39 results about "Cells/microL" patented technology

The invention discloses micro ribonucleic acids (microRNA, miRNA) carried by cell microparticles (Microparticle, MP), a method for preparing the same, and application thereof in the technical field of biotechnological pharmacy. The invention provides a combination of the micro ribonucleic acids for evaluating the physiological and / or pathological states of a participant, and the combination contains all the micro ribonucleic acids which exist stably in serum / plasma particles of the participant and are detectable. At the same time, the invention provides an experimental method for preparing the cell microparticles containing specific micro ribonucleic acids and using the cell microparticles to perform gene-level regulation and control as well as modification on other cells and tissues. The combination and the method can be used for detecting and treating various diseases, including the aspects of the diagnosis and the differential diagnosis of various tumors, various acute and chronic infectious diseases and other acute and chronic diseases, the prediction and the curative effect evaluation of the occurrences of disease complications and the recurrences of malignant diseases, as well as the active ingredient screening, the efficacy evaluation and the judicial authentication of drugs, the detection of prohibited drugs and the like; besides, the combination and the method have the advantages of wide detection pedigree, high sensitivity, low detection cost, convenient available material, easy storage of samples and the like.

The invention discloses a tumor cell microsatellite instable state detection system, and relates to quantitative fluorescence PCR (polymerase chain reaction) amplification technologies and capillary electrophoresis detection technologies. The tumor cell microsatellite instable state detection system has the advantages that six mononucleotide repeat sites NR-21, NR-24, NR-27, MONO-27, BAT-25 and BAT-26 can be amplified by the aid of the tumor cell microsatellite instable state detection system, and sites BrafV600E, UGT1A1*6 and UGT1A1*28 can be typed by the aid of the tumor cell microsatelliteinstable state detection system; reference can be provided to checking Lynch syndromes and using irinotecan medicines by detection results on the basis that MSI (microsatellite instable) states are determined. The invention further provides a detection reagent kit designed according to the tumor cell microsatellite instable state detection system.

The present invention relates a method for the enumeration of eukaryotic cell micronuclei, while simultaneously acquiring cytotoxicity and mode of action information. The method utilizes differential labeling of chromatin from dead and dying cells to distinguish the chromatin from micronuclei, nuclei, and metaphase chromosomes, and differential labeling of metaphase events to provide additional information regarding cytotoxicity and genotoxic modes of action. Counting of micronuclei events relative to the number of nuclei and quantifying perturbations to the proportion of metaphase events can be used to assess the DNA-damaging potential of a chemical agent, the DNA-damaging potential of a physical agent, the effects of an agent which can modify endogenously-induced DNA damage, the effects of an agent which can modify exogenously-induced DNA damage, and genotoxic mode of action.

The invention relates to a preparation method and application of an anti-tumor vaccine based on cell microvesicles and aims to effectively solve problems in preparation of anti-tumor vaccines high inyield, good in universality, strong in killing effect on tumor cells and capable of controlling tumors by improving tumor microenvironments, repairing an immune system and enhancing anti-tumor immuneresponse of organisms. The preparation method comprises the following steps: preparing tumor cell microvesicles loaded with immunomodulators and connecting the surfaces of the microvesicles with adjuvant-loaded liposome to stably form the anti-tumor vaccine. The prepared anti-tumor vaccine is high in yield, good in universality, strong in killing effect on tumor cells and capable of improving tumor microenvironments, repairing an immune system and enhancing anti-tumor immune response of organisms; the method is simple in process and high in efficiency; and the prepared microvesicles are derived from cells, are sufficient in quantity and wide in source, are easy for large-scale production, improve the bioavailability of adjuvants, are effectively applicable to preparation of anti-tumor vaccines based on the cell microvesicles, and can be applied to prevention and treatment of different types of tumors.

A novel growth factor specific for vascular endothelial cells has been identified in conditioned medium of bovine pituitary derived folliculo stellate cells. This factor, named folliculo stellate derived growth facto (FSdGF) or vascular endothelial growth factor (VEGF), was purified to homogeneity by a combination of heparin sepharose affinity chromatography, Bio Gel P-60 exclusion chromatography, Mono S ion exchange chromatography and hydrophobic chromatography on a C4 reverse phase HPLC column. The factor is also found in the murine AtT-20 cell line. Alternatively, the growth factor is purified by a first reverse phase HPLC using acetonitrile gradient followed by a second reverse phase HPLC using an isopropanol gradient. FSdGF, having a molecular weight of about 43,000 da, was characterized as a glycoprotein composed of two homologous sub units with MW of about 23 kDa. FSdGF was a potent mitogen for vascular endothelial cells with activity detectable at 10 pg / ml and saturation at 500 pg / ml. It did not stimulate the proliferation of other cell types such as bovine corneal endothelial cells, adrenal cortex cells, granulosa cells, BALB / MK cells or BHK-21 cells. Microsequencing revealed an amino terminal sequence containing no significant homology to any known protein. The release of FSdGF by pituitary cells and its unique target cell specificity indicate that FSdGF is useful in angiogenesis.

The present invention relates to biochemical technology, and is chemical process and formula of dissolving cell-loading microcapsule under physiological condition. The formula consists of complexoneIII, sodium citrate and sodium bicarbonate, and the present invention has the features of simple formula, mild reaction condition, fast capsule breaking speed, high cell survival rate, high capsule breaking rate and no microcapsule residue. The microcapsule breaking process maintains the cell activity and can fulfill the requirement in biomedicine application. The technology is favorable to theaccurate quantitative analysis of microcapsulated cell, the cell activity preservation, the obtaining of high purity and high yield cell or gene engineering product and the development of microcapsule to cell transportation function. The microcapsule breaking process is suitable for use in industrial production.

The invention discloses a method for preparing virus of porcine reproductive and respiratory syndrome on a large scale. In the method, the virus of the porcine reproductive and respiratory syndrome is prepared in a cell microcarrier suspension culture system by a bioreactor. The method comprises the following steps of: inoculating host cells for preparing the virus to a carrier tank containing culture solution and a microcarrier, and mixing the cells and the microcarrier uniformly to ensure that the cells are attached to the microcarrier; providing sufficient nutrients and appropriate gas environment for the cells under the appropriate culture environment to ensure that the cells are grown until the cells are in an amount which are 10 to 20 times of the inoculation concentration on the microcarrier; preparing virus suspension from the virus of the porcine reproductive and respiratory syndrome by using cell maintenance culture solution to ensure that the suspension is adsorbed to the cells; culturing the virus under the appropriate culture environment; culturing continuously for 2 to 3 days to obtain virus solution; and after the virus solution passes inspection, performing freeze thawing on the virus solution twice at the temperature of -20 DEG C, and inactivating and purifying to prepare an inactivated vaccine of the porcine reproductive and respiratory syndrome or adding a freeze-drying protective agent for freeze drying to prepare a live vaccine of the porcine reproductive and respiratory syndrome. The method has large production scale, high yield of single batch and low production cost.

The invention relates to a serum-free medium adapting to PK-15 all-suspended growth, a preparation method of serum-free medium and an all-suspended domestication method applied to a PK-15 cell. The serum-free medium comprises amino acid, Tween, linoleic acid, vitamin E, myristic acid, stearic acid, vitamin A, beta-mercaptoethanol, sodium chloride, glucose, cadmium chloride, linoleic acid, cholinechloride, inositol, ammonium metavanadate and the like. The provided serum-free medium can realize all-suspended growth of the PK-15 cell, so that the cost and digestion of a microcarrier are omitted,and the technical problem that the PK-15 cell microcarrier is difficult to digest and amplify in the suspended culture process is solved.

The invention discloses a 2D and 3D cell co-culture system capable of implementing continuous harvesting without enzyme digestion and a construction method and application thereof. The construction method of the 2D and 3D cell co-culture system comprises: (1) preparing a seal with a microarray pattern; (2) preparing a culture substrate with a microarray pattern; (3) co-culturing 2D and 3D cells onthe micro-pattern culture substrate; and (4) obtaining a 3D cell microsphere and 2D cell co-culture system. According to the cell 2D and 3D co-culture system capable of implementing continuous harvesting without enzyme digestion, which is provided by the invention, 2D and 3D coexsiting co-culture of the cells can be implemented in the same environment, and if 3D cell microspheres need to be obtained, the 3D cell microspheres can be harvested by simple blowing and beating through a simple suction pipe without enzyme digestion; In addition, the substrate cells can still continue to generate the3D cell microspheres just like that continuous harvesting can be implemented by planting plants, meanwhile, collagen, sodium alginate and other materials do not need to be added in the formation of the 3D cell microspheres, so as to reduce the influence of exogenous extracellular matrixes in research.

The invention discloses a preparation method and application of an aqueous two-phase porous pancreas islet microcapsule with a core-shell structure. According to the method, based on an electronic injection microfluidic system, a large number of microcapsules with liquid cores and hydrogel shells are obtained, a pore-forming agent polyoxyethylene (PEO) solution is added into an alginic acid (ALG) solution of the shells, so that the hydrogel of the shells becomes a porous structure; and the inner phase is a carboxymethyl cellulose (CMC) solution. The microcapsule has good biocompatibility; target cells are added into the inner phase solution to obtain the cell-loaded microcapsule; and due to the internal liquid environment and the external porous gel, the three-dimensional culture of the cells can be realized, the cell survival rate can be improved, and the cell-loaded microcapsule can be transplanted to realize the cell treatment effect. The preparation method of the core-shell structure porous pancreas islet microcapsule based on microfluidic electronic injection has the advantages of reusability, low cost, simplicity in operation, convenience, reliability and the like.

The invention relates to the technical field of microfluidic cell chip, and discloses a detection chip determining cell alpha-naphthyl butyrate esterase and a reagent thereof. Concretely, a cell chip structure contains a cell microfluidic channel, a cell-capturing nest exist in a process of a cell passing through the channel, and is reacted with alpha-naphthyl butyrate esterase after capturing granulocyte, and thus intracellular enzyme activity is determined. The beneficial effects comprise that the chip for determining cell alpha-naphthyl butyrate esterase realizes that the activity of expressing alpha-naphthyl butyrate esterase in cells is realized by using a single cell, and makes up the blank of determining cell alpha-naphthyl butyrate esterase activity. The determinable alpha-naphthyl batyrate esterase sensitivity is 20 IU / cell or more, the whole determination process is finished within 1 hour, and the method is simple, rapid and stable.

The invention discloses a method for preparing cell microvesicles based on shear stress stimulation, and belongs to the technical field of bioengineering. The method comprises the following preparation steps of: taking cells, adding the cells into a culture solution, culturing, carrying out shear stress treatment after the cells grow to 90%, collecting the cells, whirling, centrifuging, taking supernatant, and centrifuging again to obtain precipitate; and putting the precipitate into phosphate buffered saline, blowing, beating, uniformly mixing, centrifuging, taking supernatant, centrifuging again to obtain secondary precipitate, adding water, carrying out hypotonic treatment, and centrifuging to obtain the cell microvesicles. According to the method for preparing the cell microvesicles based on shear stress stimulation, EVs with high content can be conveniently and quickly obtained; meanwhile, in the preparation process, chemical reagent stimulation is not needed, heterogeneous polymer particles cannot be generated, and manpower, material resources and financial resources are saved; and the prepared cell microvesicles are uniform in size, the average particle size is 231nm, and the method has popularization and application values in the field of biological materials.

The invention provides a cell micro-carrier matrix, which is prepared from the following raw materials: methacrylated gelatin and oyster shell powder, and the mass ratio of the methacrylated gelatin to the oyster shell powder is (1-10): (0.5-2). The methacrylated gelatin is high in water content, similar to an extracellular matrix in component and good in surface activity, so that the methacrylated gelatin can be well compatible with cells and active factors, the cells are wrapped in the cell micro-carrier matrix containing the methacrylated gelatin as an effective component, a three-dimensional supporting effect can be provided for the cells, and the cells can be prevented from being damaged in the injection and transplantation processes. The methacrylated gelatin and the oyster shell powder are compounded for use, so that the swelling rate of the cell micro-carrier can be effectively reduced, and the cell micro-carrier has more excellent mechanical strength.

The invention relates to a method for large-scale culture of bionic microspheres of iPSC-derived hepatocytes. The method comprises the following steps of: preparing a GelMA cell microcarrier by utilizing a microfluidic technology, coating the surface of the GelMA cell microcarrier with Bcl-2 recombinant protein so as to enhance specific anti-apoptosis activity, observing the morphology of the GelMA cell microcarrier under a microscope, combining the GelMA cell microcarrier coated with the Bcl-2 recombinant protein with the iPSC-derived hepatocytes, screening the optimal size according to cell adhesion conditions of cell microcarriers with different diameters, observing a three-dimensional structure of the cells by virtue of a light microscope and a transmission electron microscope after large-scale culture, and detecting the activity and functional expression of the iPSC-derived hepatocytes on the cell microcarriers. The method disclosed by the invention is a bionic microcarrier culture method based on the iPSC-derived hepatocytes, and the method is capable of meeting the clinical cell number, high in cell activity, excellent in biocompatibility and high in efficiency.

The present invention provides a cell-microsphere with an eluted superparamagnetic gel coating and a preparation method thereof. The cell-microsphere comprises a gel microsphere and a super-paramagnetic sphere coating the gel microsphere. Magnetic gel coating, the gel microspheres contain cells, the superparamagnetic gel coating contains superparamagnetic particles, the superparamagnetic gel coating can be removed by elution, and the superparamagnetic gel The gel coat and gel beads cannot be removed by simultaneous elution. The present invention also provides the application of the above-mentioned cell-microspheres in the co-culture of cells containing cell-gel microspheres for clinical implantation and repair and the separation in the later stage of the co-culture. The invention can solve the problems that the existing microcarrier cannot realize the removal of magnetic material and the separation of different cells in the co-culture system after the co-culture, and cannot be used for clinical implantation and repair.

The invention provides a cell-microbead pairing capture method applied to unicellular omics analysis, and the method comprises the following steps: S1, providing a microfluidic capture device which comprises a separable particle capture layer and a substrate layer, the particle capture layer is internally provided with a plurality of capture microstructures for one-to-one pairing capture of cells-microbeads; s2, introducing a cell or microbead suspension from an inlet, and gradually completing one-to-one pairing capture of a single cell and a single microbead; s3, continuously filling the controllable curing solution from the inlet, and curing the controllable curing solution according to the characteristics of the solution, so that each cell-microbead capturing unit forms mutually isolated independent cavities; and S4, separating the substrate layer from the particle capture layer to form an open cell-microbead capture unit array, and carrying out single-cell omics analysis in the next step. According to the invention, a cell-microbead pairing capture method with high throughput, high capture efficiency, low cost and low pollution is provided for unicellular omics analysis.

The invention discloses a method for constructing a high-throughput cell mass model through droplet microfluidic gravitational positioning, multi-cell micro-droplets are prepared in a high-throughput manner by using a droplet microfluidic method, and the size of a cell mass is regulated by adjusting the cell concentration. And standing in a cell culture box, settling by virtue of gravity to form a cell accumulation body, and adhering the cultured cells to form a cell cluster. And the time for primarily forming the cell mass is only 12 hours. The gel is good in biocompatibility, physical gel is formed below the gel-sol transition temperature, and the physical gel is heated to the gel-sol transition temperature and is in a liquid state. Cell mass culture is carried out below the gel-sol transformation temperature, and cell mass recovery is carried out above the gel-sol transformation temperature. The cell cluster constructed by the method has the advantages of high flux, short time consumption, good consistency and simplicity and convenience in recovery, and pollution caused by contact with air is reduced in a closed chip environment. The construction method of the cell mass model can be really applied to the fields of drug activity, substance toxicity screening and the like.

The invention discloses construction and application of a multi-cell co-cultured three-dimensional liver microsphere model, and belongs to the technical field of cell microspheres. The construction method of the multi-cell co-culture three-dimensional model comprises the following steps: preparing a mixed cell suspension from parenchymal hepatic cells, hepatic stellate cells and endothelial cells; and adding the mixed cell suspension into an ultralow adsorption pore plate, and performing culturing for 1-2 days to obtain the multi-cell co-culture three-dimensional model, wherein the number ratio of parenchymal hepatic cells to hepatic stellate cells to endothelial cells in the mixed cell suspension is 4: 1: 1, the parenchymal hepatic cell is a human liver cancer cell HepG2, the hepatic stellate cell is LX-2, and the endothelial cell is a human umbilical vein fusion cell EA.hy926. The model disclosed by the invention can be in omnibearing contact with a culture solution and other cells, and is closer to a cell environment in a human body, so that the evaluation of the three-dimensional model on hepatotoxicity or liver injury is more accurate and reliable.

The invention discloses core-shell-structured microspheres for monitoring mechanical properties and contraction frequency of myocytes as well as application thereof, and belongs to the technical fieldof biomedicine. At present, studies on cell contraction force are mainly based on two-dimensional cell models, so that in vivo environment of myocytes cannot be completely simulated. The invention provides three-dimensional microspheres with core-shell structure. Each of the three-dimensional microspheres with core-shell structure comprises a core layer and a shell layer; the core layer is a sphere formed by performing solidification on a first gel material; the shell layer is a shell composed of cells and a second gel material, and wraps the core layer; and the core layer deforms along withpulsation of the cells in the shell layer, so that, distribution of forces produced by the shell-layer cells on the core layer can be obtained by observing the deformation of the core layer. Comparedwith two-dimensional cell culture, the three-dimensional cell culture system focuses on cell-to-cell contact and cell-to-matrix contact, so that, growth environment of organisms can be better simulated; and thus, the three-dimensional microspheres with core-shell structure can be applied in disease research cell models and drug screening models.

The invention relates to a preparation method of single-cell microgel. The preparation method of the single-cell microgel comprises the following steps: processing a cross-linking agent so that the cross-linking agent can carry positive charges to prepare the cross-linking agent with the positive charges, wherein the cross-linking agent is selected from at least one of calcium carbonate nano-particles and barium carbonate nano-particles; mixing a cross-linking agent with positive charges with cells to prepare cells adsorbed with the cross-linking agent; and mixing and reacting the cells adsorbed with the cross-linking agent with an alginate solution to prepare the single-cell microgel. The preparation method of the single-cell microgel is simple and low in cost.

The invention provides a cell microcarrier matrix. The raw materials include methacrylated gelatin and oyster shell powder. According to the mass ratio, methacrylated gelatin: oyster shell powder=1-10:0.5-2. Methacrylated gelatin has high water content, similar composition to extracellular matrix, and has good surface activity, so it can be well compatible with cells and active factors. The carrier matrix wraps the cells in it, which can provide a three-dimensional support for the cells, and can also prevent the cells from being damaged during injection and transplantation. The compound use of methacrylated gelatin and oyster shell powder can effectively reduce the swelling rate of the cell microcarriers, so that the cell microcarriers have better mechanical strength.

The invention provides a preparation method of a cell microsphere of an anchorage-dependent cell, and relates to the technical field of cell microspheres. According to the preparation method providedby the invention, through temperature reduction, collision and fluid infusion, one side of a cell layer on the hole bottom can be curled, and a cell on the whole hole bottom is naturally curled to form a point so as to form the cell microsphere under the action of a tensile force of the cell layer. A metabolic capability difference of the cell microsphere and an equal-plane growing stem cell is tested through an MTT experiment, and the result shows that the cell microsphere and the plate culture cell have no obvious difference on metabolism. The preparation method provided by the invention issimple, a large number of cell microspheres can be prepared at one time, and in addition, the diameter sizes of the cell microspheres can be further adjusted according to the area of the hole bottom of a culture plate.

The present invention relates to a serum-free medium adapted to PK-15 full-suspension growth, a preparation method thereof and a full-suspension acclimation method applied to pK-15 cells. The medium contains amino acids, Tween, linoleic acid, vitamin E, Myristic acid, stearic acid, vitamin A, β‑mercaptoethanol, sodium chloride, glucose, cadmium chloride, linoleic acid, choline chloride, inositol, and ammonium metavanadate. The serum-free medium provided by the present invention can realize the full suspension growth of PK‑15 cells, thereby omitting the cost of microcarriers and the digestion process, and solving the technical problem that it is difficult to digest and enlarge the PK‑15 cell microcarrier suspension culture process.

The present invention provides a cell reactor microcarrier cell harvesting and re-inoculation amplification method, comprising step 101: filtering and collecting the microcarrier cells; step 102: removing the medium for buffer balance; step 103: trypsinization; step 104: cell Microcarrier isolation, harvesting cells only or adding next-level microcarriers to inoculate scale-up. The invention solves the problems of the collection of production-scale microcarriers, the controllable digestion of cells, the separation of the harvested microcarriers of cells, and the amplification of spheres. The method of the invention is suitable for the preparation and production of adherent cells, and is applied to the pilot scale and production of vaccines, recombinant proteins and biological enzymes, and the method is simple and economical.

The invention provides an enhanced interception device and method for perfusion culture of cells with microcarriers, wherein a layered funnel is arranged inside the device body, and the bottom of the layered funnel is a settling chamber, and a culture medium-microcarrier inlet is opened on the settling chamber, and the sedimentation The bottom of the cavity is connected with a connecting pipe, the bottom of the connecting pipe is the medium-microcarrier return port, the top of the layered funnel is the outlet of the medium, and there is an observation window at the layered funnel on one side of the device body; the advantage is that the present invention has solid-liquid settlement The function of separating and controlling the liquid level can improve the perfusion volume and interception efficiency of cell microcarrier culture, solve the problem of microcarrier loss due to not easy sedimentation when the liquid feeding speed is too high, and cause less damage to cells during the process of microcarrier retention.

The present invention discloses a lysable cell microcapsule and a preparation method thereof and a cell culture method. The lysable cell microcapsule comprises a capsule wall and cells and a culture solution wrapped by the capsule wall, the capsule wall can create an environment for long-term stable cell proliferation, and at the same time, after a certain period of time, the microcapsule is lysedto release the cells growing adherently to the wall to meet needs of cell morphology observation. The cell microcapsule is particularly suitable for cell experiments in a space environment conductedin space.

The invention relates to a cell microcapsule and a preparation method thereof, a cell microcapsule loaded with cancer resisting medicines, and a preparation method and application of the cell microcapsule loaded with cancer resisting medicines. The preparation method of the cell microcapsule comprises the following steps of (1) culturing immunocyte, and taking cell supernatant; (2) centrifuging the cell supernatant obtained in the step (1) to remove cells and cell fragments, and taking supernatant; (3) centrifuging the supernatant obtained in the step (2) to remove apoptotic bodies, and takingsupernatant, wherein the centrifuging condition includes that the centrifugal force is 2000-3000g, and the centrifuging time is 40-100min; and (4) centrifuging the supernatant obtained in the step (3), and taking precipitate. The preparation method is simple to operate, the cell microcapsule which is good in bioactivity, complete in structure, high in purity and uniform in particle diameter can be obtained; the obtained cell microcapsule can load liposoluble cancer resisting medicines through simple incubation, and the cell microcapsule is good in effects, good in targeting properties and lowin toxic and side effects when being used for preventing or treating cancer diseases.

The invention discloses cleavable cell microcapsules, a preparation method and a cell culture method thereof. The lysable cell microcapsules of the present invention include a capsule wall and cells wrapped by the capsule wall and culture fluid. The capsule wall can create an environment for long-term stable proliferation of cells. The cells grow adherently to meet the needs of cell morphology observation. The cell microcapsule of the present invention is particularly suitable for cell experiments in space environment.