59 results about "Collagenase I" patented technology

A drug product comprising a combination of highly purified collagenase I and collagenase II from Colostridium histolyticum is disclosed. The drug product includes collagenase I and collagenase II in a ratio of about 1 to 1, with a purity of greater than at least 95%. The invention further disclosed improved fermentation and purification processes for preparing the said drug product.

The invention relates to the field of cell engineering and particularly relates to a method for separating human adipose-derived stem cells from human adipose tissues. According to the method, by adding a small amount of low-molecular-weight heparin calcium into normal saline, a mixed solution is obtained and used as a cleaning fluid to clean the human adipose tissues, residual impurities such as red blood cells in the human adipose tissues can be well removed and thus human adipose-derived stem cells with relatively higher purity can be separated, the content of the parenchyma cell is significantly reduced, the differentiation induction of the human adipose-derived stem cells is facilitated, the consumption amounts of collagenase I, trypsase and a serum-free culture medium can be reduced and the cost of the process is also decreased. In addition, by centrifuging and digesting the human adipose tissues for 1 hour at a speed of 150r / min, the human adipose-derived stem cells with significantly better activity can be obtained.

The present invention discloses an adipose mesenchymal stem cell preparation kit, the kit includes a fat storage solution, a fat washing liquid, a fat decomposition liquid, a cell washing liquid, a cell culture liquid, a cell digestion liquid and a cell cryopreservation liquid; the fat storage solution is a DMEM, DMEM / F12, MEM or RPMI-1640 medium including 100-200ug / ml of penicillin, 100-200ug / ml of streptomycin and 2.5-5 mug / ml of amphotericin; the fat washing liquid is normal saline, PBS, D-Hanks or HBS including 100-200ug / ml of penicillin, 100-200ug / ml of streptomycin and 2.5-5mug / ml of amphotericin; the fat decomposition liquid is a DMEM, DMEM / F12, MEM or RPMI-1640 medium including 0.75-2mg / ml of collagenase I; the cell washing liquid is PBS, D-Hanks or HBSS; the cell culture liquid is a mesenchymal stem cell serum-free growth medium, the cell digestion liquid is a PBS, D-Hanks or HBSS solution including 1.25-2.5mg / ml of Trypsin-EDTA; and the cell cryopreservation liquid is a mesenchymal stem cell serum-free growth medium including 60-200mg / ml of DMSO.

The invention discloses a method for isolation and serum gradient switching culture of human umbilical cord mesenchymal stem cells. The method comprises the following steps: digesting human umbilical cord with collagenase I until Wharton's jelly is fully digested, collecting digested single cells, inoculating the single cells into alpha-MEM medium, carrying out suspension culture until 80-90% of cells are fused, and digesting with trypsin; inoculating the suspension of trypsin-digested cells into alpha-MEM medium, carrying out suspension culture until 80-90% of cells are fused, digesting with trypsin, subcuturing and inoculating the subcultured cells into alpha-MEM medium, and carrying out subculture repeatedly. The concentration of fetal bovine serum in alpha-MEM medium in the steps of subculture decreases with the increase of the passage number until fetal bovine serum disappears. According to the method, the material human umbilical cords are easily accessible; the preparation of human umbilical cord mesenchymal stem cells is rapid and safe, and is not subject to ethical restrictions; and the subsequent culture is independent on the characteristics of fetal calf serum, resulting in greatly reduced culture cost and risk in clinical use. The method of the invention has a good application prospect.

The invention relates to amethod for efficientlyobtaining mesenchymal stem cells from umbilical cord tissues and aims at solving the problems of the long obtaining time and low efficiency of an existing method. The method comprises the steps that primary isolation of umbilical cord mesenchymal stem cells is performed: cleaned and cut umbilical cord tissue blocks are predigested by using trypsin and then digestive juice is removed, repeated flushing is performed with DPBS, then digestion is performed on a water bath shaker of 37 DEG C by using collagenase I digestive juice, a complete culture solution is added to the filtrate filtered by a cell strainer to terminate digestive centrifugal separation so as to obtain primary mesenchymal stem cell; residual tissue blocks are collected after filtration, and re-digestion is performed to obtain primary mesenchymal stem cells; the mesenchymal stem cells are inoculated in the complete culture solution, and standing and culture is performed in 5% CO2 culture box at the temperature of 37 DEG C; a culture solution is replaced for one time at intervals of several days; culture multiplication is performed: when the primary cells grow to be 80% and converge, TrypLE heavy suspension cells are subjected to culture multiplication in a serum-free culture solution. The method has the advantages of efficiently obtaining mesenchymal stem cells, and the obtained mesenchymal stem cells have highcell activity and differentiation potential.

The invention belongs to the technical field of cell culture engineering, and relates to a method for culturing goat precursor fat cells in vitro. The method comprises the following steps of: preparing a collagenase I culture medium and a cell culture solution, killing a goat, taking goat precursor intercostal fatty tissues out, quickly soaking the fatty tissues into alcohol, repeatedly flushing the fatty tissues by using sterile normal saline, transferring the fatty tissues to a sterilization phosphate buffer solution (PBS), putting the fatty tissues in a sterile room, rinsing the fatty tissues by using the PBS, removing blood vessels and connective tissues by using ophthalmic scissors to obtain a fatty tissue sample, quickly cutting the sample into small blocks, transferring the small blocks to a centrifugal tube, adding the collagenase I culture medium, mixing uniformly and digesting; and performing filtration by using a cell sieve to remove undigested tissue blocks and fatty cells, transferring the digested cell culture solution to the centrifugal tube, centrifuging, removing the supernate, adding the cell culture solution, cleaning, centrifuging, preparing a cell suspension, inoculating cells to a culture dish, and culturing the cells in an incubator. The method is simple, reliable in principle, controllable in culture, good in culture effect and environment-friendly.

The invention discloses an umbilical cord mesenchymal stem cell preparation and a preparation method and application thereof. Through exploration and verification by a large quantity of experiments, the 3rd-10th-generation umbilical cord mesenchymal stem cells are selected as raw materials, and by a method of dispersing tissue by a mechanical method and then performing treatment with mixed enzymes containing collagenase I, collagenase II and trypsin for a short time for many times, the vitality of the separated umbilical cord mesenchymal stem cells is guaranteed. A culture medium special for the umbilical cord mesenchymal stem cell preparation, containing DMEM / F12 (without phenol red), 10% FBS, 20 kinds of amino acids and 8 kinds of vitamins, is adopted, mixed liquor of repeated cell culture supernatant and cell lysate supernatant is collected, more umbilical cord mesenchymal stem cell active protein and active factors are obtained, the preparation cost is reduced, and industrial requirements for a stem cell product preparation are met. The prepared umbilical cord mesenchymal stem cell preparation can promote proliferation of epidermic cells and skin fibroblast, is safe and is free from toxic and side effects, an allergy does not exist, and when the umbilical cord mesenchymal stem cell preparation is applied to feature-beautifying skin-care products, the effects are notable.

The present invention provides a method for producing a drug product comprising a combination of highly purified collagenase I and collagenase II from Clostridium histolyticum. The method utilizes an improved medium for the cultivation of Clostridium histolyticum which includes a non-meat-derived (i.e., non-mammalian) peptone or vegetable peptone. The method includes one or more of: (1) reducing glucose content in the meat-free or vegetable-derived media; and (2) increasing the salt concentration in the meat-free or vegetable-derived media. Also provided is a drug product which includes collagenase I and collagenase II at an optimized fixed mass ratio, and which has a purity of greater than at least 95%.

The invention discloses an extraction and culture method of human umbilical cord mesenchymal stem cells. The umbilical cord tissue is cut up and digested by collagenase I and then transferred into a culture bottle containing a culture medium for continuous culture. The culture medium is a low-sugar DMEM (Dulbecco modified eagle medium) and is added with fetal bovine serum, fibroblast growth factor, epithelial cell growth factor, cell transcription factor and cholesterol. The extraction and culture method disclosed by the invention can culture human umbilical cord mesenchymal stem cells for a long time while maintaining the activity of the stem cells. Through the invention, the problems of excessively fast cell aging and differentiation in current culture of human umbilical cord mesenchymal stem cells are solved, and the human umbilical cord mesenchymal stem cells with the characteristics of stem cells can be obtained for a long time.

The invention relates to a multi-enzyme system for separating different tissues-derived primary culture cells of a normal human and a mammal. The system comprises a plurality of 0.0125 to 0.125 weight percent of trypsin, 0.02 to 0.2 weight percent of collagenase I-IV, 0.0015 to 0.015 weight percent of hyaluronidase, 0.02 to 0.2 weight percent of neutral protease, 0.0015 to 0.015 weight percent of deoxyribonuclease I, 1 to 10 U / ml papain, 0.015 to 0.15 weight percent of pronase, 5 to 50 U / ml elastase and 0.01 to 0.1 weight percent of separase. The invention also relates to the application of the multi-enzyme system and a kit formed by the multi-enzyme system. The multi-enzyme system of the invention has the characteristics of reasonable design and capacity of separating the different tissues-derived primary culture cells of the normal human and the mammal highly specifically and highly sensitively; and the separated primary culture cells have the characteristics of high survival rate, high purity, easy cryopreservation and recovery, can be used for medicament experimental evaluation, medicament toxicity test and medicament therapeutic judgment, and are suitable for large-scale popularization and application.

The invention relates to a multi-enzyme system used for separating patient tumor tissue-originated primary cells. The system comprises components of, by weight: 0.02 to 0.2% of collagenase I-IV, 0.001 to 0.01% of hyaluronidase, 0.01 to 0.1% of trypsin, 0.02 to 0.2% of proteinase neutral, 0.001 to 0.01% of deoxyribonuclease I, and 5 to 50U / ml of elastase. The invention also relates to an application method of the system, an application of the system, and a kit formed by the system. The multi-enzyme system is advantaged in reasonable design. With the system, patient tumor tissue-originated primary cells can be separated with high specificity and high sensitivity. The separated primary cells are characterized by high survival rate, high purity, easy cryopreservation and easy resuscitation. The system and the kit can be used in medicine experimental evaluation, medicine toxicity testing, and medicine therapeutic performance determination. The system, the method and the kit are suitable to be widely popularized.

The invention discloses a method for separating and purifying rat dermal microvascular endothelium cells. The method for separating and purifying the rat dermal microvascular endothelium cells relates to the technical field of animal blood vessel endothelium cell culture in vitro, in particular to a method for culturing the rat dermal microvascular endothelium cells in vitro. The method is characterized by the following steps of digesting rat skin through neutral protease II, separating to obtain a dermal layer, digesting through collagenase I and II, obtaining single-cell suspension, and utilizing a CD31 immunomagnetic beads sorting technology to obtain purified target cells. Through the method, the pollution of parenchyma cells in microvascular endothelium cell culture can be simply, conveniently and effectively solved, the rat dermal microvascular endothelium cells with high purity can be obtained through separating, and no obvious morphological feature change occurred after continuous cell culture for 20 times.

The invention relates to a method for separating and culturing a human placental-derived mesenchymal stem cell. Aiming at problems and defects in an existing common PMSCs separation method, the technical scheme for solving the problems comprises the following steps: 1, taking out placenta by big tweezers, and cutting the placenta; 2, peeling the maternal surface and the infant surface of the placenta; 3, digesting placental villus; 4, diluting the digestive juice with 1000ml of PBS, filtering and centrifuging to collect cells for later use; 5, resuspending the cells with PBS, and centrifugally precipitating the cells; 6, subculturing the cells; and 7, freezing the cells. According to the method, PMSCs is separated by a one step digestive treatment with collagenase I, the operation steps can be reduced, cell damage can be lightened, the proportion of target cells can be effectively improved, and pollution of other cells can be reduced, so that the time for cell primary culture can be shortened, the cell purity can be improved, the cost can be greatly reduced, the original state of PMSCs can be well maintained, and a firm foundation is laid for massive production and wide clinical application of PMSCs in the future.

The invention belongs to the technical field of biology, and particularly relates to a micropterus salmoides myocardial fibroblast cell line and application. According to the cell line, aiming at thecharacteristics of micropterus salmoides heart tissue cells, brain tissue cells are separated by adopting a collagenase I and pancreatin combined digestion method, primary culture is performed, the micropterus salmoides myocardial fibroblast cell line is successfully constructed and is continuously passed to more than 60 generations, a large number of micropterus salmoides heart-derived cells canbe provided, and the cells can maintain a good growth state and can be cryopreserved. The micropterus salmoides myocardial fibroblast cell line has sensitivity to various fish viruses, cytopathy is generated after virus inoculation, the cell line can be directly applied to pathogen characteristic research and vaccine development, and the micropterus salmoides myocardial fibroblast cell line can also be used for expressing exogenous genes for gene function research.

The invention discloses a primary culture method for alveolar epithelial cells of Microhyla ornata. The primary culture method comprises the following steps: subjecting adult Microhyla ornate to surface sterilization; dissecting the adult Microhyla ornate, taking out the lung and carrying out cleaning with a HBSS balanced salt solution; adding a collagenase I solution for digestion; subjecting a digested solution to refrigeration and centrifugation and adding a complete cell culture medium; blowing and beating the fragments of the lung tissue, and then carrying out filtering by using a cell filter screen with a pore diameter of 100 [mu]m so as to obtain a cell suspension; and subjecting the cell suspension to culture at a constant temperature of 27 DEG C and replacing the previous completecell culture medium with a new complete cell culture medium every 3 to 4 d. The primary culture method of the invention can rapidly and repeatedly establish primary culture of the alveolar epithelialcells of Microhyla ornate; needed cell culture equipment is simple and has good operability; and acquired cells have obviously faster growth speed than the alveolar epithelial cells of mammals. The prepared alveolar epithelial cells of the invention significantly supplement existing alveolar epithelial cells of mammals and provide a novel cell material for research on major biological and medicalproblems such as terrestrial adaptability, lung development, pulmonary fibrosis and tuberculosis.

The invention discloses a method for performing in-vitro culture to bovine preadipocytes, belonging to the technical field of cell and tissue engineering. The method is based on the traditional subculture method, a tissue homogenizer is utilized and combines with the collagenase I culture medium to treat the fatty tissue, a DMEM (Dulbecco's Modified Eagle Medium) / F12 culture solution containing fetal bovine serum is used to perform in-vitro culture to bovine preadipocytes, and a new model of the in-vitro culture of bovine preadipocytes is successfully built. The technology is advanced and simple, the preparation of the culture medium is scientific and reasonable, manpower and material resources are saved, and the treatment time of the test sample is greatly shortened; the obtained cell morphology is best, the RNA (Ribonucleic Acid) culture and extraction efficiency is maximum, the preadipocyte culture method is convenient and efficient in a long term and lays the foundation for the gene expression and protein expression and the signal pathway in the later intensive study of the proliferation and differentiation process of preadipocytes; and a new idea and a new method are provided to regulate body fatness deposition of beef cow and prevent diseases in the perinatal period and lactation peak period of milk cow, etc.

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 discloses a nucleus pulposus cell separation and in-vitro culture method. When nucleus pulposus cells are separated, nucleus pulposus tissues are obtained, and bloodstains and cartilagetissues need to be cleaned; the influence of miscellaneous cells on attachment and subsequent culture of nucleus pulposus cells is prevented, collagenase I is used as digestive juice, a digestion process is milder, the digested cells and tissues are higher in activity and easy to adhere to the wall, the digestion is terminated with a mixed solution containing fetal calf serum with the volume fraction of 20% and DPBS, the obtained cells are not prone to adhesion, bottle laying is more uniform, 1% of penicillin / streptomycin is added into a used reagent, and pollution to a sample or in collectionand transportation is prevented; during in-vitro culture, placenta mesenchymal stem cell culture supernatant freeze-dried powder reconstitution fluid is added, the adding amount is 10-40%, growth factors lacking in a culture medium are made up, and growth of nucleus pulposus cells is promoted; and the cultured cells are high in survival rate, easy to adhere to the wall, good in cell morphology and high and stable in quality.

The invention provides a method for separate-culturing cardiomyocytes. The method includes the following steps that (1) heart tissue is taken, and PBS liquid is used to wash away residual blood; the heart tissue is cut into tissue blocks of 0.1-1mm<3> with ophthalmic scissors, 0.5-5ml of collagenase I is added, and shaking digesting is carried out at 36-37.5 DEG C for 0.5-3 hours; after digestion,2.5-50mL of PBS is added for dilution and stop, centrifuging is carried out at 1200rpm / min for 5 min, and a supernatant is discarded to obtain a cell pellet; (2) the cell pellet is resuspended with 0.5-5mL pancreatin, an elbow pipette is used for blowing-beating for 10-300 times, after digestion fluid is turbid, a culture medium is added to stop pancreatin digestion, and centrifuging is carried out at 1200rpm / min for 5 min, and a supernatant is discarded to obtain primary cardiomyocytes; and (3) after the primary cardiomyocytes is resuspended in the culture medium, the primary cardiomyocytesare inoculated into the culture medium according to 0.1-10x10<5> cells / cm<2>, and the obtained mixture is placed in a culture incubator of 37 DEG C and 5%CO2 for culturing to obtain the cardiomyocytes.

The invention relates to the technical field of dedifferentiation and induced dedifferentiation in cell biology and discloses a method for inducing and differentiating a mature fat cell from an adult rabbit into cardiomyocyte. A dedifferentiated fat cell (DFAT) can be obtained from a white fat tissue; and the DFAT can be differentiated into multiple mesenchyma cell systems through a certain induced culture method and has ossification, lipid formation, gristle formation and muscle formation capacities. The invention aims to culture the DFAT of the rabbit from the mature fat cell of the adult rabbit and induce and differentiate the DFAT into the cardiomyocyte. According to the invention, the white fat tissue of the adult rabbit is selected, isopyknic digestion is carried out through collagenase I, a 20% complete culture medium is adopted, the mature fat cell is dedifferentiated into a DFTA cell through a ceiling culture method, and the morphology of the cardiomyocyte and the expression of an identification molecular are formed. By using the method disclosed by the invention, a seed cell is provided for tissue engineering, convenience is brought for basic research, and a prospect is brought for clinical application.

The invention discloses digestive juice for separating mouse fat stem cells. The digestive juice comprises 10% of fetal calf serum, 0.09% of collagenase I and the balance of DMEM / F12 (dulbecco's modified eagle medium / nutrient mixture F-12). The digestive juice is simple in components, convenient in source and low in cost, and can effectively separate the fat stem cells from fat tissues of a mouse, well maintain the activity of the cells and lay a foundation for late-stage culture of the cells and further scientific research.

The invention discloses a preparation method of a dental pulp stem cell polymer for exfoliating deciduous teeth, the dental pulp stem cell polymer prepared by the preparation method and an applicationof the dental pulp stem cell polymer in preparation of a preparation for permanent tooth reimplantation. The preparation method comprises the following steps: (1) cleaning dental pulp tissue from exfoliated deciduous teeth, (2) digesting the dental pulp tissue by collagenase I and Dispase to obtain a cell suspension, (3) adding a VC culture solution and culturing to obtain a dental pulp stem cellpolymer. The dental pulp stem cells of exfoliated deciduous teeth not only have the characteristics of mesenchymal stem cells, but also express related genes of embryonic stem cells, have higher induced differentiation capacity than the dental pulp stem cells, and have ideal effects on dental pulp regeneration and periodontal healing of completely-dislocated young permanent teeth.

The invention discloses a method for extracting human stromal vascular fraction (SVF). The method mainly comprises the following steps: collecting adipose tissues, washing and centrifuging the adiposetissues, adding an SVF extract liquid containing a DMEM low-sugar medium, collagenase I, trehalose and gelatin, carrying out oscillation digestion, performing centrifuging to obtain a precipitate, and washing and centrifuging the precipitate to obtain SVF cells. The trehalose can protect the cell membrane structure; and the gelatin can promote cell proliferation and migration, and can improve theviscosity of a solution and reduce the damages of a shear force to the cells in order to achieve the purposes of protecting the cells and improving the survival rate of the cells in the adipose tissues. The method is adopted to make the number of the SVF cells reach 4.21 * 10<6> / ml and the viability rate reach 95% and make the number of adipose stem cells in the SVF reach 10.67 * 10<6> and the viability rate reach 95% on the ninth day of culture.

The invention discloses a primary culture method for skeletal muscle cells of Microhyla ornata. The primary culture method comprises the following steps: subjecting adult Microhyla ornate to surface sterilization; dissecting the adult Microhyla ornate, taking out leg muscle and carrying out cleaning with a HBSS balanced salt solution; successively adding collagenase I and trypsin for digestion; subjecting a tissue suspension to blowing, beating and extruding with a sharpened tip and a non-sharpened tip, and then carrying out filtering by using cell filter screens with pore diameters of 100 [mu]m and 40 [mu]m respectively so as to obtain a cell suspension; and subjecting the cell suspension to culture at a constant temperature of 27 DEG C and replacing a previous complete cell culture medium with a new complete cell culture medium every 3 to 4 d. The primary culture method of the invention can rapidly and repeatedly establish primary culture of the skeletal muscle cells of Microhyla ornate; and needed cell culture equipment is simple and has good operability. The prepared skeletal muscle cells of the invention significantly supplement existing skeletal muscle cells and provide a novel cell material for research on major biological and medical problems such as terrestrial adaptability, development by metamorphosis, muscle development and muscle injury.

The invention relates to a method for extracting, separating and identifying chilo suppressalis midgut stem cells. The method comprises the following steps: carrying out enzymolysis on midgut cells by collagenase I so that the midgut cells fall off from the midgut wall, and then separating the stem cells and enteric cells by adopting a density gradient centrifugal process or a flow cytometry. By virtue of the method, high-purity chilo suppressalis midgut stem cells are efficiently obtained and can be applied to the subsequent stem cell function study.

The invention relates to a method for reversibly separating human adipose mesenchymal stem cells through monoclonal antibody L-NGFR immunomagnetic beads. The method comprises the following steps of: (1) preparing the immunomagnetic beads; (2) coupling the immunomagnetic beads with biotin analogues; (3) combining the biotin analogues with streptavidin; (4) coupling the streptavidin with monoclonal antibody L-NGFR; (5) separating the mesenchymal stem cells from adipose suspension digested by collagenase I; and (6) eluting the adipose mesenchymal stem cells. According to the method, the mesenchymal stem cells can be effectively separated from human adipose tissues through utilizing the specific monoclonal antibody L-NGFR of the adipose mesenchymal stem cells and adopting a reversible protein combination method with controllable pH and an immunomagnetic bead enrichment method; and the adipose mesenchymal stem cell separating method is simple, environment-friendly, and low in cost, ensures high activity of the stem cells and has better application prospect.

The invention relates to the technical field of tissue culture, and discloses a culture medium for preparing endometrial stem cells as well as a preparation method. On the basis of a serum-free culture medium, Pall serum substitute, penicillin / streptomycin, gentamicin sulfate, glutamine, MEM NEAA, EGF, bEGF, sodium pyruvate and VEGF are added as nutritional ingredients of the endometrial stem cells, so that the survival rate of the endometrial stem cells can be significantly improved; and by combining with the mixed digestion of collagenase I and Dispase II, the survival rate of the endometrial stem cells can be further improved, and the development application of the endometrial stem cells can be promoted.