34 results about "Trypsin Digestion" patented technology

The invention relates to the technical field of aquatic animal cell culture, in particular to a rapid enrichment and separation method for gonad somatic cells of plectropomus leopardus. Comprising the following steps: selecting healthy plectropomus leopardus for anesthesia; the method comprises the following steps: taking gonad tissues, performing sterile treatment, cutting, centrifugal washing and filtering, inoculating the gonad tissues into a culture medium for primary culture to obtain gonad cells emigrated from tissue blocks, performing trypsin-EDTA digestion and centrifugal washing, and treating the gonad cells with 1 * PBS containing polysorbate; after closing, antibody incubation and washing, the marking effect is verified through a fluorescence microscope, and specific enrichment of gonad somatic cells is achieved; and filtering and purifying the enriched fluorescence-labeled cells, placing the cells under a fluorescence microscope, accurately picking fluorescence-positive cells under a fluorescence visual field, transferring the fluorescence-positive cells into a culture dish containing a complete medium, inoculating the separated fluorescence-positive cells into the optimized complete medium, and carrying out purification culture, so that the cells grow in a wall-attached manner and stable passage is realized.

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 invention discloses a urate oxidase mutant (rAfUOX) with trypsin and chymotrypsin tolerance and application of the urate oxidase mutant (rAfUOX). The invention provides a urate oxidase mutant with trypsin and chymotrypsin tolerance by modifying a key amino acid residue in a wild type aspergillus flavus urate oxidase molecule through a protein engineering technology. The activity half-life period of the recombinant aspergillus flavus urate oxidase mutant rAfUOXK49Q after trypsin digestion is increased by 244% compared with the activity half-life period of the wild type rAfUOX, and the activity half-life period of the recombinant aspergillus flavus urate oxidase mutant rAfUOXK49Q after chymotrypsin digestion is increased by 191% compared with the activity half-life period of the wild type rAfUOX. Other enzymatic properties are basically consistent with those of the wild type rAfUOX.

The invention belongs to the field of tendon tissue scaffold preparation, and provides a tendon tissue scaffold preparation method based on a decellularization technology. Although the existing decellularization technology can realize removal of cell components and retention of a matrix structure, contradiction still exists in the aspects of considering decellularization efficiency and matrix integrity. The method comprises three steps of freeze thawing treatment, trypsin digestion ultrasonic treatment and nuclease treatment, and perfect balance between decellularization efficiency and matrix retention is realized by optimizing freeze thawing parameters, regulating ultrasonic conditions and reasonably matching enzyme types and concentrations. According to the method, the cell structure can be accurately destroyed, the release of cell contents is promoted, and meanwhile, the extracellular matrix structure and bioactive molecules are protected. Experiments show that the tendon tissue scaffold prepared by the invention is thorough in cell removal, retains an extracellular matrix structure of natural tendons to the greatest extent, is closer to natural tissues in mechanical properties and biological functions, and provides a new thought for tendon repair research and application.

The invention discloses a magnetic suspension three-dimensional cell culture method which comprises the following steps: performing pancreatin digestion, centrifugation and resuspension on cells, inoculating the cells into a pore plate, adding a magnetic nanoparticle solution, performing co-incubation, performing pancreatin digestion, stopping digestion, centrifuging to remove residual magnetic nanoparticles in a culture medium, and adding the culture medium for resuspension; placing a magnetic plate above the pore plate, and carrying out three-dimensional culture in an incubator; a magnet is loaded on the magnetic plate, and one side loaded with the magnet is attached to the pore plate, so that the magnet is always located above the culture medium. According to the invention, the three-dimensional magnetic suspension culture of cells is realized by preparing ferroferric oxide magnetic nanoparticles Fe3O4 (at) DA-PEG (at) PLL for enhancing cell adhesion and designing a three-dimensional culture device for large-scale magnetic suspension cell culture, and the homogenized cell aggregate with a three-dimensional structure is constructed.

The invention relates to the technical field of cell culture, in particular to an efficient separation culture method for hepatocytes of small yellow croakers and a hepatocyte source identification method. The invention provides a method for efficiently separating and culturing hepatocytes of small yellow croakers, which comprises the following steps: selecting liver tissues of juvenile small yellow croakers, cleaning with a PBS (Phosphate Buffer Solution) containing high-concentration double antibodies, and digesting with pancreatin to quickly obtain hepatocytes; optimizing cell culture to obtain an L-15 culture medium containing 10% of FBS, 1% of sodium pyruvate and 0.2% of NaCl; and cell source identification is carried out by amplifying mitochondrial COI gene and 16S rRNA gene sequences. According to the method, the separation efficiency of the hepatocytes of the small yellow croakers is remarkably improved, the pollution rate is reduced, the result reliability is improved through double-gene molecular identification, the technical blank of construction of hepatocyte lines of the small yellow croakers is filled, and the method has wide application potential in the fields of aquaculture, fish physiology research, environmental toxicology detection and the like.

The invention discloses a method for extracting and separating cord blood and placenta stem cells of sika deer, and belongs to the technical field of biomedicine. The problems that an existing stem cell extraction and separation method is low in efficiency, poor in cell activity, low in purity and large in pollution risk are solved. The method comprises the following steps: collecting cord blood and placenta after delivery of sika deer; diluting the cord blood, performing density gradient centrifugation, collecting the albuginea layer enriched with the stem cells, adding an erythrocyte lysate to remove residual erythrocytes, and centrifuging and collecting the stem cells; the method comprises the following steps: cutting placenta tissues into pieces, putting the placenta tissues into trypsin-EDTA digestive juice containing collagenase, carrying out ultrasonic-assisted digestion, and centrifuging to obtain stem cells enriched at the bottom of a centrifugal tube; and finally, resuspending the collected stem cells in a culture medium containing fetal calf serum and growth factors, adjusting the cell concentration, and inoculating into a cell culture bottle for culture. According to the method, the extraction efficiency and quality of the sika deer cord blood and placenta stem cells can be remarkably improved, and the activity and safety of the cells are ensured.

The invention discloses a sheep female stem cell separation and in-vitro culture method, and relates to the technical field of biologication.According to the technical scheme, the sheep female stem cell separation and in-vitro culture method is characterized in that 0.25% pancreatin is adopted for digestion for 1 hour at the room temperature, mechanical cutting operation is combined, intercellular connexin can be effectively decomposed, and the survival rate of the sheep female stem cells is increased; the ovarian granular cells are quickly released. Medical alcohol and a culture medium containing double antibodies are used for cleaning ovaries for many times, so that the probability of bacterial and fungal contamination is remarkably reduced. A DPBS buffer solution preheated at 37 DEG C and a DMEM / F12 culture medium containing 10% of FBS are used in the whole process, and stress damage to cells caused by temperature shock is avoided. Complete granular cells and tissue fragments are efficiently separated through filtration and centrifugation, and the purity of a cell suspension is improved. The method is developed on the basis of ovaries of sheep, does not depend on feeder layer cells or a complex three-dimensional culture system, can be operated only by conventional laboratory equipment, and is suitable for research scenes with limited resources.

The invention relates to a method for separating, culturing and purifying human amniotic mesenchymal stem cells. The method comprises the following steps: collecting and pretreating a human amniotic membrane, washing, digesting pancreatin, neutralizing and washing, digesting type II collagenase, performing primary culture, performing passage purification and the like. Compared with the prior art, in the pancreatin digestion stage, by increasing the number of times in the pancreatin digestion process, mixing of hybrid cells can be greatly reduced, meanwhile, a large number of stem cells with good activity are obtained, and the extraction success rate of the stem cells is remarkably increased; due to the optimized operation process, tedious steps are reduced, and operability is improved; through multiple purification steps, it is ensured that the finally obtained cells have high purity and a good state.

The application provides a preparation method and application of a retinal nerve vascular unit in-vitro 3D printing model, and relates to the field of biomedical technology, and comprises the following steps: preparing a composite hydrogel precursor solution by using GelMA, HAMA and LAP; selecting RECs, RGCs and RMCs in an exponential growth phase, respectively resuspending after trypsin digestion, mixing the three single-cell suspensions to obtain a cell mixture; mixing the composite hydrogel precursor solution and the cell mixture to obtain a biological ink; printing and packaging the biological ink according to preset parameters to form a preliminary 3D-RNVU model; and culturing the preliminary 3D-RNVU model again. The 3D-RNVU model constructed in the application realizes the reproduction of the physical and chemical properties of ECM and the neural-glial-vascular three-way interaction in a three-dimensional structure, and provides an in-vitro platform with high physiological correlation for mechanism research and drug screening of diseases such as diabetic retinopathy.

The invention is applicable to the technical field of biological medicine, and provides a metformin-engineered umbilical cord mesenchymal stem cell and a preparation method and application thereof, and the preparation method comprises the following steps: taking fresh umbilical cord tissue, cleaning, cutting into small blocks, performing adherent culture, and performing fusion passage when the cell grows to 80%; and co-incubating the P5-generation human umbilical cord mesenchymal stem cells and metformin, and centrifugally collecting the cells after pancreatin digestion to obtain the metformin-engineered umbilical cord mesenchymal stem cells. The concentration of the metformin is 500 [mu] mol / L. Metformin is used for pretreating human umbilical cord mesenchymal stem cells to construct Met-MSC with metabolism enhancement and function optimization characteristics, so that the Met-MSC has stronger anti-apoptosis, anti-oxidation, angiogenesis promotion and immunoregulation capabilities, a safe, simple, convenient and convertible new cell treatment strategy is provided, and the application bottleneck of traditional MSC treatment in diabetic foot ulcer is broken through.

This invention relates to a mass spectrometry method for detecting seven sesame allergenic proteins in processed foods. The method uses 12 sesame processed products (roasted, ground, boiled, baked, fried, and steamed) as samples. Protein extraction and trypsin digestion are performed, and the hydrolyzed peptides are analyzed by high-resolution mass spectrometry. Characteristic peptides of sesame allergenic proteins are initially screened based on principles such as response intensity and the number of amino acids (6-15). The specificity of these peptides is analyzed using BLAST, and specificity verification is performed to identify nine characteristic peptides with processing stability. Mass spectrometry parameters are optimized for each ion pair of peptides, and a multiple reaction monitoring (MRM) method is established. This method can sensitively and with high throughput detect sesame allergens in processed foods, which not only helps to standardize allergen labeling but also helps to protect the health of allergy sufferers, and is of great significance.

The present application relates to the technical field of aquatic animal cell culture, and particularly relates to a rapid enrichment and separation method of Plectrypops leopardus gonadal somatic cells. The method comprises the following steps: selecting healthy Plectrypops leopardus for anesthesia; taking gonadal tissue, and after aseptic treatment, cutting, centrifugal washing and filtration, inoculating in a culture medium for primary culture to obtain gonadal cells migrated from the tissue block, and after trypsin-EDTA digestion and centrifugal washing, using 1xPBS containing polysorbate for treatment; after blocking, antibody incubation and washing, verifying the labeling effect by a fluorescence microscope to realize specific enrichment of gonadal somatic cells; after filtration and purification of the enriched fluorescent labeled cells, placing the cells under a fluorescence microscope, accurately picking fluorescent positive cells under a fluorescence field, and transferring the cells to a culture dish containing complete culture medium, inoculating the separated fluorescent positive cells in optimized complete culture medium for purification culture, so that the cells adhere to the wall and realize stable subculture.

The application discloses a production process of recombinant antibody protein produced by hybridoma cell suspension culture, and comprises the following steps: 1) resuscitation culture, 2) static expansion culture, and 3) suspension domestication and expansion culture. Compared with the semi-adherent culture method of hybridoma cells, the hybridoma cell suspension culture method has the following advantages: no trypsin digestion is needed during cell passage; the cell survival rate is higher, and the culture is easy to enlarge.

This invention provides a sample pretreatment kit and its application for the simultaneous detection of Aβ40, Aβ42, and lencanezumab, belonging to the field of biodetection technology. The kit includes: mixed magnetic beads, internal standard solution, trypsin digestion solution, enzyme digestion enhancer, and ammonia; the mixed magnetic beads are composed of protein A magnetic beads and streptavidin magnetic beads; the internal standard solution is composed of… 15 N-labeled Aβ40, 15 This kit consists of N-labeled Aβ42 and lencanezumab internal standard peptides. The pretreatment kit eliminates the need for anti-IgG1 antibodies, requiring only Protein A for detection, thus shortening sample processing time, greatly simplifying the sample handling process, and increasing throughput. The kit can simultaneously detect Aβ40, Aβ42, and lencanezumab, providing more reliable data support for clinical diagnosis and treatment efficacy evaluation, and significantly promoting the development of companion detection technologies for Alzheimer's disease biomarkers and therapeutic drugs.

The invention provides a preparation method and application of a retinal nerve and blood vessel unit in-vitro 3D printing model, and relates to the technical field of biomedicines.The preparation method comprises the steps that GelMA, HAMA and LAP are used for preparing a composite hydrogel precursor solution; the method comprises the following steps: selecting REC, RGC and RMC in an exponential growth phase, digesting the REC, RGC and RMC with trypsin respectively, resuspending, and mixing three single-cell suspensions to obtain a cell mixture; mixing the composite hydrogel precursor solution with the cell mixture to obtain biological ink; the bio-ink is printed and packaged according to preset parameters, and a preliminary 3D-RNVU model is formed; and the preliminary 3D-RNVU model is cultured again. According to the constructed 3D-RNVU model, the physical and chemical characteristics of ECM and nerve-colloid-blood vessel ternary interaction are represented in a three-dimensional structure, and an in-vitro platform with high physiological correlation is provided for mechanism research and drug screening of diseases such as diabetic retinopathy.

The application discloses application of human amniotic epithelial stem cells in preparation of a medicine for treating depressive diseases. The human amniotic epithelial stem cells are obtained by mechanically separating the inner surface amnion of discarded placental tissue, and then subjected to trypsin digestion, centrifugation, resuspension and harvesting in sequence after washing; and a human amniotic epithelial stem cell scheme for treating depression; the cells are widely sourced, free of ethical controversy, low in immunogenicity and non-tumorigenic, and provide a new safe and effective approach for treating depression; the cell preparation can be administered through intravenous injection, intrathecal injection or nasal delivery, and the mechanism of action is to inhibit the overactivation of glial cells, reduce the depressive-related neuroinflammatory response, and thus improve the neuronal microenvironment. The application can effectively promote hippocampal neurogenesis (increase in DCX positive cells), regulate 5-hydroxytryptaminergic neurons and glutamatergic neuron activity, restore the steady state of neural networks, and significantly improve the depressive behavior of model mice.

The invention relates to the field of diabetic foot ulcer, and discloses a diabetic foot ulcer wound edge skin-derived extracellular vesicle as well as a preparation method and application thereof. The preparation method comprises the following steps: pretreating diabetic foot ulcer wound edge skin tissues to obtain tissue fragments; the method comprises the following steps: mixing type IV collagenase and DNaseI enzyme in a DMEM (Dulbecco Modified Eagle Medium) high-glucose culture medium to prepare a tissue dissociation solution; carrying out primary dissociation by using pancreatin digestive juice, and then carrying out secondary dissociation by using tissue dissociation liquid; sequentially and alternately carrying out gradient size filtration and differential centrifugation on the filtrate, and collecting supernate; performing ultra-high-speed centrifugation, ultra-filtration and gradient needle type filtration on the supernate; and performing ultra-high-speed centrifugation again to obtain the diabetic foot ulcer wound edge skin-derived extracellular vesicles. The concentration of the obtained EVs is 3.29 mg / mL, and the characteristics of delayed healing and insufficient collagen deposition can be stably obtained by injecting the EVs into normal mice.

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.

The application discloses a kind of Vibration Mountain beard toad hind leg / toe tissue cells and its quick acquisition method and application.The method comprises: the body surface disinfection of Vibration Mountain beard individual, cut and take hind leg or toe tissue, and put back after wound disinfection;After tissue is soaked in 75% alcohol and washed with PBS containing penicillin, streptomycin and amphotericin B, it is temporarily stored in 4 ℃ temporary storage solution;After tissue is cut, it is sequentially digested with trypsin and type I collagenase;In the optimized low permeability medium, primary culture is carried out at 25-27 ℃;When cell confluence reaches 60%-70%, it is passaged using trypsin-EDTA digestion;Freezing and recovery.The application realizes the non-lethal cell of Vibration Mountain beard for the first time Quick acquisition, cell first time migration time is only 3 days, grow into monolayer only 12-17 days, pollution rate is reduced to 5.6%, recovery survival rate reaches 76.19%, and provides efficient and reliable technical support for endangered amphibian germplasm resource preservation and biological research.

The disclosed methods relate to a strategy for producing ground truth quantification ratios (both synthetic knockouts and simulated dose response gradients) using synthetic peptides mixed with a human proteome background. These, synthetic, bespoke proteins serve as a built in quality control for sample preparation for proteomics workflows. Additionally, mixtures of synthetic peptides are combined to form assembled synthetic proteins that can be spiked into samples at the start of processing (e.g., trypsin digesting, purification, etc.). In this way, problems in processing methods can be discovered. Sequences can be engineered to inform how well samples are being processed.

The invention relates to a micropterus salmoides skeletal muscle cell line and an establishment method thereof, and belongs to the technical field of aquatic organism cells. At present, research on molecular metabolism pathways of fish for regulating and controlling muscle quality is less due to large individual difference of fish, the method for continuously and efficiently culturing the micropterus salmoides cell line comprises the following steps: treating dorsal muscle tissues of micropterus salmoides, sequentially flushing, digesting, stopping digesting and cleaning, and culturing the micropterus salmoides cell line in a culture medium to obtain the micropterus salmoides cell line. Pasting the tissue block into a T25-coated cell culture bottle for standing, adding a complete culture medium to start primary culture, observing the cell emigration condition at the edge of the tissue block, carrying out subculture by using a pancreatin digestion method containing EDTA when the emigrated cells are fused and spread at the bottom of the bottle, and replacing a subculture medium after the subculture exceeds 15 generations, and cryopreserving the cells which are good and stable in culture state for later use, and resuscitating the cells when the cells are used. According to the method, the cell migration efficiency is greatly improved, and a new path is opened up for continuously culturing the skeletal muscle cell line of the micropterus salmoides.

The invention relates to a method for measuring sesame content based on labeled peptide combined with a liquid chromatography-mass spectrometry technology. The method comprises the following steps: selecting wheat flour, cocoa powder and mixed nut powder as food matrixes, respectively adding sesame powder with different masses, mixing to obtain a sample, extracting protein by adopting a buffer solution with specific conditions, performing reduction and alkylation reaction on the protein, and performing trypsin digestion to obtain a to-be-detected solution; based on targeted proteomics, the enzymolysis peptide fragment is analyzed through high performance liquid chromatography-triple quadrupole tandem mass spectrometry, the linear relation between the ratio of the peak area of the sesame quantitative peptide fragment to the peak area of the internal standard peptide fragment and the sesame content is explored, and sesame quantitative curves under three matrixes are established. The content of sesame in food can be detected according to the quantitative curve, and the quantitative limits of sesame under three matrixes of wheat, cocoa and nut powder are respectively 1mg / kg, 1.5 mg / kg and 2mg / kg. According to the method, the labeled peptide fragment is introduced, the loss caused by instability in the analysis process of a mass spectrometer is corrected by using the peak area ratio of the sample enzymolysis peptide fragment to the labeled peptide fragment, the sesame content in the food can be sensitively and accurately detected, the precision is good, and a certain technical support is provided for food allergen label identification.

A method for increasing peptide fragmentation by labelling the peptide at the C-terminal end with a guanidinium group or other basic functional group and distinguishing isobaric amino acids and amino acid combinations of asparagine and glycine-glycine; glutamine and glycine-alanine; and / or glutamine and alanine-glycine, during polypeptide sequencing. The method involves: obtaining a peptide of interest and / or digesting a polypeptide of interest with a protease, such as pepsin, chymotrypsin or trypsin, or by chemical cleavage to produce shorter peptides; reacting the obtained and / or generated peptides with a coupling reagent to derivatize the free C-terminal carboxylic acid function of the peptides, thus adding a basic functional group rendering C-terminal peptide fragment ions detectable by mass spectrometry; selecting a charge state of 2+ or more, and fragmenting the derivatized peptides in a mass spectrometer under conditions effective to generate at least w ions; and detecting the w ions by mass spectrometry, and identifying derivatized peptides which incorporate the additional mass of the basic functional group.

The invention belongs to the technical field of in-vitro culture of germline stem cells, and particularly relates to a separation and in-vitro culture method of spermatogonial stem cells of grass carp. The method comprises the following steps: by taking male grass carps as donors, anesthetizing, killing, cleaning, disinfecting, dissecting and separating gonad tissues, crushing into fragments by adopting a mode of combining magnetic bead crushing with shearing, digesting with trypsin, filtering and centrifuging to obtain high-purity spermatogonial stem cells, and performing in-vitro culture on the spermatogonial stem cells by adopting a culture solution. By optimizing the age of the male grass carp, an enzyme digestion process and an in-vitro culture system and adopting a one-step enzyme digestion method, the digestion time is shortened, the operation process is simplified, and the industrial problems that the spermatogonial stem cells of the grass carp are low in separation efficiency and culture is easy to differentiate are solved. According to the invention, a separation, purification and primary culture technical system of the spermatogonial stem cells of the grass carp is systematically established for the first time, and important cell materials and technical supports are provided for breeding technologies such as transplantation of germline stem cells of the grass carp, gene editing and the like.

The invention provides a separation and preparation method of dental pulp stem cells. The method comprises the following steps: using dental stem cells (DSC) separated from dental pulp tissues of human permanent teeth or deciduous teeth; obtaining a single-cell suspension through a double-enzyme digestion method; washing the single-cell suspension and the phosphoric acid buffer salt solution with the same volume for three times; carrying out adherent culture on the undigested tissues; culturing in an improved MEM culture medium (MEM-alpha) supplemented with fetal calf serum (FBS) with the volume fraction of 20%, penicillin with the volume fraction of 100 g / ml and streptomycin with the volume fraction of 100 g / ml in an incubator at the temperature of 37 DEG C under the condition of 5% CO2; according to the double-enzyme digestion method, the culture medium is replaced every two days; the culture medium is replaced once every 4-5 days by the tissue adherence method; after 90% confluence is achieved, performing trypsin digestion to harvest adherent cells; under the condition, the content of CD117 positive cells exceeds 96.67%. The dental pulp stem cells obtained by the method do not induce premature differentiation of cells, the cell proliferation speed is greatly increased, more sufficient nutrition is provided for the cells, synthesis of protein is promoted, proliferation of the cells is promoted, and differentiation of the cells is inhibited; the separation speed and the separation effect are improved, and the operation difficulty is also reduced. By utilizing the method disclosed by the invention, a rapid and stable separation method and an efficient amplification system can be established, and the method has a very wide clinical application prospect in the fields of life science and medicine. Along with continuous development of research, hDPSCs can play a greater role in the medical field.

This invention provides a method for culturing adherent cells that generate tight junction structures, comprising: seeding adherent cells resuspended in culture medium onto a bottom-supporting liquid in a culture container for culture to obtain a membrane-like cell sheet; wherein the density of the bottom-supporting liquid is greater than that of the culture medium and is immiscible with the culture medium. This invention is the first to apply a bottom-supporting liquid to the culture of adherent cells, which can promote the formation of tight junctions between adherent cells and the secretion of a large amount of extracellular matrix to form a high-density membrane-like cell sheet, effectively mimicking the in vivo tissue structure; at the same time, it eliminates the need for phosphate-buffered saline washing and trypsin digestion steps in traditional adherent cell culture methods, preserving the extracellular matrix of adherent cells during growth, better restoring the cell growth microenvironment, making it more suitable for life science and clinical medical research; moreover, this culture method is simple to operate, has low additional costs, and has significant commercial value.

The utility model discloses a bioreactor microcarrier adherent cell pancreatin digestion device, which belongs to the technical field of cell pancreatin digestion, and comprises a pancreatin digestion unit connected with a bioreactor, and the pancreatin digestion unit is connected with a bottom valve of the bioreactor through a second connecting pipe; the micro-carrier adherent cells are digested by pancreatin in the bioreactor. The bioreactor microcarrier adherent cell pancreatin digestion device provided by the utility model can be used for observing the cell digestion state in real time and terminating digestion in time, carrying out data statistics and analysis on cell counting, calculating the recovery rate of digested cells, and observing the cell attachment rate and the cell growth condition of the cells in a next-stage bioreactor, so that the digestion efficiency of the cells in the next-stage bioreactor is improved. Compared with culture parameter data of a production batch for cell digestion by using a butterfly-shaped pancreatin digester before improvement, the recovery rate and the adherence rate are stably increased, and the working efficiency and the product yield are better improved.

The present invention provides a novel method for determining a long-chain non-coding ribonucleic acid interaction protein. The present invention provides a fusion protein formed by BASU and dCasRx, a mammalian expression vector for expressing said fusion protein. The method for determining the lncRNA interaction protein according to the present invention comprises: co-transfecting a mammalian expression vector that expresses the fusion protein and a gRNA that specifically targets the target lncRNA into target cells, thereby BASU specifically biotin-labeling effector proteins nearby; isolating the biotinylated proteins by using a streptavidin affinity coupled magnetic bead and then eluting, and digesting by trypsin and quantitatively analyzing by a label-free mass spectrometry. The present invention can highly credibly determine the proteins that interact with lncRNA.

The present invention relates to the technical field of cell culture. Specifically disclosed is a recombinant trypsin cell digestion solution for maintaining high cell viability. The recombinant trypsin cell digestion solution consists of the following concentrations of components: 5000-8000 mg / L sodium chloride, 500-1500 mg / L glucose, 500-1500 mg / L disodium hydrogen phosphate, 100-400 mg / L sodium bicarbonate, 1000-2000 mg / L potassium dihydrogen phosphate, 1000-2000 mg / L potassium chloride, 0.5-2 mM of EDTA, 100-500 mg / L poloxamer 188 PRO, 100-500 mg / L HEPES, and 100-500 mg / L recombinant trypsin, with the balance being pure water. The provided recombinant trypsin cell digestion solution greatly improves cell viability after digestion, and solves the problem of reduced cell viability after conventional trypsin digestion.