314 results about "Adherent Culture" patented technology

The invention belongs to the field of allogenic mesenchymal stem cells, and particularly relates to a preparation method of allogenic mesenchymal stem cells by CRISPR (clustered regularly interspaced short palindromic repeats) technique editing and IGF (insulin-like growth factor) optimization and application of the allogenic mesenchymal stem cells in treating myocardial infarction. The preparation method comprises the following steps: carrying out separation by density gradient centrifugation to obtain allogenic single karyocytes, and carrying out adherent culture to obtain mesenchymal stem cells; designing a mesenchymal stem cell surface antigen B2M-gRNA and an inflammatory factor TNF-alpha-gRNA; establishing recombinant slow virus particles, and transfecting the mesenchymal stem cells; optimizing the mesenchymal stem cells by using IGF-1; and preparing drugs for treating myocardial infarctions by using the modified and optimized mesenchymal stem cells. The CRISPR/Cas9 technique is utilized to remove the antigens capable of causing immunological rejection and the inflammatory factors capable of causing inflammatory reaction on the mesenchymal stem cell surface, and the IGF-1 is utilized to enhance the apoptosis resistance of the mesenchymal stem cells and promote the homing of the mesenchymal stem cells, thereby providing a new technical scheme for preparing drugs for treating cardiovascular diseases in clinic. The prepared allogenic mesenchymal stem cells can not cause immunological rejection after cell transplantation.

The invention relates to the culture medium research and development technical field of modern biological technology and provides a serum-free medium for MDCK cell large-scale adherent culture and single-cell suspension culture, which comprises 21 amino acids, 6 vitamins, 8 salts, 8 lipids, 4 trace elements, 2 buffers, 1 protein hydrolysate, 1 acid-base indicator and 6 other additives. The serum-free medium can be prepared by the conventional preparation method, and an application method thereof is the conventional method. The serum-free medium has the beneficial effects that: the serum-free medium does not contain serum, has clear components, is beneficial for separating and purifying the product and improves the product quality; the serum-free medium supports long-term subculture of MDCK cells and does not require long-term and complex adaptation process; and the serum-free medium can well support the adherent growth and single-cell suspension growth of the MDCK cells, has clear components and easy preparation and utilization, and is suitable for mass production of biological products.

The invention provides BHK-21 cells obtained by high-density suspension culture in a low-serum and serum-free culture medium and a culture method thereof. The culture method comprises the following steps: 1) resuscitating frozen BHK-21 cells and performing adherent culture to obtain adherently cultured BHK-21 cells; 2) subculturing the adherently cultured BHK-21 cells in culture solution to obtain suspension-cultured BHK-21 cells and freezing the BHK-21 cells; 3) performing resuscitation culture of the suspension-cultured BHK-21 cells and performing biological property identification; and 4) performing bioreactor adaptive culture of the suspension-cultured BHK-21 cells identified to be qualified. The invention has the advantages that: a cell domesticating method provided by the invention and the obtained BHK-21 cells which can be cultured in a suspended manner save culture solution and bovine serum, are in accordance of current energy-saving, environment-protection and low-carbon concepts for the application of suspended-cultured cells into actual production saves floor area and labor investment greatly, and create considerable economic benefit and social benefit.

The invention discloses a method for differentiating human neural stem cells into dopaminergic neuron by in-vitro directional induction. The method comprises the following steps of adherent culture of human neural stem cells, induction of dopaminergic neuron precursor cells, directional induction of dopaminergic neuron and cryopreservation and anabiosis of the human neural stem cells and the dopaminergic neuron precursor cells. By virtue of the steps of the method disclosed by the invention, abundant dopaminergic neuron can be obtained in vitro; compared with the plurality of dopaminergic neuron production ways at present, the method disclosed by the invention has characteristics of low cost, high safety, high production efficiency and strong operability. The in-vitro dopaminergic neuron production way can provide a novel idea for applying dopaminergic neuron to clinical transplant treatment of the Parkinson disease.

The invention belongs to the field of microalgae culture, and particularly relates to an inserting-plate type microalgae semi-dry solid adherent culture device. The inserting-plate type microalgae semi-dry solid adherent culture device is characterized by comprising a supporting truss, culture plates, liquid distributing pipes, liquid collecting tanks and a liquid storage bottle, wherein the supporting truss is provided with at least one culture plate, and one liquid distributing pipe is arranged above each culture plate; the liquid collecting tank arranged on the supporting truss is arranged under each culture plate; all the liquid collecting tanks are communicated with the liquid storage bottle by the main liquid pipe; the bottom of the liquid storage bottle is respectively provided with a circulating pump and a carbon-supplementing gas distributing pipe communicated with a carbon dioxide source, and the circulating pump is communicated with a liquid distributing pipe by a pipeline; and each culture plate is provided with a picking scraper. The inserting-plate type microalgae semi-dry solid adherent culture device has the advantages that the yield in unit occupied area is effectively increased, the culture period is shortened, the water consumption for culture is greatly reduced, the cost and the operating cost of the culture device are reduced and the industrial culture on large scale is easily realized.

The invention provides a separation method for human amniotic mesenchymal stem cells (hAMSCs) in a process of cultivating the hAMSCs through enzyme digestion and epidermal growth factor (EGF). The cultivating process comprises the steps of separation of hAMSCs, primary culture of hAMSCs, hAMSCs subculturing and amplification, hAMSCs cryopreservation and recovery, and hAMSCs immunophenotyping detection. According to the method disclosed by the invention, step-by-step digestion is implemented for multiple times through trypsin and collagenase to collect amnion cells, and adherent culture and digestion time control are combined through adding of 4ng/ml of EGF (Epidermal Growth Factor), so as to obtain human amniotic mesenchymal stem cells (hAMSCs) which are relatively high in both purity and activity. The method disclosed by the invention, which combines enzyme digestion and EGF, has the advantages of being simple to operate, rapid, practical and the like, being an effective method for separating and purifying hAMSCs.

The invention discloses a serum-free medium suitable for large-scale single-cell suspension culture of young hamster kidney cells (BHK cells), including 21 kinds of amino acids, 11 kinds of inorganic salts, 12 kinds of vitamins, 1 kind of protein hydrolyzate, 2 kinds of Lipids, 2 buffer components and 6 additives. The BHK cell serum-free medium of the present invention produces biological products by single-cell suspension culture, which can not only avoid various disadvantages of serum culture, but also eliminate the problem of difficult scale-up of roller bottle adherent culture, thereby improving the production efficiency of BHK cell culture And reduce production costs, while ensuring product quality, has good application value and huge market prospects.

The invention provides a method for culturing human umbilical cord mesenchymal stem cells through combination of an adherence density gradient method and as EGF (Epidermal Growth Factor). The method comprises the following steps of: seperation of the UC-MSCs (Umbilical Cord Mesenchymal Stem Cells), primary culture of the UC-MSCs, subculture and augmentation of the UC-MSCs, cryopreservation and resuscitation of the UC-MSCs and detection of UC-MSCs immunophenotyping. According to the method, 4ng/ml of EGF is added by adopting the adherence density gradient method, adherent culture and digestion time control are combined, so that the UC-MSCs with higher purity and activity is obtained; and the method combining the adherence density gradient method and the EGF has the advantages of simple operation, rapidness, practicability and the like and is an effective method for separating and purifying the UC-MSCs.

The invention discloses a method for separating MVs (microvesicles) and MV exosomes from a tumor cell supernatant. The method comprises the following steps: after adherent culture of tumor cells, a culture solution is taken and subjected to centrifugation, the supernatant is mixed with a coupling compound, the mixture is incubated at 4 DEG C under the 100-300-rpm horizontal vibration condition for 3-16 h, a liquid is removed, an incubated coupling compound is obtained and cleaned with PBS (phosphate buffered saline), and coupling microspheres with MVs adsorbed are obtained. The total MVs extracted with the method retain the structures and components of all the MVs, and the yield is high. Secondary sorting can be performed, specific MVs can be analyzed, and the extraction efficiency and the research value are increased. The total extraction time requires only at least 4 hours to finish purification of the MVs and requires only at least 5 hours to finish purification of the exosomes. The extraction purity is higher, and experiments show that the extracted MVs account for 90% or higher of the total extracted proteins while organic precipitate accounts for lower than 20%.

The invention discloses an identification and preparation method and application CD106-positive mesenchymal stem cells. The method comprises the following steps: identifying and isolating CD106-positive cells from mononuclear cells isolated from bone marrow, placenta or other tissues through an immunological method, carrying out adherent culture, and collecting adherent CD106-positive cells; or carrying out adherent culture of mononuclear cells isolated from bone marrow, placenta or other tissues, collecting adherent mesenchymal cells, and isolating the CD106-positive mesenchymal stem cells when the count of the obtained cells is large enough. The CD106-positive cells are mesenchymal stem cells with adherent growth, express mesenchymal stem cell-related immunological phenotype, and have osteogenesis, adipogenesis and other differentiation potencies. Compared with CD106-negative mesenchymal stem cells, the CD106-positive cells have higher immunosuppression capacity, can better inhibit proliferation of IFN-gamma (interferon-gamma) secretion of lymphocytes, and can be used for treating graft versus host disease (GVHD) and autoimmune diseases.

The invention provides a domestication method of a full suspension culture type Marc-145 cell line. The domestication method comprises the following steps: (1) cultivating adherent-culture Marc-145 cells; (2) domesticating a low-serum adherent-culture Marc-145 cell line; (3) domesticating low-serum full suspension culture type Marc-145 cells; and (4) domesticating serum-free full suspension culture type Marc-145 cells. With the application of the cell domestication method disclosed by the invention, the Marc-145 cells of suspension culture can be obtained; the cells are adaptive to low-serum and serum-free full suspension culture; the density of the cells, when subjected to serum-free culture in a 7.5L reactor, can reach 8*10<6> /ml, and at least 4.4*10<10> cells can be obtained, equal to the number of cells obtained from 80-90 15L roller bottles or 18-20 40-layer cell plants; and the domestication method disclosed by the invention is significant in effect.

The invention relates to the field of cell engineering, and discloses a method for culturing mesenchymal stem cell in vitro. The method comprises the following steps: separating from marrow, umbilical cord blood or umbilical cord through a gradient density centrifugation by using 1.073g/cm<3> of Percoll separation medium to obtain a mononuclear cell, inoculating the mononuclear cell to alpha-MEM nutrient solution containing fetal calf serum with volume percentage of 10%, and performing adherent culture under 5% of CO2 concentration at 37 DEG C until the anchorage-dependent cell is appeared and is in fibroblast to obtain the mesenchymal stem cell. The culture method provided by the invention is low in cost and simple for configuration without adding the components such as equine serum, I-inositol, I-glutamine, mercaptoethanl and the like, the cell activity is higher, the required period of cell expansion is shortened; and meanwhile, the method is superior to the existing Dexter-LTC culture system, and is capable of providing high-activity cell source for the corresponding therapy and research.

The invention relates to an autologous adipose-derived mesenchymal stem cell culture method and a used culture medium. Specifically, the culture method disclosed by the invention comprises the following steps of culture pretreatment, adherent culture, fluid replacement and fluid change, one time or optional multiple times of cell passage, optionally preparing an adipose-derived mesenchymal stem cell preparation and optionally performing cell cryopreservation and revival on the obtained adipose-derived mesenchymal stem cells. The the culture medium disclosed by the invention comprises DMEM and FBS. When the culture medium disclosed by the invention is utilized to culture the autologous adipose-derived mesenchymal stem cells, especially the autologous adipose-derived mesenchymal stem cells of eye adipose cells, an excellent technological effect of the culture method disclosed by the invention can be presented; the obtained autologous adipose-derived mesenchymal stem cells can be applied to autologous application; for example, the autologous adipose-derived mesenchymal stem cells obtained from eye adipose can be applied to face injection such as injection filling at parts of face wrinkles, temple and lacrimal sulcus.

The invention relates to a serum-free medium used for culturing Vero cells. The serum-free medium is composed of, by volume, 85 to 95% of a base culture solution, 2 to 5% of an amino acid solution, 2 to 7% of a serum alternative factor solution, 0.1 to 0.5% of an antioxidant solution, 0.8 to 2% of a yeast extract solution, and 0.1 to 0.5% of an ethanol amine solution. The invention also provides a preparation method of the serum-free medium. In the preparation method, the base culture solution, the amino acid solution, the serum alternative factor solution, the antioxidant solution, the yeast extract solution, and the ethanol amine solution are mixed at the above ratio so as to obtain the serum-free medium. The serum-free medium used for culturing Vero cells is capable of promoting rapid attachment of Vero cells; cell morphology of the Vero cells cultured with the serum-free medium can be maintainer preferably, and cell proliferation rate is higher; the composition of the serum-free medium is simple; cost is reduced; and preparation is convenient.

The invention relates to an ovary cancer specific tumor antigen peptide and preparation method thereof. The preparation method comprises the following steps: carrying out an adherent culture of cell HTB161A2 with a supplemental medium, and collecting a supernatant; collecting 0.5ml magnetic beads and washing with a coating buffer; suspending the magnetic beads again, filtering and eluting; collecting eluted substances in a centrifuge tube, and collecting a filtrate, and the filtrate is a solution of the antigen peptide. The method provided by the invention is a direct biochemical method, that is using an immuno-affinity purification method for purifying MHC/polypeptide compound molecules from tumor cells or tissues, and eluting the antigen peptides combined with MHC from the MHC/polypeptide compound molecules. These antigen peptides can be used for antibody treatment potentially, or producing ovary cancer specific CTLs, or preparing tumor vaccine; the invention also be developed for producing diagnosis kit.

The construction of human corneal endothelium cell line with corneal endothelium as material includes the steps of: the first adherent culture of human corneal endothelium cell inside DMEM/F12 culture liquid with ox embryo serum in 20 wt%, chondroitin sulfate oxidizing and degrading matter, epidermal cell growth factor, basic fibroblast growth factor and ox eye auxin to obtain pure corneal endothelium cell; and the subsequent secondary culture in trypsinization process. The technological process is scientific and reasonable, and up to now, passage cell of 106-th generation has been obtained. The human corneal endothelium cell line of the present invention has no any virus or cancer genetic transfection, has no any tumorigenicity, and is expected to be used in direct artificial cornea production and clinical application.

The invention relates to a method for inducing a human pluripotent stem cell to differentiate into an RPE cell. According to the method, an inhibitor is used for inducing the human pluripotent stem cell to differentiate into a neural precursor; after Shh, IGF1 and EGF growth factors are utilized to activate ERK 1/2, JNK, Sonic hedgehog signal routes, the cell is presented as the shape of neuroepithelium, Activen A and Chir99021 are added to activate TGFbeta and Wnt signal routes respectively to induce a neuro-epithelial cell into a retinal progenitor cell; adherent culture is conducted on theretinal progenitor cell to obtain an RPE cell at an underdeveloped state. The invention further studies the field of applying the underdeveloped RPE cell to treating retinal degenerative diseases. Compared with the prior art, the method firstly makes three stages of differentiating the human pluripotent stem cell to the underdeveloped RPE cell and related small molecular substances and growth factors used therein clear. Compared with a matured RPE, the underdeveloped RPE obtained through differentiation can treat the retinal degenerative diseases more effectively after being transplanted.

The invention relates to a canine stem cell isolated culture technology, and especially relates to a method for in vitro simultaneous insolated culture of canine bone marrow mesenchymal stem cells and multifunctional hematopoietic stem cells. The method is characterized in that adherent culture of primary mesenchymal stem cells is carried out based on the separation of bone marrow mononuclear cells, half medium change is adopted, and a mononuclear cell culture suspension is added, that is, hematopoiesis microenvironment provided by the adherent-growth mesenchymal stem cells is used to culture the suspending-growth multifunctional hematopoietic stem cells, so the achieve the simultaneous isolated culture of the canine bone marrow mesenchymal stem cells and the multifunctional hematopoietic stem cells is realized.

The invention discloses a method for preparing a heat-resisting attenuated virus live vaccine for goatpox by using a BHK21-C13 passage cell. Based on the existing attenuated virus AV41 for goatpox, which has excellent immunogenicity, the method comprises the following steps: culturing 25-28th generations of virus solution with a heterologous BHK21 cell, and then adding an appropriate heat-resisting freeze-drying protective additive to obtain the heat-resisting attenuated virus live vaccine for goatpox. The passage cell vaccine used by the method is superior to the primary cell vaccine in the aspects of virus yield, homogeneity, purity and the like, not only guarantees the effective level of the vaccine and has a better heat-resisting protection effect, but also can ensure the safety of a purebred pregnant goat. As the existing BHK21 cell adopts a mature suspension culture process, the exploration of various parameters of adherent culture lays a solid foundation for the next promotion of suspension process.