164 results about "Serum free medium" patented technology

The invention discloses a serum-free medium for culturing placenta mesenchymal stem cells. The serum-free medium takes a DMEM (Dulbecco Modified Eagle Medium) culture solution as a basis and also contains a fibroblast growth factor receptor 2, growth hormone, insulin, transferrin, glutathione, BMP-4, L-glutamine, sodium pyruvate, non-essential amino acids and beta-mercaptoethanol. According to various serum-free media provided by the invention, growth and proliferation of the placenta mesenchymal stem cells in a serum-free medium system can be effectively promoted, the placenta mesenchymal stem cells have higher growth and proliferation rate in the serum-free medium system compared with a serum cell culture medium, the characteristics of the stem cells are preserved, the serum-free medium has multiple differential potentials, and the stem cells can be directionally induced into fat cells and osteoblasts.

A method for transferring a gene into target cells by a retrovirus with the use of serum-free medium. This method comprises infecting target cells with a retrovirus in serum-free medium optionally containing low-density lipoprotein and/or cytokines in the presence of a functional substance such as fibronectin in an amount effective in elevating the gene transfer efficiency of the retrovirus into the target cells by co-localizing the retrovirus and the target cells.

The invention discloses a serum-free medium for full suspension cultivation of a BHK (Baby Hamster Kidney)-21 cell. The serum-free medium for full suspension cultivation of the BHK-21 cell comprises an amino acid part, a vitamin part, an inorganic salt part and a part of other additives. The medium disclosed by the invention has the beneficial effects that the serum-free medium for full suspension cultivation of the BHK-21 cell is high in culture cell density, clear in component and free from animal serum, facilitates purification of downstream products, and improves the product quality. The medium is convenient to prepare and use, and is applicable to production of biological products on a large scale.

The invention provides a method for preparing mesenchymal stem cells of menstrual blood. The method comprises steps as follows: female menstrual blood is collected and taken as a raw material and is subjected to sterile processing; stem cells in the menstrual blood are separated; the mesenchymal stem cells of the menstrual blood are cultured; the mesenchymal stem cells of the menstrual blood are cryopreserved; and the mesenchymal stem cells of the menstrual blood are recovered. The method has the technical characteristics as follows: the female menstrual blood is collected and stored in a preservative fluid; by means of a bacterial pollution prevention method, the pollution probability is reduced from a collection source, a collecting cup is repeatedly washed by sterile water, and the pollution probability is effectively reduced; with the adoption of a differential centrifugation method, bacteria in the menstrual blood are removed as far as possible; a sample is repeatedly separated by HES (hydroxyethyl starch), and the maximum quantity of mesenchymal stem cells of the menstrual blood can be obtained; a serum-free medium is utilized for culturing, components of animal origin are reduced, the cell performance is stable, the in-vitro long-term culturing process of the mesenchymal stem cells of the menstrual blood can be kept, and cellular morphology, multiplication capacity, MSC (mesenchymal stem cell) surface marker expression, differentiation capacity and the like are maintained. The method is simple, practical and convenient to operate, the maximum quantity of required stem cells can be obtained, and the stem cells are successfully cultured.

The invention relates to the technical field of culture medium development and research of biotechnology and discloses a serum-free medium suitable for large-scale production of influenza vaccines. The serum-free medium comprises 23 basic metabolism nutrients, two nucleic acid compounds, 6 vitamins, 9 inorganic salt compounds, a shear force protective agent, two acidity and alkalinity buffer agents, an acidity and alkalinity indicator, 10 virus reproduction promoters and three additives. The conventional preparation method of the serum-free medium comprises the following steps of: dissolving the components in ultrapure water without a heat source so as to prepare the serum-free medium. The using method is a conventional method. The serum-free medium has the beneficial effects that the serum is not contained, the total protein content is lower than 10mg per liter, separation and purification of the product are promoted, and the serum-free medium is suitable for production of influenza vaccines, supports the normal growth and long-term continuous cell culture of animal cells and can be used without adaption; and moreover, because the components are clear, the serum-free medium is convenient to prepare, controllable in cost and suitable for large-scale production of the influenza vaccines.

The invention provides a separation and purification method of a human umbilical cord mesenchymal stem cell exosome and an application of the human umbilical cord mesenchymal stem cell exosome, and belongs to the technical field of medicines. The human umbilical cord mesenchymal stem cell exosome provided by the invention is prepared by cultivating human umbilical cord mesenchymal stem cells via aserum-free medium, collecting supernatant liquid, implementing centrifuging as well as ultra-filtration and concentration, transferring a concentrated solution onto a 30% sucrose-heavy water densitypad and implementing further purification through sucrose density centrifuging, so that the human umbilical cord mesenchymal stem cell exosome is obtained. According to the separation and purificationmethod that the human umbilical cord mesenchymal stem cell exosome is obtained through separation and purification, immunoreactivity is effectively reduced, and a controllable dosage when the human umbilical cord mesenchymal stem cell exosome is used is guaranteed. The human umbilical cord mesenchymal stem cell exosome, by improving a degree of activating an insulin signaling pathway of a type IIdiabetes animal model, can inhibit composition and decomposition of hepatic glycogen, so that glucose metabolism homeostasis can be achieved; and meanwhile, the sensitivity of the type II diabetes animal model to insulin and an insulin secretion function of pancreatic [beta] cells can be improved and a blood glucose concentration can be reduced, so that the application of the human umbilical cordmesenchymal stem cell exosome to the preparation of medicines for treating type II diabetes can be achieved.

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 serum-free medium for human adipose-derived stem cells, comprising a DMEM basic medium and further comprising L-glutamine, insulin-like growth factors, epidermal growth factors, transforming growth factors, platelet-derived growth factors, fibroblast growth factors, transferrin, vitamin C, human serum albumin and 1, 2, 3, 4, 6-O-pentagalloylglucose. The invention belongs to the technical field of stem cells; the serum-free medium for human adipose-derived stem cells provided by the invention is capable of promoting quick adherence and proliferation of the human adipose-derived stem cells and keeping good stem cell pedomorphism and stem cell characteristics.

The invention discloses a serum free medium for expressing erythropoietin in CHO (Chinese hamster ovary) cells, and a culture method for high expression of erythropoietin in CHO cells. The serum free medium contains additives D-glucose and sodium butyrate, and also contains one or more of D-galactose, D-mannitose and N-acetylglucosamine. The culture method comprises the steps of: performing serumculture on activated seed cells by using a serum medium, and then performing serum free culture by the serum free medium. By adopting the medium and culture method disclosed by the invention, recombinant human erythropoietin protein of high and stable expression can be obtained, and the glycosylation degree of erythropoietin is improved, i.e. the specific gravity of EPO (erythropoietin) sialic acid is improved. As high as 1.0*107IU recombinant human erythropoietin can be obtained from one liter of culture fluid by the culture method provided by the invention, the expression is stable, and theculture method is simple and suitable for large-scale production.

The invention discloses a method for separating primary adult hepatocytes. The method comprises the following steps: (1) carrying out multi-point puncture on surface of isolated adult hepatic tissue through a needle syringe, and injecting a preperfusate, wherein connective tissues are removed from the isolated adult hepatic tissue; (2) carrying out the multi-point puncture on the surface of the isolated adult hepatic tissue through the needle syringe again, and injecting a IV collagenase solution; (3) separating the isolated adult hepatic tissue, followed by adding the IV collagenase solution and carrying out digesting through vibration at a temperature of 37 DEG C to obtain digest; (4) carrying out filtering for the digest, followed by centrifuging and collecting cell aggregate in the underlayer, then resuspending the adult hepatocyte aggregate through a hepatocyte wash buffer, followed by filtering and centrifuging, then abandoning supernatant and collecting the adult hepatocyte aggregate in the underlayer; (5) washing the adult hepatocyte aggregate in the underlayer from the step (4) through a serum-free DMEM medium to obtain the primary adult hepatocytes. The invention further discloses a disposable special sterile apparatus box for separating the primary adult hepatocytes. With the present invention, the disposable special sterile apparatus box is adopted, the primary adult hepatocytes are separated through the multi-point puncture on the surface of the tissue and the injection, such that operation is simplified, cost is reduced, and the method and the apparatus box are applicable for extracting the hepatocytes from small pieces of the irregular isolated adult hepatic tissues of recovery of liver resection.

The invention belongs to the field of use of a stem cell conditioned medium in medicinal cosmetics, and especially relates to an application of the conditioned medium in skin ageing resistance. Researches find that a large amount of complex active substances secreted by stem cells are stored in a medium in the in vivo culture process of mesenchymal stem cells, and is called as the stem cell conditioned medium, and an obtaining method of the medium comprises the following steps: carrying out digestive passage on p2 generation mesenchymal stem cells by using 0.05% pancreatin, inoculating 2*10<6> into a 150mm culture dish, and culturing to obtain p3 generation cells; and sucking the obtained culture supernatant when 80% of the p3 generation cells fuse, washing by using PBS twice, adding a new serum-free medium, putting the obtained cells in a 37DEG C 5% CO2 incubator, culturing for 48h, and collecting the obtained culture supernatant to obtain the stem cell conditioned medium. The stem cell conditioned medium obtained in the invention can supplement skin cell metabolism promotion substances lacked in skins and also can induce new collagen synthesis in order to maintain elasticity of skins and reduce wrinkles.

The invention relates to the technical field of cell culture, and particularly relates to a culture method of mesenchymal stem cells. In the culture method, a serum-free culture medium optimized by the invention is used for culturing the mesenchymal stem cells. In addition, the invention further provides a better culture method for 3D culture. The cells obtained by the culture method disclosed bythe invention are strong in proliferation capacity, intact in shape and strong in secretion capacity, and are in accord with the international quality control standards of the mesenchymal stem cells.Meanwhile, according to the culture method disclosed by the invention, a large number of mesenchymal stem cells (particularly human umbilical cord mesenchymal stem cells) can be obtained by amplifyinga small number of mesenchymal stem cells under the conditions of small space occupation, less culture medium consumption, simpler and more convenient operation and greatly reduced workload; and a solid technical scheme and a theoretical basis are provided for obtaining a large number of high-quality mesenchymal stem cells (especially human umbilical cord mesenchymal stem cells) and secretions thereof in the clinical field.

A method of producing a protein having free cysteine with the use of a serum-free medium, characterized in that the protein is produced in the activated state; a method of producing a protein by culturing in a serum-free medium in accordance with the above method; a method of activating a protein having free cysteine which has been produced in the inactivated state; and a protein obtained by any of the above methods. This protein shows physicochemical or biological properties comparable to a protein obtained by using a serum medium.

The invention belongs to the technical field of stem cells and relates to a serum-free medium for placenta-derived mesenchymal stem cells and a preparation method thereof. The serum-free medium comprises a DMEM basic medium and further comprises vitamin H, glutathione, recombinant human insulin, human serum albumin, transferrin, fibroblast growth factors, epidermal growth factors, stem cell growth factors, Human stem cell factors and magnolol. The serum-free medium provided by the invention has the advantages of high safety and capability for significant improvement of proliferative capability of placenta-derived mesenchymal stem cells, the serum-free medium is capable of well keeping cell forms and stem cell characteristics of the placenta-derived mesenchymal stem cells and is convenient to popularize and apply.

The invention relates to umbilical cord mesenchymal stem cells (MSCs) and a culture method and application thereof, and belongs to the technical field of biological medicine. The culture method includes MSCs culture steps and phenotypic examination. The MSCs culture steps include primary culture and sub-culturing; during primary culture, Huatong gum tissue of the umbilical cord is obtained and cultured in a serum-free culture medium under hypoxic conditions; during sub-culturing, P1 primary cells are collected, a single cell suspension is prepared, cell precipitates are obtained after centrifuging, the serum-free culture medium is added into the cell precipitates, culturing is conducted under hypoxic conditions until next generations come out, continuous culturing is performed to obtain P2-P3 generations, ligustrazine hydrochloride and Shenmai injections are added in each sub-culturing stage, when the cells grow to a predetermined fusion degree, digestion is performed and the cells arecollected until the P6 generations come out. During phenotypic examination, the collected cells are subjected to phenotypic examination for use. An MSCs preparation obtained through culturing in themethod reduces the easy-to-aggregation characteristics of the stem cells, the occurrence of cell adhesion, erythrocyte rouleaux formation and cell mass embolism after intravenous infusion into the human body is thus avoided, and the stem cell (MSCs) technology can be better applied to clinical practice.

The invention discloses a combined medium for preparing insulin-like secreting cells, which consists of a medium I, a medium II and a medium III which are independent respectively, wherein, the medium I is a DMEM (Dulbecco's modified Eagle's medium) high-glucose medium prepared from fetal calf sera, basic fibroblast growth factors (bFGF), dimethyl sulfoxide (DMSO) and glucose; the medium II is a serum-free DMEM/F12 medium prepared from glucose, niacinamide, epidermal growth factors (EGF), bFGF, exendin-4, a B27 additive and an N2 additive; and the medium III is an RPMI (Roswell Park Memorial Institute) 1640 medium prepared from glucose, niacinamide, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, hepatocyte growth factors (HGF), activin A and exendin-4. The invention further discloses a method using the combined medium for preparing insulin-like secreting cells at the same time. The invention can facilitate the differentiation of hBM-MSC (human bone marrow mesenchymal stem cell) into insulin-like secreting cells having the effect of correcting diabetes hyperglycemia.

Disclosed is a method for production of recombinant human FSH in high yield and a high purity. The method comprises the steps of: (a) culturing recombinant human FSH-producing mammalian cells in a serum-free medium, (b) collecting culture supernatant, (c) subjecting the culture supernatant to cation-exchange column chromatography, (d) dye affinity column chromatography, (e) hydrophobic column chromatography, and (f) gel filtration column chromatography to collect recombinant human FSH-active fractions, in the order.

The invention discloses a method for culturing animal cells for producing anti CD20 antibody, and specifically discloses inoculation quantity, medium formula and various culture process control parameters. The medium is a serum-free medium, so that no side effect is caused by serum in the process of cell culture, and antibody extraction and purification are further facilitated. By using the method for producing the anti CD20 antibody, differences between different batches are small, antibody expression quantity is high, production cost is low, and safety is good.

The invention provides a protein-free serum-free medium suitable for diploid cell culture and a preparing method thereof. The medium contains calcium chloride, magnesium sulfate, potassium chloride, sodium dihydrogen phosphate, L-arginine hydrochloride, L-asparaginate, L-cystine dihydrochloride, L-histidine hydrochloride, L-leucine, L-isoleucine and the like. The medium can meet the requirement of the growth or maintenance stage of diploid cells and prevents contact with exogenous viruses possibly existing in protein and serum to reduce potential safety hazards; as the components are simple and definite, standard products can be purchased in the market, the difference between different product batches is small, and the step of purifying down-stream products is simplified; a scientific basis is provided for large-scale standard production. In addition, the protein-free serum-free medium can be applied in the growth or maintenance stage of the diploid cells.

The invention discloses a serum-free medium used for culture of elderly people cartilage cells, the-free medium comprises a basal medium and an additive comprising the components of the following concentration: 233muM-287 muM of vitamin C, 3.7mu M-8.9 muM of linoleic acid, 9muM-17 muM of cholesterol, 6nM-15 nM of dexamethasone, 43 muM-58mu M of acetyl cysteine, 18 mug / mL-32 mu g / mL of transferrin, 22nM-52 nM of sodium selenite, 13 muM-24 muM of sodium pantothenate, 28 muM-43 muM of biotin, 7 mug / mL-18 mu g / mL of insulin, 2 ng / mL-9 ng / mL of epidermal growth factor, 2 ng / mL-9 ng / mL of fiber growth factor, 2 ng / mL-9 ng / mL of platelet derived growth factor, and 0.5%-2.5% of human serum albumin. The cell culture time is short, and the cell biological performance is good.

The invention discloses a serum-free medium for mesenchymal stem cells (MSCs). The serum-free medium comprises a basic medium, recombinant human insulin, cholesterol, recombinant fibronectin, sodium linoleate, recombinant human transferrin, perilla seed protein, stilbene glycoside, resveratrol and a ginkgo biloba extract. According to the serum-free medium, the recombinant human transferrin and the plant protein perilla seed protein are added to be compounded, the stilbene glycoside, the resveratrol and the ginkgo biloba extract are compounded for use, on one hand, the serum-free medium has effects of resisting oxidation and scavenging free radicals and contributes to keeping cell activity; and on the other hand, the serum-free medium still keeps a higher proliferation speed and a better growth state of cells under the condition that exogenous serum is not added. The serum-free medium comprises clear components, the repeatability, accuracy and stability of an experiment can be improved, raw materials are easy to obtain, and the preparation cost of the serum-free medium is effectively reduced. The invention further provides a preparation method of the serum-free medium, and the preparation process is convenient and easy to operate.

The invention provides a method for fast separating and extracting human Umbilical Cord mesenchymal stem cells (hUC-MSC) from umbilical cord outer layer amnion tissue. The method includes the following steps that fresh collected umbilical cord tissue of a healthy newborn is taken, transported on ice in double-antibody-containing umbilical cord storing transporting liquid, washed and disinfected with 75% ethyl alcohol and normal saline, outer layer amnions are separated bluntly after blood vessels are rejected, mechanically smashed and treated with erythrocyte lysate for 3 min, IV type collagenase digestion is used, and a digestion product is cultured in a suspension mode with a serum-free culture medium after being screened through a 100-200-mesh sieve; medium change is carried out every three to five days, supernatant is taken to detect cell contamination, after the attachment rate in a plate reaches 30-70% and trypsinization is used, cell passage amplification is collected centrifugally, and confluence is carried out when the cell confluence rate reaches 90% or above, the cells are collected for cryopreservation, and the biological characteristics of hUC-MSC are detected.

The invention provides a method and system for separating, culturing and amplifying human umbilical cord mesenchymal stem cells. The method comprises the following steps: step I, pretreating a sample; step II, obtaining Wharton's jelly, cutting the obtained Wharton's jelly into small tissue, and adding a filtered red blood cell lysis buffer solution of which the volume is 3.5 times that of the small tissues; step III, digesting collagenase, wherein a digestion solution is a balanced salt solution containing 1% of IV type collagenase, 0.5% of hyaluronidase, 300U/ml DNA enzyme and 2% of a serum substitute; step IV, performing primary cell isolated culture: adding a serum-free culture medium of mesenchymal stem cells to obtain a cell suspension, and adding a special primary cell culture solution for umbilical cord mesenchymal stem cells for primary culture; step V, subculturing the cells, and inoculating the collected cells according to 5000 cells/cm < 2 > by using trypsin with the mass percent concentration of 0.215%; and step VI, collecting the umbilical cord mesenchymal stem cells, and performing osteogenic differentiation, adipogenic differentiation and chondrogenic differentiation experiments.