31 results about "Dmem f12" patented technology

The invention discloses a method and application for inducing human umbilical cord mesenchyme stem cells to be differentiated into testicular interstitial cells. The method comprises the following step of culturing human umbilical cord mesenchyme stem cells of patients suffering from adenovirus and carrying mice steroidogenic factor-1 genes in a DMEM-F12 culture solution containing 0.3-3ng/ml of luteinizing hormone, 200-800mu M of dibutyryl cyclic adenosine monophosphate, 5*10<-6>-5*10<-4>M of all-trans retinoic acid (ATRA), 10mU/ml of human chorionic gonadotropin and 2.4uM of adrenocorticotrophic hormone for a week. Induced by the method in the invention, the human umbilical cord mesenchyme stem cells can be differentiated into testicular interstitial cells in vitro and provides important sources of cells for treating testosterone shortage by the cell replacing method or the genetic method.

The invention relates to a reprogrammed culture medium and a culture method of a reprogrammed induced multipotential stem cell. The reprogrammed culture medium is composed of a DMEM-F12 basal culturemedium and additive components, wherein the additive components comprise 60-180 microgram/mL of L-ascorbic acid, 5.3-74 micromole/L of hydrocortisone, 3-89 nanogram/mL of sodium selenite, 8-23 micromole/L of Optiferrin, 0.5-7.4 micromole/L of retinyl acetate, 40-60 nanogram/mL plant-derived recombinant human alkaline growth factors, 8-12 microgram/mL of IGF, 0.2-0.6 microgram/mL A-83, 2-6 micromole/L of CHIR99021 and 100-450 micromole/L of sodium butyrate. The use of the reprogrammed culture medium not only can improve the safety of clinical use, but also can improve the efficiency of adult cell reprogramming to induce the multipotential stem cell.

The invention discloses a mesenchymal stem cell serum-free culture medium. The culture medium takes an L-DMEM, DMEM-F12 or alpha-MEM culture medium as a basic culture medium and also includes the following addition components: fibronectin, laminin, transferrin, trypsin, aprotinin, growth hormone, insulin, hydrocortisone, dexamethasone, bFGF, EGF, B27, IGF-I, IGF-II, choline, linoleic acid, sodium selenite, phosphorylethanolamine, glutathione, vitamin C, vitamin E, vitamin B12 and biotin. The culture medium has the advantages of defined composition and controllable quality, and mesenchymal stem cells cultured by the culture medium grow normal; and after multiple passages, the 'dryness' of the mesenchymal stem cells and the ability of the mesenchymal stem cells to be differentiated into osteoblasts, chondrocytes, adipocytes, neurons and other types of cells are still kept.

The invention discloses a novel serum-free medium. The serum-free medium comprises, by volume, 0.05-0.2 part of beta-mercaptoethanol, 0.5-2 parts of a non-essential amino acid aqueous solution, 4-6 parts of human mesenchymal stem cell cultural concentrated supernatant and 90-95 parts of a-MEM/DMEM-F12 and recombination human basic fibroblast growth factors with the final concentration of 5-15 ng/ml. A preparing method of the cultural concentrated supernatant includes the following steps that umbilical cord mesenchymal stem cell cultural supernatant is collected; cells, cell debris, impurities and the like are centrifugally removed; filtering is carried out through a micro-filtration membrane; ultrafiltration and concentration are carried out. When cultured through the medium, stem cells still keep the multi-directional differentiation potential and the high multiplication capacity under the long-time culturing condition.

The invention provides connexin core sequence-containing amphiphilic polypeptide AcN-RADARADARADARADAGGDHLSDNYTLDHDRAIH-CONH2, wherein the amphiphilic polypeptide and the amphiphilic polypeptide with the structure of AcN-RADARADARADARADA-CONH2 are triggered by Ca2+or a cell culture medium DMEM-F12 to automatically form into a compound, i.e. a nanometer-grade fiber material, and the nanometer-grade fiber material is taken as a bracket of the bone mesenchymal stem cells, so that the biological behavior of the cells can be obviously improved, the degenerated intervertebral disc can be repaired, and the original height can be recovered.

The invention provides digestive enzyme liquid for poultry tibial growth plate cartilage primary cell separation. A complete medium is used as basic liquid, and is prepared from the following ingredients including collagenase IV with the mass concentration being 80 to 100mg/mL and hyaluronidase with the mass concentration being 80 to 100mg/mL; the complete medium takes a high-glucose DMEM-F12 culture medium as a basic culture medium, and contains the following ingredients including fetal calf serum with the volume concentration being 8 to 12 percent, vitamin C with the mass concentration being 4.5 to 5.5mg/mL and mycillin with the mass concentration being 1 to 10mg/mL. Through the selection on digestive enzyme and the concentration optimization, the cell yield reaches 85 to 95 percent or higher; during the further culture on the digested cells, the cell upward trend is good; meanwhile, the cell viability of the digested primary cell reaches 85 to 95 percent or higher; the cell viability is relatively stable.

The present invention encompasses methods and compositions for enhancing the growth of neural stem cells. Methods for modulating MHC molecule expression on a neural stem cell (NSC) are also included in the invention. Figure 1 is a graph depicting the proliferation of BMSCs in BMSC medium (DMEM-low glucose, 10% lot tested fetal bovine serum) or NSC medium (DMEM-F12, N2-Supplement, EGF 20 ng/ml, bFGF 10 ng/ml, heparin 8 ug/ml, penicillin-streptomycin).

The present invention relates to a method for isolating mesenchymal stem cells from placental vessels and used digestive enzyme compositions. The method comprises the following steps: the placenta issterilized with alcohol; Dissecting placental blood vessels from the placenta; Cut into pieces, wash with PBS, filter the residual blood stains to obtain placental vascular tissue; Adding mixed enzymesolution to digest; End digestion, filter and collect tissue fluid; The placental mesenchymal stem cells (P0) were obtained by centrifugation and cultured with DMEM-F12 basal medium was resuspended and the number and viability of nucleated cells were counted. The obtained cells are cryopreserved with cryopreservation protection solution so as to be re-cultured before use, or the passages and/or the mesenchymal stem cells are continued to be subjected to cell identification and/or detection, cryopreservation, library establishment and the like. The method of the invention can effectively improve the efficiency of isolating mesenchymal stem cells from the blood vessels of the placenta.

The invention discloses a cell freezing medium. The cell freezing medium comprises a basal culture medium, glycerin, fetal calf serum and a non-permeable protective agent, and the contents of the components are 80-94 v/v% of the basal culture medium, 5-10 v/v% of glycerol, 1-10 v/v% of the fetal calf serum and 0.01-0.10 w/v% of the non-permeable protective agent, and the basal culture medium comprises RPMI-1640, MEM, high-sugar DMEM, low-sugar DMEM or DMEM-F12; and the non-permeable protective agent comprises sodium alginate, sodium hyaluronate or gamma-polyglutamic acid. Dimethyl sulfoxide isnot contained, a good freezing effect is achieved through different matching of the non-permeable protective agent with glycerin and fetal calf serum, a strong water molecule complexing ability is achieved, the freezing function is similar to the freezing function of dimethyl sulfoxide, but formation of intracellular ice crystals is reduced during freezing; the cell freezing medium has clear added ingredients, high safety, small damage to cells, consistent morphology before and after freezing, a high cell survival rate after cell resuscitation and good proliferative capacity.

The invention discloses a novel kit for serum-free stem cell culture. The kit comprises a serum-free culture medium, wherein the serum-free culture medium contains 0.05-0.2 part of beta-mercaptoethanol, 0.5-2 parts of a non-essential amino acid aqueous solution, 4-6 parts of human mesenchymal stem cell culture supernatant concentrate, 90-95 parts of a-MEM/DMEM-F12 and human recombinant basic fibroblast growth factors with the final concentration of 5-15 ng/ml by volume. The culture supernatant concentrate is prepared by adopting the following steps of collecting the umbilical cord mesenchymal stem cell culture supernatant, centrifugally removing cells, cellular debris, impurities and the like, performing filtration by using a micro-filtration membrane and performing ultra-filtration concentration. By utilizing the kit for stem cell culture, the multi-directional differentiation potential and stronger proliferation ability of the cells are still kept under the condition of long-term culture.

Provided is a serum-free culture medium, the ingredients of the culture medium comprising 0.05-0.2 parts by volume of β-mercaptoethanol, 0.5-2 parts by volume of non-essential amino acid aqueous solution, 4-6 parts by volume of human mesenchymal stem cell culture supernatant concentrate, and 90-95 parts by volume of a-MEM/DMEM-F12 and recombinant human alkaline fibroblast growth factor of a final concentration of 5-5 ng/ml. The present culture medium is used for carrying out stem cell culture.

The invention relates to a method for cryopreservation and resuscitation of mesenchymal stem cells. A method for preparation and cryopreservation of the mesenchymal stem cells comprises the followingsteps of disinfection and cleaning, digestion treatment, cell culture, cell passage, addition of a cell cryopreservation solution to the mesenchymal stem cells and cryopreservation in liquid nitrogen.The cell cryopreservation solution contains DMEM-F12, dimethyl sulfoxide, canine blood albumin and the like. The method for cryopreservation and resuscitation of the mesenchymal stem cells comprisesthe following steps that the prepared mesenchymal stem cells and the cell cryopreservation solution are uniformly mixed, then the cell solution is placed in the liquid nitrogen for freezing, and cryopreservation is conducted until the cells are needed; when the cells need to be resuscitated, the cryopreserved cells are taken out of the liquid nitrogen and quickly put into a water bath of 37 DEG Cto make the cryopreservation solution completely thawed, and after the cells are thawed, the cells are immediately transferred to an ice bath to complete the resuscitation of the cells. The inventionfurther relates to the cell cryopreservation solution for the cryopreservation and resuscitation of the mesenchymal stem cells. The method and the cryopreservation solution have excellent technical advantages shown in the description.

The invention relates to a method for improving the survival of mesenchymal stem cells after resuscitation and an adopted cryopreservation solution. Particularly, the invention relates to the method for cryopreservation and resuscitation of the mesenchymal stem cells. The method comprises the following steps that the prepared mesenchymal stem cells are uniformly mixed with the cell cryopreservation solution, and then the cell solution is placed in liquid nitrogen for freezing until the cells are needed; when the cells need to be resuscitated, the cryopreserved cells are taken out of the liquidnitrogen and quickly put into a water bath of 37 DEG C to make the cryopreservation solution completely thawed, and after the cells are thawed, the cells are immediately transferred to an ice bath of4 DEG C to complete the resuscitation of the cells. The invention further relates to the cell cryopreservation solution for the cryopreservation and resuscitation of the mesenchymal stem cells. The cell cryopreservation solution contains DMEM-F12, dimethyl sulfoxide, canine blood albumin and the like. The invention further relates to a method for preparation of the mesenchymal stem cells from thecanine umbilical cord and cryopreservation of the mesenchymal stem cells. The methods and the cryopreservation solution have excellent technical advantages shown in the description.

The invention discloses a serum-free medium for a monkey embryonic stem cell. The serum-free medium comprises a basic medium and additives, wherein the additives are dissolved in the basic medium; the basic medium comprises a DMEM-F12 medium; and the additive comprises L-glutamine, non-essential amino acid, L-ascorbic acid, sodium selenite, beta-mercaptoethanol, human serum albumin, heparin sodium, human transferring, insulin human, a basic fibroblast growth factor, a transforming growth factor beta 1 and an epidermal growth factor. The serum-free medium is capable of maintaining an undifferentiation state and totipotency of the monkey embryonic stem cell within a relatively long time, and is applicable to culture of the monkey embryonic stem cell in both a free-feeding layer system and a feeding layer system.

The invention discloses a human induced multipotential stem cells cryopreserved reagent and a cryopreserving method thereof. The cryopreserved reagent comprises the following components: 0.03-100 ml of recombinant human albumin, 0.0125 mg/100 ml of recombinant human epidermal growth protein, 1 ml/100 ml of beta-mercaptoethanol, 91-100 ml of a DMEM-F12 medium, 8-100 ml of dimethyl sulfoxide, and 5-100 ml of trehalose. The method has the beneficial effects that 1) the component is completely clear and is convenient for quality control; 2) the recombinant human albumin is used for substituting the fetal calf serum of the animal component, can avoid an animal serum-containing component, the cryopreserved reagent is safe and reliable, and is adapted to clinic application; 3) the recombinant human epidermal growth protein, beta-mercaptoethanol, and trehalose in a formula can better protect the cells in a state of dormancy under low temperature environment, ice crystal is not generated in tissue of the cells, cell activity of the cells can be kept, the cell differentiation can be effectively prevented, and the recovery rate of the cryopreserved cell can reach more than 96%; and 4) the cryopreserved reagent belongs to a ready-to-use type, and the usage is convenient.

The invention belongs to the field of biological medicines, and particularly relates to application of exosomes derived from menstrual blood stem cells on preparation of a medicament for treating intrauterine adhesion. After primary isolation, culture and passage of the menstrual blood stem cells are carried out, 3rd-6th generations of menstrual blood stem cells are selected for exosomes preparation. When the density of the menstrual blood stem cells reaches 80%, serum components are removed by PBS washing, serum-free DMEM-F12 culture medium is added, and the menstrual blood stem cells are cultured in an incubator at the conditions of 37 DEG C, 5% CO2 and saturated humidity for 24 hours. The supernate is collected, the exosomes are prepared by ultracentrifugation under GMP conditions, andsterile PBS is collected and stored at the temperature of 80 DEG C. The cells have the advantages of being high in survival rate, stable in characters and easy to acquire and expand, and having an obvious treatment effect on intrauterine adhesion. The exosomes can even completely simulate the biological function of the cells to achieve the effects of promoting tissue repair and treating diseases.The exosomes are safer than the cells where the exosomes are derived from, do not lose functions after cryopreservation, can be used for allotransplantation and are suitable for mass production, and have important clinical application values and social and economic benefits.

The invention discloses a special thawing reagent and a thawing method for human induced pluripotent stem cells. The thawing reagent is free of animal components and comprises 100ng-300ng of recombinant human epidermal growth factors, 1000ng-2000ng of recombinant human blood albumin, 0.1ml-0.25ml of b-mercaptoethanol and the balance DMEM-F12 culture medium, wherein the total volume is 10.0ml. The special thawing reagent and the thawing method for the human induced pluripotent stem cells have advantages that (1) high survival rate (thawing rate) and high activity of the human induced pluripotent stem cells can be guaranteed, and high functional versatility of the cells is guaranteed as well; (2) due to freeness of the animal components, complete exactness and controllability of components can be realized, and high safety in use is achieved; (3) the thawing method is convenient to operate, easy to implement and suitable for wide popularization and utilization.

The invention discloses an isolation and culture method of human adipose-derived stem cells. A culture medium is a DMEM-F12 medium added with fetal calf serum with final concentration of 1-20%, bFGF of 1-100 ng/ml and EGF of 1-100 ng/ml. The isolation and culture method includes fat primary operation steps. The fat primary operation steps include steps of crushing, cleaning, digestion, isolation and culturing. The isolation and culture method also includes subculture operation steps. The subculture operation steps include cleaning, digestion, cleaning, centrifugation and culturing. The isolation and culture method of the human adipose-derived stem cells is simple in culture medium composition, short in culture time in the isolation and culture method, fast in growth speed and high in stemcell purity. No stem cell differentiation occurs during the culture.

The present invention relates to a method for culturing corneal stem cell-like cell lines by inducing differentiation of induced pluripotent stem cells (iPSC). The method comprises the following steps:

The invention relates to a method for high-efficiency amplification of Tembusu virus by chicken hepatocytes, wherein the method includes the following steps: (1) cell culture: culturing the chicken hepatocytes in a DMEM-F12 culture medium containing fetal bovine serum with the volume concentration of 10%, after the cells grow on a single layer fully, washing with PBS once, digesting with pancreatic enzymes with the mass concentration of 0.25%, and passaging to a cell culture plate; and (2) high-efficiency amplification of Tembusu virus in the chicken hepatocytes: inoculating the chicken hepatocytes into the cell culture plate and culturing, when the confluence degree is 60% to 70%, inoculating the chicken hepatocytes with the Tembusu virus AHRD2018 strain with MOI of 0.01, culturing in 2 ml of a culture medium containing fetal bovine serum with the volume concentration of 2% for 4 days, collecting 200 [mu]l of a supernatant at a fixed time and cryopreserving in an -80 DEG C refrigerator for standby application, and at the same time, supplementing 200 [mu]l of the culture medium containing fetal bovine serum with the volume concentration of 2% into each hole. The characteristic thatthe chicken hepatocytes can produce high titer virus more prematurely is utilized, and the Tembusu virus is amplified by the chicken hepatocytes having the virus replication rate significantly higherthan that of DF-1 cells and BHK-21 cells.