tooth is crushed with a physical method to obtain the dental pulp tissue, the collagenase type I and the dispase are used for digestion, the single-cell suspension is obtained, primary planting and culturing are performed for 10-15 days, and then passage is performed; and the problems that the primary cells are mixed with other components of the tooth and aging occurs probably in a cell mass culture process are solved effectively.

The invention discloses a human normal bronchial epithelial cell and preliminary isolated culture and subculture methods and purposes of the human normal bronchial epithelial cell. The cell is named as the human normal bronchial epithelial cell HNBEC / HL-001, the preservation number of the cell is CCTCCNO: C201311. The preliminary isolated culture method includes the steps that fat in a para-carcinoma tissue sample excised from a patient with lung cancer through an operation is removed, dispase and DNasel are added to the fat for action after the fat is digested, the cell is collected through filtering and centrifugal operations, and the cell is suspended again with an HL culture medium for inoculated culture. The subculture method includes the steps that when the cell proliferates to 70-90% abundance, pancreatin-EDTA is used for digestion, and then DMEM is used for neutralization; the cell is collected through a centrifugal operation, and the cell is suspended again with an HL culture medium for inoculated culture. The human normal bronchial epithelial cell can be used for physiological research of human normal cells, drug toxicity research and detection of external normal cells, and research on pathogenesis of bronchia and lung diseases including bronchogenic carcinoma.

The invention discloses a method for culturing and amplifying odontogenic epithelial cells, and belongs to the technical field of cell culture. The method for culturing and amplifying the odontogenic epithelial cells of the invention comprises the steps of obtaining and culturing primary epithelial cells and amplifying the epithelial cells, wherein the primary culture comprises the steps of digesting odontogenic chyle-shaped tissues by using mixed enzyme digestive juice consisting of dispersing enzyme, I-type collagenase and DNA enzyme, collecting cells and culturing; and the mixed enzyme comprises the dispersing enzyme, the I-type collagenase and the DNA enzyme; and sub-culturing comprises the steps of digesting the cells by combining a cell scraper scrapping method and a 0.025 to 0.25 percent trypsin digestive juice, suspending the cells again with a culture medium and culturing. By the method, the cells are sub-cultured for 2 to 3 times to form a great number of purified odontogenic epithelial cells which are identified to be epithelial cells by an epithelial cell expression marker CK14. The method solves the problem of difficulty in culturing the odontogenic epithelial cells, and a great number of stable and purified odontogenic epithelial cells are obtained and the source of the epithelial cells is provided for the teeth development and regeneration research.

The invention provides a method for preparing a placenta hematopoietic stem cell preparation. The method comprises the following steps: (1) digesting placenta tissues with an enzyme solution so as to obtain a digestive product and removing tissue residues in the digestive product to obtain enzymatic hydrolysate; (2) mixing the enzymatic hydrolysate with a hydroxyethyl starch solution, and layering the mixed materials to obtain a nuclear cell suspension; and (3) mixing the nuclear cell suspension with a frozen stock solution, wherein the enzyme solution contains type I collagenase, DNA enzyme I, dispase and hyaluronidase, and the frozen stock solution contains dimethyl sulfoxide, hydroxyethyl starch, human albumin and dextran. According to the technical scheme, the separation efficiency of the placenta hematopoietic stem cell can be improved; and the frozen placenta hematopoietic stem cell can be directly applied to stem cell treatment when heated to a room temperature.

A method for preparing antler polypeptide or tortoise plastron polypeptide includes: dissolving antler gel or tortoise plastron gel, boiling, cooling and placing at 45-58deg.C constant temperature, regulating pH to 7.5-8; adding pancreatin and enzymolyzing, killing pancreatin activity by enzymolyzed liquid, re-placing at 45-58deg.C constant temperature; re-adding dispase and second enzymolyzing, killing dispase activity by second enzymolyzed liquid; filtering, concentrating, vacuum drying and grinding to powder. It can be used to improve digestive and absorbing ability and workability for food, drink and beverage.

The invention provides a method of manufacturing tissue engineering skin out of the body with epithelial stem cells. The method includes the following steps: (1) preparation of the epithelial stem cells: epithelial cells are made into cell suspension liquid, adhered and screened by IV collagen, and cultivated with epithelial stem cell media; (2) preparation of deepitheliarizing derma; (3) construction of the tissue engineering skin: epithelial stem cells for 2nd to 5th generation cultivation are selected, Dispase enzyme is adopted to digest the bossy cell patches in the size of rice to soya beam, the patches are inoculated to the deepitheliarizing derma, the tissue media is added, to cultivate the tissue engineering skin underwater or on the water. The tissue engineering skin constructed by the method of the invention is more suitable for the biological characteristics of human skin.

The invention provides composite dermal matrix biological dressing and a preparation method thereof. The composite decellularized dermal matrix biological dressing is prepared through the steps of mixing fatty alcohol polyoxyethylene ether with polyoxy ethylene nonyl phenyl ether and sodium dodecyl sulfate in an appropriate proportion, defatting skin, performing decellularizing treatment on skin through complex enzymes of Dispase and Trypsin-EDTA in an appropriate proportion, performing soaking in a mixed solution of carboxymethyl chitosan, hyaluronic acid, hydroxyethyl cellulose, calcium chloride and keratan sulfate, and performing a cross-linking reaction through oxidized sodium alginate. Based on high defatting rate, the characteristic of original skin collagen content is maintained, the united synergetic action of the carboxymethyl chitosan, the hyaluronic acid, the hydroxyethyl cellulose, the calcium chloride and the keratan sulfate is used for cooperation, and the composite decellularized dermal matrix biological dressing is prepared. The composite decellularized dermal matrix biological dressing has favorable efficacy of resisting bacteria and promoting healing, can concurrently have favorable mechanical properties, is easy to tile and apply on wound surfaces and is not liable to damage.

The invention discloses a method for quickly separating and culturing fibroblast from human skin tissues. The method comprises the following steps of cleaning and shearing fresh foreskin tissues afteroperation of adults or children, and sequentially adopting solutions of dispase II and dispase I to digest under the condition of oscillation in a water bath at the temperature of 37 DEG C; and collecting skin tissue blocks through centrifuging; enabling a culture medium to resuspend, and culturing in a culture box with CO2 (carbon dioxide) volume concentration of 5% at the temperature of 37 DEGC. The method has the advantages that the temperature of the digestion function of the dispase I and the dispase II is increased from 4 DEG C to 37 DEG C, the activities of the dispase I and the dispase II are respectively improved, the contact areas between the dispase I as well as the dispase II and a corresponding primer are increased by the oscillation in the water bath, the enzymatic reaction is favorably performed, the digestion time of the dispase I as well as the dispase II is greatly shortened, and the separating and culture speed of the fibroblast is improved; the damage of enzyme to tissue cells is decreased, the wall attaching rate of the cells is improved, and the primary fibroblast can complete passage after culturing for 6 to 8 days.

The invention relates to digestive juice and method for dispersing pancreatic cancer tissue of human into single living cells, and belongs to the field of unicellular analysis and cell culture. The digestive juice prepared by mixing three enzymes of Collagenase Type IV, Dispase and DNase I is mainly used for treating pancreatic cancer tissue, and then operations of performing centrifugation, removing dead cells and the like are performed, so that single pancreatic cancer cells high in purity and high in activity can be obtained within a short time. The used reagent is a frequently-used molecule cell biology reagent. Used consumptive materials are also common cell culture consumptive materials, are low in price and are easy to obtain, so that the cost is effectively reduced. The entire operation process is simple, special instruments and equipment are not needed, the operation process can also be performed in a general purpose laboratory, and the method has high practicality.

The invention belongs to the field of cell biology and relates to a method for separating and purifying schwann cells. The method sequentially comprises the following steps of: firstly, taking a nerve segment under the aseptic condition and removing an epineurium; secondly, digesting a nerve tract by using a NB4 composite collagenase and dispase mixed digestive enzyme solution and discarding the solution; thirdly, placing the mixture in a trypsin and an ethylene diamine tetraacetic acid (EDTA) for 5 to 10 minutes to obtain a nerve fiber from which a perineurium is removed; fourthly, placing the obtained nerve fiber in the mixed enzyme solution, and oscillating the mixture to obtain a cell suspension; and finally, performing centrifugation and heavy suspension. By using the method, a greatquantity of high-purity schwann cells are obtained in a low-cost and high-efficiency mode so that the abundant and high-quality schwann cells are supplied for repairing the damaged nerve.

The invention discloses a method of non-exogenous induction of a pluripotent stem cell to be a GABA neuron. The method includes the following steps of 1 cultivating a pluripotent stem cell in a Matrigel or a Vitronectin matrigel system to the density of 60 % -80 %; 2 digesting the pluripotent stem cell obtained in step 1 with Dispase or EDTA (Ethylene Diamine Tetraacetic Acid), differentiating the suspension culture through neural induction on that day, and recording the day as the 0 day; 3 adding neural induction differentiation solution for further cultivation when a spherical cell formed by the pluripotent stem cell can be observed; 4 performing adherence on the seventh day; 5 adding SHH or a small molecule compound Purmophine on the tenth day for continuous induction; 6 blowing off a formed neural tube structure on the sixteenth day and continuing to cultivate with the neural induction solution; 7 obtaining the GABA neuron on the twenty-second day. The method of non-exogenous induction of the pluripotent stem cell to be the GABA neuron can significantly improve cultivation solution complication and low differentiation rate and eliminate the interference of exogenous factors.

The invention discloses a preparing method of zebrafish embryo single-cell suspension. The preparing method sequentially comprises the following steps of 1, taking a zebrafish embryo, and adding an aqueous solution of Pronase E; 2, conducting incubation at 37 DEG C for 4-5 min; 3, utilizing a 2-mL disposable dropper for gently blow-sucking the embryo so that the chorion can be peeled off, moving the embryo into a 1.5-mL non-enzyme centrifugal tube, and quickly sucking away Pronase E; 4, adding an E3 culture medium for washing, and then removing the culture medium, wherein the step is repeated3-5 times; 5, adding dispase which is pre-cooled on ice; 6, conducting incubation at 37 DEG C for 2-5 min, wherein intermittent vibration is conducted in the incubation period; 7, adding FBS for terminating digestion; 8, making suspension obtained in step 7 pass through a 70-micrometer screen, and then making a filtrate pass through a 40-micrometer screen; 9, conducting 200-500*g centrifuging on the screened suspension for 3-5 min; 10, taking HBSS re-suspension cells which are pre-cooled on ice and contain 1% BSA; 11, conducting 200-500*g centrifuging on the suspension obtained in step 10 for3-5 min; 12, repeating steps 10 and 11 and conducting washing once; 13, taking IESC re-suspension cells pre-cooled on ice to obtain the zebrafish embryo single-cell suspension.

The invention discloses an in-vitro construction method of bioengineering full-layer human cornea. Cell culture comprises preparation of single cell, cultivation of corneal stroma cells and preparation of corneal endothelial cells. The method comprises the following steps: washing a corneosclera ring of a donor with 100 U / ml penicillin and 80 U / ml streptomycin solution wash for three times, soaking into a 2.4 U / ml Dispase 2 solution, performing incubation in an incubator for 1 hour, lightly stripping limbal epithelium through a dissecting microscope and microscopic tweezers, soaking the stripped epithelial sheet into a 0.25 percent pancreatin-0.02 percent EDTA solution, and digesting in the incubator for 5 minutes again to obtain single cell. The in-vitro construction method of the bioengineering full-layer human cornea can be applied to an in-vitro medicine toxicity experiment, more importantly, sufficient biomechanical property is achieved, traction of a suture in penetrating keratoplasty and pressure in eyeballs after operation can be tolerated, and high biocompatibility and biological security are achieved, so the bioengineering full-layer human cornea can replace the donor's cornea to be applied to corneal transplantation.

The invention discloses a primary separation and culture method for periodontal ligament stem cells, which comprises the following steps: separating periodontal ligaments, carrying out enzymolysis on periodontal ligament tissues by a mixed enzyme of collagenase V of which the mass-volume concentration is 0.05-0.25% and pancreatin of which the mass-volume concentration is 0.05-0.25%, carrying out primary culture on periodontal ligament stem cells, and separating the periodontal ligament stem cells by adopting a limiting dilution method. The primary separation and culture method disclosed by the invention has the following advantages: (1) the cost is reduced because the cost of the pancreatin is lower than that of dispase; (2) the digestion time is shortened, the periodontal ligaments are dense connective tissues, the affinity of the collagenase V to the connective tissues is superior to that of collagenase I, and the collagenase V and the pancreatin are combined, so that the enzymolysis effect is obviously improved.

The invention belongs to the technical field of the construction in vitro of tissue engineered cartilage and in particular relates to an experimental method for anzymolysis, digestion and acquisition in vitro of a rabbit knee cartilage unit. The experimental method comprises the following steps of: taking a rabbit knee joint, repeatedly shearing joint cartilages into fragment tissues, washing the fragment tissues, removing supernate and putting the fragment tissues in an aseptic cone bottle for later use; adding dispase enzyme and II type collagenase into the cone bottle in the proportion of 12ml DMEM-F12 culture solution per gram of cartilages, stirring the mixture and digesting to form digesting suspension; and filtering and centrifugating the digesting suspension and adding primary chondrocyte culture solution into the obtained product to prepare aseptic cartilage unit suspension. The experimental method has the advantages that: the acquisition method is simple and the repetitiveness is high; the experimental method more accords with the growing environment and the biological characteristics of the chondrocyte in vitro; and compared with the pure cell, the cartilage unit has obviously improved biomechanics characteristics and the defect of poor biomechanics characteristics of the chondrocyte serving as a seeded cell is overcome.

The invention belongs to the technical field of in-vitro establishment of tissue engineering cartilages, more particularly to an experimental method for establishing tissue engineering cartilage in vitro by taking a knee-joint cartilage unit of a rabbit as a seed cell. The experimental method comprises the following steps of: taking a keen joint of a rabbit, repeatedly cutting the joint cartilage into fragmentized tissues, washing, removing supernatant away and putting in a sterile conical flask for later use; adding dispase and II-type collagenase into the conical flask according to 12mL DMEM-F12 culture solution / g cartilage, stirring and digesting to form a digested suspension solution; filtering and centrifuging the digested suspension solution, adding a primary chondrocyte culture solution to prepare a sterile chondrocyte suspension solution, and carrying out sodium alginate gel three-dimensional culture on the obtained cartilage unit to construct the tissue engineering cartilage. The invention has the beneficial effects that: the cartilage unit has stable form and multiplication situation in the in-vitro long-term culturing process, the physiological function of substrate ingredients, such as secreted II-type collagen, and the like is improved, and the effect of repairing cartilage injury by adopting a tissue engineering method is expectedly accelerated.

Digestive juice used for separating anchorage-dependent cells is disclosed. The digestive juice includes a component A, a component B and a component C. The component A includes NaCl, KCl, glucose, KH2PO4 and Na2HPO4. The component B includes dispase, papain, NaCl, KCl, glucose, KH2PO4 and Na2HPO4. The component C includes NaCl, KCl, CaCl2, MgCl2, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acidand glucose. The anchorage-dependent cell detachment efficiency of the digestive juice is high, and the digestive juice has low damage on cells and low toxicity and allows the cell survival rate to be high. A plurality times of cell passage can be performed without influences on cell vitality. The digestive juice can be used for cell detachment experiments for anchorage-dependent cells cultured in a serum-free manner. The method is simple to operate, and experiment result differences among different operators are reduced.

The invention provides a compound enzyme digestive juice as well as a preparation method and an application thereof. The digestive juice comprises the following components with final concentrations: 100-200U / mL of type IV collagenase, 1-10U / ml of type II dispersive enzyme, 50-200U / ml of deoxyribonuclease I, 0.1-0.5 mM of PMSF, 10wt%-20wt% of ethylene glycol, 5wt%-10wt% of polysucrose, and sterilewater serving as a solvent, wherein the pH value of the digestive juice is 7.4-8.0. The preparation method comprises the following steps: preparing materials according to the final concentration of each component of the digestive juice; dissolving type IV collagenase, type II dispersive enzyme, deoxyribonuclease I, PMSF and polysucrose in sterile water, adding ethylene glycol, and uniformly mixing; and adjusting the pH value to 7.4-8.0, filtering and sterilizing to obtain the product. The compound enzyme digestive juice and the preparation method thereof are applied to the organoids technology. The digestive juice disclosed by the invention not only can be used for obtaining complete organoid, but also can be used for obtaining cell clusters with uniform dispersion sizes, and is short in digestion time, convenient to operate and stable in effect.

The invention discloses RNCEC (rabbit normal corneal epithelial cells) and an application thereof. The cell is named as RNCEC / HL-032, and the collection number is CCTCC NO: C201677. A primary isolation culture method of the cell is as follows: a normal corneal limbus tissue sample of a New Zealand rabbit is digested, then dispase and DNase I are added after digestion, cells are filtered and collected in a centrifugal manner and then resuspended with an HL culture medium for inoculated culture. The subculturing method of RNCEC comprises steps as follows: the cells are digested with pancreatin-EDTA when proliferating to have the abundance of 70%-90% and then neutralized with a DMEM (dulbecco's modified eagle medium); the cells are collected in a centrifugal manner and resuspended with the HL culture medium for inoculated culture. A 2D and 3D culturing system established for the cell can be used for animal substitutive experiments of corneal drugs and used for detecting pharmacology and toxicology of drugs and establishing a new experimental animal substitution model for corneal drug research.

The invention discloses tumor tissue digestive juice and a method thereof. The digestive juice comprises the following components: type II collagenase of 2 mg / ml, type IV collagenase of 2 mg / ml, Dispase (neutral protease) of 0.2 ml / ml, DNA enzyme of 12units / ml, FBS of which the volume fraction of is 10%, DMEM of 0.8ml / ml, and CaCl2 of 3mM. According to the tumor tissue digestive juice provided bythe invention, the Dispase (neutral protease) is added, the protease can better digest and dissociate protein connection between compact tumor cells, besides, the Dispase enzyme content is increased,and the digestion efficiency is remarkably improved. Besides, the type IV collagenase is added to the tumor tissue digestive juice provided by the invention, so that the digestive efficiency can be remarkably improved, the digestive time can be shortened, the concentrations of the type IV collagenase and the type II collagenase in the digestive juice are relatively low, the cell damage can be reduced, and the cell viability in a cell suspension can be improved.

The present invention relates to a methods, compositions, and admixtures which prevent the aggregation of a population of adipose stromal cells (ASCs), such as an isolated population of adipose stromal cells. In some embodiments, the present invention relates to admixtures and methods of use thereof comprising a population of ASC and a modulator of ASC aggregation. In some embodiments, a modulator of ASC aggregation includes, for example, ionic agents (e.g., heparin), chelating agents (e.g., EDTA), proteolytic agents.(e.g., trypsin or dispase), and agents which inhibit the expression of cell surface receptors and molecules on the surface of ASCs (e.g., inhibitors of integrins expression). In some embodiments, the methods to block ASC aggregation and the admixtures are useful in use of the ASCs to treat various diseases and / or conditions, and increases the safety and / or efficiency of ASCs. The methods, compositions and admixtures have utility in treating a number of conditions including, but not limited to, the treatment of any vascularized organs, such as heart, kidney, liver and the like.

The invention discloses a rapid separating method for cyprinid fish hepatocytes. The rapid separating method comprises the steps of chopping fish livers to obtain fish liver fragments and adding a Hank's balanced salt solution into the fish liver fragments, and performing oscillation and centrifugation to obtain precipitates; adding digestive juice consisting of CaCl2, bovine serum albumin, collagenase, dispase, a trypsin inhibitor, deoxyribonuclease I and the Hank's balanced salt solution into the precipitates, and performing oscillation, beating and sieving with a cell strainer to obtain large tissue precipitates and a cell suspension; adding a cleaning solution consisting of the CaCl2, the bovine serum albumin and the Hank's balanced salt solution into the large tissue precipitates, andperforming beating and sieving with the cell strainer to obtain a cell suspension, repeating the step for 3 times to obtain a merged cell suspension, performing centrifugation on the merged cell suspension to obtain cell precipitates, adding the cleaning solution into the cell precipitates, and performing beating and centrifugation to prepare primary hepatocytes. The method effectively solves theproblems that cost is high and the sizes of fishes are required to be large due to the fact that the separating time is long and multiple enzyme preparations are required by employing an existing method

The invention relates to the field of stem cell organoid research, and discloses a method for separating crypts from colonic mucosa tissues and inducing to generate colonic organoid. Compared with the prior art, the method has the following advantages and effects that the elastin is degraded through Dispase, the connection between the colon epithelium and the intrinsic layer is loosened on the premise that cells are intact, the three micromolecules of Thiazovivin (TZV), SB431542 and CHIR99021 are added into the OGM culture medium to improve the organoid induction success rate, and the method has the advantages that the method is simple and convenient to operate, and the cost is low. The human colon-like organs can be reliably obtained with relatively low cost and relatively high efficiency; meanwhile, the method has the advantages that the method does not depend on a high-skill epithelial layer stripping technology; more primary colonic progenitor cells are obtained; the induction success rate of the primary culture medium on the colon-like organs is improved; and popularization and application are facilitated.