57 results about "Endothelial cell culture" patented technology

A laminate comprising a transparent collagen I type sheet and a human corneal endothelial cell culture layer provided on the sheet. An endothelial cell culture layer laminate usable in transplantation is provided.

The invention relates to a preparation method of a mechanically-enhanced cell-loaded microchannel hydrogel, which comprises the following steps: preparing a mixed solution of hydrogel sol and cell culture fluid, and pouring the sol-cell mixed liquid into a polymethyl methacrylate die into which capillary tubes or fibers are inserted, thereby enabling primary crosslinking; drawing out the capillary tubes or fibers, introducing the hydrogel sol into microchannels of the cell-loaded hydrogel, and reinserting the capillary tubes or fibers, or sucking excess hydrogel sol out of the microchannels and carrying out ultraviolet irradiation, thereby enabling secondary crosslinking, so that the cell-loaded hydrogel with thick hydrogel layer is formed in the wall regions of the microchannels; and drawing out the capillary tubes or fibers, introducing endothelial cell culture fluid into the microchannels of the cell-loaded hydrogel, and culturing so that the endothelial cells grow and spread to form endothelial microchannels. The introduction of the thick hydrogel layer can maintain the stability of the microchannels in the hydrogel, and prevent shedding and intrusion of the endothelial cells;and the culture fluid in the microchannels can be filtered and screened.

The invention provides a human vascular endothelial cell culture solution. The human vascular endothelial cell culture solution is prepared from a basic culture medium, fetal calf serum, human recombined epidermal growth factors, human recombined fibroblast growth factors, vascular endothelial growth factors, insulin-like growth factors, vitamin C, cortisol, cholera toxin, aminoethanol and ethanolamine phosphate. Compared with a traditional vascular endothelial cell culture medium, the human vascular endothelial cell culture solution has the advantages that the culture solution can be suitable for multiple kinds of vascular endothelial cells, cell proliferation is effectively promoted, the division time is effectively shortened, the characteristics of the vascular endothelial cells are maintained, the passage number of the in-vitro human vascular endothelial cells is greatly increased, and the survival time of the in-vitro human vascular endothelial cells is greatly prolonged; meanwhile, all the ingredients of the cell culture solution can be obtained in a general laboratory, preparation is convenient, a special finished product culture medium is not needed, therefore, the cost for culturing the endothelial cells is greatly reduced, and the human vascular endothelial cell culture solution is suitable for application and popularization.

The invention provides an endothelial cell culture medium and an endothelial cell culture method, wherein the endothelial cell culture medium comprises a serum-free basal culture medium, insulin, transferin, vitamin C, bovine serum albumin, a basic fibroblast growth factor, a blood vessel endothelium growth factor, a stem cell growth factor, laminin, an epidermal growth factor, non-essential amino acid and melbinum hydrochloride. By virtue of compatibility of ingredients, the endothelial cell culture medium provided by the invention is capable of improving the endothelial cell multiplication capacity. In addition, the endothelial cell culture medium provided by the invention is further capable of improving the blood vessel formation capability of endothelial cells. The experimental result indicates that when endothelial cells are cultured to the P10 generation by virtue of the endothelial cell culture medium provided by the invention, the cells still grow vigorously; the P10-generation endothelial cell phenotype CD309 and CD31 double-positive expression is as high as 97.2%.

The invention relates to a human corneal endothelial cell culture solution as well as a preparation method and an application thereof. The solution comprises bovine corneal endothelial cell lysis solution with 10-200 mug / ml of protein as the main active component, fetal calf serum with the content being 10%, 100U / ml of penicillin, 100U / ml of streptomycin and the remainder being supplemented by D / F12 basal culture solution. The method comprises of: firstly culturing bovine corneal endothelial cell in vitro; preparing bovine corneal endothelial cell lysis solution and formulating in a disinfection chamber to obtain the product. The invention, for the first time, prepares bovine corneal endothelial cell lysis solution into cell culture solution and applies to in vitro culture of human corneal endothelial cells, can promote great augmentation of human corneal endothelial cells cultured in vitro, maintain form feature of corneal endothelial cells, inhibit cells from ageing and apoptosis, and facilitate the subculture of endothelial cells; wherein, the raw material bovine corneal endothelial cell has abundant source and is easy for in vitro culture, the preparation method of the culture solution is simple, easy to implement, conducive for industrialization, and has wide application and development prospect.

The invention discloses a method for promoting directional differentiation of human multipotent stem cells into endothelial cells. The method includes: from day 0 to day 1, performing induced differentiation on human multipotent stem cells with cell density reaching 95% or above by use of a CDM3 differential medium containing 4-8micron of GSK3I; on the second day, performing further induced differentiation on the cells subjected to induced culture in the last step by use of a CDM3 differential medium containing 40-60ng / ml of bFGF; from day 3 to day 5, performing further induced differentiationon the cells subjected to induced culture in the last step by use of a CDM3 differential medium containing 40-60ng / ml of VEGF and 20-30ng / ml of BMP4; on the sixth day, digesting the cells subjected to induced culture in the last step, and adopting an endothelial cell culture medium to continue culture for 3-4 days. The method is economical and effective, and convenience and quickness in a differentiation process are achieved; serum consumption is avoided in a differentiation process, interferences of various factors are eliminated, and differentiation results are high in reliability; by adding of RA, endothelial differentiation efficiency can be promoted, and more endothelial cells can be obtained in once differentiation.

The invention discloses a vascular endothelial cell culture method. The method comprises the following steps: 1) differentiating pluripotent stem cells into mesendoderm precursor cells in a culture medium A; 2) differentiating the mesendoderm precursor cells into progenitor cells of vascular endothelial cells in a culture medium B; 3) differentiating the progenitor cells of the vascular endothelial cells into the vascular endothelial cells in a culture medium C. The culture medium A contains a DMEM, an F12 culture medium, sodium selenate, sodium bicarbonate, vitamin C, insulin, an activin A , bone morphogenetic protein-4 and a glycogen synthase kinase-3 inhibitor; the culture medium B contains a DMEM, an F12 culture medium, sodium selenate, sodium bicarbonate, vitamin C, insulin, a vascular endothelial growth factor and a transforming growth factor beta signaling pathway inhibitor; the culture medium C contains a DMEM, an F12 culture medium, sodium selenate, sodium bicarbonate, vitamin C, insulin, a vascular endothelial growth factor, an epidermal growth factor and a fibroblast growth factor. According to the method, the pluripotent stem cells can be differentiated into the vascular endothelial cells.

Provided herein relates to devices for simulating a function of a tissue and methods of using the same. In some embodiments, the devices can be used to simulate a function of a human liver tissue. In some embodiments, the devices can be used to simulate a function of a dog liver tissue. Endothelial cell culture media for long-term culture of endothelial cells are also described herein.

The invention provides a method of inhibiting neovascularization in a subject. The method comprises administering to the subject an agent that interferes with fibronectin (Fn) matrix assembly in an amount effective to inhibit neovascularization. The invention also provides a method of identifying an agent that inhibits neovascularization. The method comprises detecting fibronectin (Fn) matrix assembly by stimulated endothelial cells cultured in three-dimensional culture gel in the presence and absence of an agent. A decrease in Fn matrix assembly in the presence of the agent compared to Fn matrix assembly in the absence of the agent is indicative of an agent that inhibits neovascularization. Alternatively, the method of identifying an agent that inhibits neovascularization comprises detecting changes in nuclear architecture in stimulated endothelial cells cultured in three-dimensional culture gel in the presence and absence of an agent. A reduction in nuclear architecture organization identifies an agent that inhibits neovascularization.

The invention provides a method for constructing a milk cow blood milk barrier three-dimensional model in vitro. The method comprises the steps of cultivation of mammary epithelial cells and fibroblast of a milk cow, cultivation of venous endothelial cells of the milk cow, immunofluorescent identification of the mammary epithelial cells and the venous endothelial cells, cocultivation of Transwell,preparation of a cocultivation medium by taking prolactin, insulin, an epidermal growth factor, cow growth hormone, a vascular endothelial growth factor and a fibroblast growth factor as hormone andcell factors related to growth of the mammary epithelial cells, the fibroblast and the endothelial cells; and finally the epithelial cells, the fibroblast and the endothelial cells are inoculated on atranswell chamber, and accordingly the milk cow blood milk barrier model is successfully constructed. The method can be used for researching a molecular mechanism by which nutrient substances, drugsand toxic substances enter the mammary tissue and the milk in real-time and effectively, not only fills in the gap both domestically and internationally, but also provides a research platform for research on the molecular mechanism by which lipid, vitamins, the drugs and the toxic substances pass through the blood milk barrier.

The invention provides a method for evaluating lung injury caused by nanoparticles based on organ-on-a-chip technology. A chip is mainly prepared by bonding and sealing an upper PDMS layer and a lowerPDMS layer and comprises a matrix inlet pool, two cell inlet pools, a waste liquid pool, a cell culture chamber and a matrix chamber. The chip is vertically inoculated with vascular endothelial cellsand alveolar epithelial cells separately, and culture is carried out for a certain period of time so as to form a lung air-blood barrier. Nanometers are added into an alveolar epithelial cell culturechamber, and monocytes are added into a vascular endothelial cell culture chamber in the manner of perfusion to simulate monocytes in the circulating blood of people, so an in-vitro nanoparticle lunginjury evaluation model is established. The nanoparticle lung injury evaluation model based on the chip can simulate the injury of the nanoparticles to the lung air-blood barrier, monitors destroy tothe integrity of the barrier in real time, realizes observation of the responses of two types of cells of the air-blood barrier, and traces the motion of the monocytes in real time.

The invention relates to an aged rat brain vascular endothelial cell culture fluid. On the basis of each 100 ml of DMEM (Dulbecco Modified Eagle Medium) culture medium, components including plasma derived serum (PDS), penicillin-streptomycin, L-glutamine, gentamicin, heparin, an alkaline fiber growth factor, an endothelial cell growth replenishing factor, vitamin C, a vascular endothelial growth factor, an insulin-like growth factor-1 and an endothelium growth factor are added. According to the aged rat brain vascular endothelial cell culture fluid, the PDS is adopted for replacing FBS (Fetal Bovine Serum), beef calf serum or horse serum in the traditional culture fluid to ensure that the purity of brain vascular endothelial cells is increased by 30 percent; and the vitamin C and puromycin are added to ensure that the brain vascular endothelial cells grow more rapidly and purely.

Disclosed herein is an RGD-enriched solubilized extracellular matrix composition derived from endothelial cell culture that can be used to modify the immunogenicity or thrombogenicity of an organ intended for transplant. The RGD-enriched solubilized extracellular matrix composition is applied to the lumen of the vasculature of the organ, thereby placing a barrier between the antigens on the lumenal surfaces of the transplanted organ and the blood of the recipient.

The invention relates to a human glomerular endothelial cell culture and drug detection toxicity evaluation method based on a micro-fluidic chip, and the specific culture method comprises the following steps: carrying out cell culture and test by using a micro-fluidic cell chip analyzer, and specifically comprises the following steps: 1, preprocessing the chip; step 2, cell inoculation; step 3, cell culture; after cell culture is completed, successfully cultured cells are used for subsequent drug evaluation detection, and the method specifically comprises the following steps: step 1, preparation for detection; 2, detecting the concentration of LDH; step 3, activity detection; in addition, living and dead cell staining, immunofluorescence detection and inflammatory cytokine detection can also be used, the temperature and gas components of a cell growth area can be accurately regulated and controlled by implementing the steps, an external incubator is omitted, long-term continuous observation of cells under a microscope is achieved, and the detection accuracy is improved. The microfluidic cell model can better simulate the human body microenvironment, and the drug evaluation effect is more accurate.

The invention provides a separation, purification and culture method of rat cornea endothelial cells, and belongs to the field of cell biology. The method comprises the following steps: 1, separating a cornea tissue sample to obtain single cells; 2, preparing immunomagnetic beads for specifically adsorbing cornea endothelial cells, and purifying the cornea endothelial cells from the separated single cells; and 3, culturing the rat cornea endothelial cells with an optimized cell culture liquid. The separation process of the rat cornea endothelial cells has the advantages of simplicity in operation, high cell output and high cell survival rate, the purification process of the rat cornea endothelial cells has the advantages of simplicity in operation, very high yield and very high purification rate, and the culture process of the rat cornea endothelial cells has the advantages of fast cell proliferation, short period and high economic benefit.

The invention relates to a preparation method of a cell-loaded microchannel hydrogel based on sucrose fiber template, which comprises the following steps: preparing sucrose fibers, coating a polymer film on the surface of the sucrose fibers, and putting the sucrose fibers with the polymer film in the middle of a polymethyl methacrylate die; preparing a mixed solution of hydrogel sol and cell culture fluid, and pouring the sol-cell mixed liquid into the polymethyl methacrylate die; changing the temperature or carrying out ultraviolet irradiation so that the hydrogel sol is crosslinked to form a hydrogel; dissolving the sucrose fibers, sucrose powder or polyvinylpyrrolidone out of the hydrogel in a phosphate buffer solution to form a cell-loaded microchannel hydrogel; and introducing endothelial cell culture fluid into microchannels of the cell-loaded microchannel hydrogel, and culturing so that the endothelial cells grow and spread to form endothelial microchannels. The cell-loaded microchannel hydrogel prepared by the method has stable microchannel structure, the endothelial cells can not easily shed, and the cell-loaded microchannel hydrogel can wrap the cells in the hydrogel crosslinking process; and the endothelial cell culture liquid is implanted after the microchannels are formed, so as to form the endothelial microchannels.

The invention belongs to the technical field of biology, and particularly relates to endothelial cell culture solution. The endothelial cell culture solution comprises a basic culture medium DMEM (dulbecco modified eagle medium), human platelet lysate, L-glutamine, vitamin C, laminin 10, laminin 11, fibronectin, penicillin and streptomycin. The endothelial cell culture solution has the advantages that a pH (potential of hydrogen) value of the endothelial cell culture solution is 6.8-7.0, and the osmotic pressure of the endothelial cell culture solution is 310 mOsm / kg; endothelial cells cultured by the endothelial cell culture solution are high in adherence and proliferation speed, purity, migration capacity and lumen formation rate, invasion effects of the endothelial cells can be inhibited, and accordingly the endothelial cell culture solution is a perfect endothelial cell culture system.

Methods of promoting liver morphogenesis prior to the functioning of blood vessels by culturing liver cells with endothelial cells is provided. Also provided are cell cultures and method of promoting vasculogenesis of liver tissue by contacting liver cells with endothelial cells.

The invention provides a method for in-vitro construction of vascular endodermis with flow shear stress resistant and platelet aggregation resistant functions. According to the method, flow shear stress increased gradually is slowly applied to vascular endothelial cells on the surface of an artificial blood vessel inner cavity so that the vascular endothelial cells gradually adapt to the shear force environment. The method comprises the following steps of connecting an artificial blood vessel with vascular endothelial cells growing on the surface of an inner cavity to a pipe of a shear stress inducible system, starting a liquid driving pump of the shear stress inducible system so that an initial shear force of 0-2dyne / cm<2> is applied to the vascular endothelial cells, adjusting the liquid driving pump every once in a while so that the shear stress on the vascular endothelial cells is improved by a certain ratio than shear stress in the previous stage until the shear stress on the vascular endothelial cells is equal to average or peak shear stress resisted by the arterial endothelial cells in a transplantation site, keeping the shear stress in a level of the average or peak shear stress resisted by the arterial endothelial cells in the transplantation site and carrying out endothelial cell culture for 24-72h.

The invention relates to the technical field of drug experiments, and provides a bionic intestinal liver microfluidic cell culture-drug screening integrated chip which at least comprises a core plate layer, a bottom plate layer and a top plate layer. The core plate layer comprises a bionic intestinal cell region and a bionic hepatocyte region which do not communicate with each other and are separately arranged on two sides of the chip; the bionic intestinal cell region comprises an intestinal cell culture chamber and a vascular endothelial cell culture chamber; the extending directions of the intestinal cell culture chamber and the vascular endothelial cell culture chamber are on the same axis, and the vascular endothelial cell culture chamber is positioned on the outer side of the intestinal cell culture chamber; a bending structure is arranged between the intestinal cell culture chamber and the vascular endothelial cell culture chamber; and the bionic hepatocyte region comprises a plurality of hepatocyte culture chambers which are arranged in a surrounding manner in the circumferential direction, and bionic capillary channels which are arranged between the adjacent hepatocyte culture chambers. According to the scheme provided by the invention, more accurate metabolism data of the organ cells on the medicine can be obtained.

The present disclosure provides methods for forming stable three-dimensional vascular structures, such as blood vessels and uses thereof. More specifically, the present disclosure provides methods for culturing differentiated endothelial cells that include an exogenous nucleic acid encoding ETV2 transcription factor on a matrix under conditions that express exogenous ETV2 protein in the endothelial cell to form stable three-dimensional artificial blood vessels without the use of a scaffold, pericytes or perfusion. The present disclosure also provides stable three-dimensional blood vessels that are capable of autonomously forming a functional three-dimensional vascular network, and uses thereof. In addition, the present disclosure includes methods for vascularizing an organoid and a decellularized organ by culturing the organoid or decellularized organ with endothelial cells that include an exogenous nucleic acid encoding ETV2 transcription factor under conditions that express exogenous ETV2 protein in the endothelial cell to vascularize the organoid or decellularized organ.

The invention provides an endothelial cell culture medium and an endothelial cell culture method, wherein the endothelial cell culture medium comprises a serum-free basal culture medium, insulin, transferin, vitamin C, bovine serum albumin, a basic fibroblast growth factor, a blood vessel endothelium growth factor, a stem cell growth factor, laminin, an epidermal growth factor, non-essential amino acid and melbinum hydrochloride. By virtue of compatibility of ingredients, the endothelial cell culture medium provided by the invention is capable of improving the endothelial cell multiplication capacity. In addition, the endothelial cell culture medium provided by the invention is further capable of improving the blood vessel formation capability of endothelial cells. The experimental result indicates that when endothelial cells are cultured to the P10 generation by virtue of the endothelial cell culture medium provided by the invention, the cells still grow vigorously; the P10-generation endothelial cell phenotype CD309 and CD31 double-positive expression is as high as 97.2%.

The invention discloses a method for separating and cultivating endothelial cells of common carotid arteries of SD (Sprague Dawley) rats. The method includes steps of 1), incising the common carotid arteries of the SD rats under sterile conditions; 2), cutting and segmenting the common carotid arteries, then stripping outer membranes from the common carotid arteries in PBS (poly butylenes succinate) liquor, longitudinally cutting the common carotid arteries, downwardly flatly spreading inner membranes in containers filled with digestive enzyme liquor and enabling the inner membranes of the common carotid arteries to be in sufficient contact with the digestive enzyme liquor in the containers; 3), vibrating the containers to allow the endothelial cells to fall off, removing the common carotid arteries after digestion is completed, adding fetal bovine serum into the containers and uniformly stirring the fetal bovine serum to obtain mixed liquid; 4), centrifuging the mixed liquid, removing supernatants to obtain the endothelial cells of the common carotid arteries, cultivating the endothelial cells of the common carotid arteries in endothelial cell cultivation media, transferring the endothelial cells to cultivation tanks to cultivate the endothelial cells after the endothelial cells reach log phases and then carrying out generation cultivation on the endothelial cells. The method has the advantages of high survival rate, little cell injury, economic efficiency, practicality, simplicity, feasibility and applicability to mass production in factories and the like.

The present disclosure provides a method of deriving endothelial cells, comprising (a) culturing lateral plate mesoderm cells under oxygen-deprived condition to obtain endothelial lineage cells; and (b) culturing endothelial cells from (a) on an extracellular matrix to maintain and expand the endothelial lineage cells. Also disclosed herein is a cell co-culture system comprising an endothelial cell culture and a hepatocyte cell culture, as well as a microfluidic-based system comprising said cell co-culture system. Also disclosed herein is a method of disease modelling or drug testing using said cell co-culture system or said microfluidic-based system.

The invention provides a primary mouse thoracic aortic endothelial cell separation and culture method, which solves the problems of great difficulty in primary VECs (vascular endothelial cell) in-vitro culture, low cell activity and little output in the prior art. The method comprises: (Step 1) a mouse thoracic aortic section is obtained, and after separation and purification, the mouse thoracic aortic section is produced into an aortic ring; (Step 2) a matrigel is used for pretreating the bottom of a culture vessel; (Step 3) the aortic ring is inoculated to the surface of the matrigel, an endothelial cell culture medium which contains an endothelial cell growth-assisting factor and heparin is then added into the culture vessel for culture; (Step 4) after 3 to 7 days of culture, the aortic ring is removed, and culture is continued. The method is easy and convenient to implement, economical and applicable, and has the advantages of decreasing the probability of impurity cell contamination, increasing the survival rate and output of cells, enabling VECs to have high activity and high multiplication rate, etc..

The invention provides a human vascular endothelial cell culture solution. The human vascular endothelial cell culture solution is prepared from a basic culture medium, fetal calf serum, human recombined epidermal growth factors, human recombined fibroblast growth factors, vascular endothelial growth factors, insulin-like growth factors, vitamin C, cortisol, cholera toxin, aminoethanol and ethanolamine phosphate. Compared with a traditional vascular endothelial cell culture medium, the human vascular endothelial cell culture solution has the advantages that the culture solution can be suitable for multiple kinds of vascular endothelial cells, cell proliferation is effectively promoted, the division time is effectively shortened, the characteristics of the vascular endothelial cells are maintained, the passage number of the in-vitro human vascular endothelial cells is greatly increased, and the survival time of the in-vitro human vascular endothelial cells is greatly prolonged; meanwhile, all the ingredients of the cell culture solution can be obtained in a general laboratory, preparation is convenient, a special finished product culture medium is not needed, therefore, the cost for culturing the endothelial cells is greatly reduced, and the human vascular endothelial cell culture solution is suitable for application and popularization.

The invention discloses a method for screening medicaments injuring endothelial cells by active nitrogen. Exogenous active nitrogen inducers are added to endothelial cells cultured in vitro in a certain concentration, and after reacting for a certain time, the active nitrogen inducers injure the endothelial cells so as to establish a model of injuring the endothelial cells by active nitrogen. By utilizing the cell model, different medicaments are added, and medicaments which can lighten the injury of the active nitrogen to the endothelial cells are screened in vitro by observing generating conditions of corresponding ROS and 3-NT and using a cell survival rate as a target spot. The invention avoids the blindness of the integral level screening of the cells, reduces the workload, has specific medicament action mechanism and the advantages of immediacy, rapidness and convenience and is suitable for mass screening of heart cerebrovascular candidate medicaments.