13 results about "Collagenase I" patented technology

The invention discloses a preparation method of vascular tissue-derived extracellular vesicles, and belongs to the field of cytobiology. According to the preparation method of the extracellular vesicles, the artery blood vessel tissue is directly subjected to enzymolysis through collagenase, the extracellular vesicles are obtained by combining low-speed centrifugation and ultra-speed centrifugation methods and utilizing a differential centrifugation mode, and the technical blank of obtaining the extracellular vesicles from the blood vessel tissue is filled. Experimental results show that the extracellular vesicles can be successfully prepared from collagenase I, collagenase II, collagenase IV, collagenase V or collagenase IV combined with elastin, the prepared extracellular vesicles are different in particle size, form, yield, protein quantity, purity and the like, and the quality of the extracellular vesicles obtained by enzymolysis of collagenase IV combined with elastin is optimal. The extracellular vesicles are extracted from vascular tissues with normal physiological characteristics for the first time, a new source is provided for preparation of the extracellular vesicles, and the extracellular vesicles have great significance in expanding application of the extracellular vesicles.

The application discloses a construction method and application of an ovary cell line of Quasipaa spinosa. The method comprises the following steps: selecting metamorphosis period larva ovary tissue, disinfecting and obtaining the ovary tissue, and then using collagenase I digestion solution to digest the ovary tissue at 26 DEG C for 1 hour; after the tissue block adheres to the wall, using a low-osmotic-pressure complete culture medium to carry out primary culture at 25-27 DEG C, wherein the culture medium is 60% diluted DMEM / F12 culture medium containing 10%-15% fetal bovine serum; when the cell confluence degree is higher than 70%, using trypsin-EDTA to digest the cells for 1-2 minutes to subculture, and the subculture culture medium contains 30% cell conditional culture medium; and finally, the cells are cryopreserved through programmed cooling. The ovary epithelial cell line of Quasipaa spinosa is successfully established for the first time, the survival rate of the cells after recovery is high, and the karyotype is stable. The constructed cell line can be used for amphibian reproductive biology research, compound screening and in-vitro conservation of germplasm resources.

The present invention relates to the fields of collagenase production and collagenase products, and particularly to improving the reproducibility, purity, and stability of collagenase I and collagenase II compositions, where the compositions are pure to at least 95% by area as measured by reverse phase high pressure liquid chromatography (RP-HPLC) and essentially free of neutral protease.

The invention discloses a construction method and application of an ovarian cell line of hapalogenys nitens. The method comprises the following steps: selecting a metamorphosis stage larva ovarian tissue, disinfecting and obtaining, and digesting for 1 hour at 26 DEG C by using collagenase I digestive juice; after the tissue block adheres to the wall, a low-osmotic-pressure complete medium is used for primary culture at the temperature of 25-27 DEG C, and the medium is a 60% diluted DMEM / F12 medium containing 10%-15% of fetal calf serum; after the cell confluence degree exceeds 70%, trypsin-EDTA (Ethylene Diamine Tetraacetic Acid) is used for digesting for 1-2 minutes for passage, and a passage culture medium contains 30% of a cell condition culture medium; and finally performing programmed cooling and cryopreservation. According to the method, the ovarian epithelial cell line of the hapalogenys nitens is successfully established for the first time, the cell recovery survival rate is high, and the karyotype is stable. The constructed cell line can be used for amphibian reproductive biology research, compound screening and germplasm resource in-vitro preservation.

This invention discloses a highly active duck embryo liver primary cell and its low-cost preparation method and applications. The preparation method includes: obtaining and pretreatment of duck embryo liver tissue, separation by complex enzyme digestion, preparation of cell suspension, primary cell culture, and maintenance of cell viability. The core of this method lies in the gentle digestion using a complex digestion solution composed of collagenase I, trypsin, and DNase I in specific weight ratios, followed by culture in a well-defined, non-commercial growth factor-free complete culture medium. This complete culture medium contains synergistic components such as DMEM / F12, fetal bovine serum, epidermal growth factor, hepatocyte growth factor, and an insulin-transferrin-selenium mixture. The duck embryo liver primary cells prepared by this invention exhibit high yield, high viability, and excellent liver-specific function, and can maintain their activity and function well after passage and cryopreservation.

The invention relates to a pretreatment reagent and method for a tissue sample and microorganisms difficult to break walls and application. The pretreatment reagent comprises a first enzyme mixed solution and a second enzyme mixed solution, the first enzyme mixed solution contains protease K and collagenase I, and the second enzyme mixed solution contains lywallzyme and beta-glucanase. A specific pretreatment reagent and a treatment method are designed, a tissue sample is efficiently dissociated into a dispersed cell state by utilizing a first enzyme mixed solution, subsequent grinding and second enzyme mixed solution treatment are facilitated, and the second enzyme mixed solution can greatly improve the release effect of nucleic acid in the sample and has a relatively great promotion effect on subsequent nucleic acid extraction; in addition, specific grinding condition parameters are designed, nucleic acid release can be further promoted, the sample detection performance can be improved, and the probability of false negative in detection is reduced.

This invention proposes a method for preparing a single-cell suspension of porcine pancreatic cells and its application. The method includes the following steps: S1, taking porcine pancreatic tissue, removing surface fat, and mincing the tissue; S2, washing the tissue fragments, and then placing the tissue into a mixed enzyme dissociation solution for the first digestion; S3, after the first digestion, collecting the supernatant, and adding the remaining tissue to the mixed enzyme dissociation solution for a second digestion; S4, after the second digestion, collecting the supernatant and combining it with the supernatant from step S3, and filtering; S5, centrifuging the filtered dissociation solution, discarding the supernatant, and resuspending the cell pellet to obtain a cell suspension; the mixed enzyme dissociation solution includes trypsin, collagenase I, elastase, lipase, DNase I, heparin, sodium sulfate, and DH Kans buffer. The enzyme mixture of this invention has the effects of removing intercellular matrix, reducing cell aggregation, protecting cell membrane morphology, and reducing cell damage.

The embodiment of the invention provides an enzyme-based tissue dissociation method which comprises the following steps: sequentially carrying out first digestion, second digestion and third digestion on a tissue by using a first enzyme, a second enzyme and a third enzyme so as to dissociate the tissue, the second enzyme is a mixed enzyme and comprises a mixed enzyme of collagenase I and collagenase IV; the third enzyme comprises trypsin. According to the embodiment of the invention, the enzyme-based tissue dissociation method which is low in cost, high in dissociation efficiency, short in treatment time and few in generated cell debris impurities is successfully established for the first time, and the human scalp tissue single-cell suspension with complete cell types can be obtained; and good technical support is provided for single-cell-based researches such as single-cell sequencing, single-cell apparent group research and the like.

The invention discloses a kit and a method for preparing alligator sinensis tissue single cells, and belongs to the technical field of single cell sequencing. The kit comprises Collagenase I, DNase I, Dispase and a DMEM (Dulbecco Modified Eagle Medium) culture medium. The kit and the method provided by the invention fill the blank of the Yangtze alligator tissue single cell preparation technology. The kit is simple in component and easy to prepare, the method has very high repeatability and success rate, a large number of high-motility and low-impurity single cells can be obtained, and the kit can be applied to single cell sequencing and more application fields.

The application belongs to the technical field of biotechnology, and particularly discloses a method for extracting primary cancer-associated fibroblasts from renal clear cell carcinoma tissue. The method comprises the following steps: treating fresh renal clear cell carcinoma tissue at 0-4 DEG C, and only retaining a region rich in tumor cells and interstitial mixed; low-speed shearing the region rich in tumor cells and interstitial mixed into fragments with a volume of 1-3 mm 3 ; placing the fragments in a collagenase I solution, collecting cell precipitates after digestion is completed; inoculating the cell precipitates into a first culture medium, and replacing the fresh first culture medium every day within the first 3 days after inoculation, and then replacing the first culture medium every 2-3 days. The method overcomes the problems of low cell adhesion rate, serious contamination, low cell activity and low purity of the conventional extraction technology of primary cancer-associated fibroblasts when applied to renal clear cell carcinoma tissue, and can stably obtain primary cancer-associated fibroblasts with high purity and high survival rate within 7-10 days.

The present invention relates to the fields of collagenase production and collagenase products, and particularly to improving the reproducibility, purity, and stability of collagenase I and collagenase II compositions, where the compositions are pure to at least 95% by area as measured by reverse phase high pressure liquid chromatography (RP-HPLC) and essentially free of neutral protease.

The invention relates to the technical field of cell biology, in particular to a preparation method and application of high-quality single cells of synovial tissues. An enzymolysis method in the preparation method comprises the following steps: carrying out enzymolysis on synovial tissues by adopting a mixed enzyme until the number of cells meets the requirement; the mixed enzyme is prepared from the following components in parts by weight: 1 to 3 parts of streptavidin, 0.2 to 1 part of neutral protease, 0.5 to 2 parts of collagenase I, 0.2 to 0.8 part of hyaluronidase, 0.1 to 0.4 part of elastase, 2 to 4 parts of collagenase II and 0.1 to 0.3 part of DNA (Deoxyribose Nucleic Acid) enzyme. According to the method for preparing the single-cell suspension based on enzymolysis of the synovial tissue of the specific mixed enzyme, the prepared single-cell suspension has high quality and can be applied to the fields of synovial cell in-vitro experiments of various cell levels, synovial single cell sequencing, animal model construction, cell line construction, CAT treatment and the like.

The invention relates to the technical field of organoids, and discloses a multi-enzyme digestion system for construction of pig lung organoids, the digestion system comprises collagenase I, collagenase II, collagenase IV, DNaseI, neutral protease and an Advanced DMEM / F12 culture medium; wherein the working concentrations of the components are as follows: the collagenase I is 400 to 500 U / mL, the collagenase II is 100 to 200 U / mL, the collagenase IV is 40 to 60 U / mL, the DNaseI is 8 to 12 U / mL, and the neutral protease is 4 to 6 U / mL. The invention provides a multi-enzyme digestion system, the system is composed of collagenase I, collagenase II, collagenase IV, DNaseI and neutral protease, the lung tissue is efficiently dissociated through the synergistic effect, and high-activity single cells or cell groups are obtained. Under the synergistic effect of the multi-enzyme digestion composition, the cell release rate and stem / progenitor cell survival rate of the pig lung tissue can be remarkably improved, a stable cell source can be provided for efficient formation of subsequent organoid, and a solid foundation is laid for construction of the pig lung organoid with complete functions.