63 results about "Trypsinization" patented technology

The invention discloses a human placenta, umbilical cord mesenchyme stem cell bank and its making method. The method includes the following steps: the first is taking human placenta, umbilical cord to test; smashing after cleaning by phosphate buffer; adding phosphate buffer to dilute; the second is adding collagenase to digest; the third is adding phosphate buffer; the fourth is adding trypsinization; the fifth is mixing the cell from the second and fourth, centrifuging, removing supernatant, cleaning by the phosphate buffer, centrifuging, and removing supernatant; the mesenchyme stem cell are gained; the sixth is freezing them in liquid nitrogen; saving by ABO/Rh subtype and HLA subtype; setting recallable cell information file. The invention is newborn storage placenta and umbilical cord mesenchyme stem cell. It can offer mesenchyme stem cell to treat the disease for personal, family and others.

The invention provides a stem cell culture medium and a method for culturing endometrium stem cells by using the stem cell culture medium. The method comprises the following steps: separately collecting menstrual blood and endometrium tissues, respectively culturing the menstrual blood and endometrium tissues in the stem cell culture medium provided by the invention to respectively obtain menstrual blood adherent cells and endometrium adherent cells, culturing the menstrual blood adherent cells and endometrium adherent cells in a cell culture bottle, collecting the adherent cells by trypsinization, inoculating the adherent cells in a cell coculture dish, and culturing the adherent cells in the stem cell culture medium provided by the invention. The stem cell culture medium has the advantages of simple components, fewer added components and lower cost. After more than 20 generations of in-vitro culture, the cells can not easily have the phenomenon of aging or degeneration, and can maintain the activity and stem property of the stem cells for a long time. The stem cell culture method is simple and effective, the cell proliferation efficiency is high, and the in-vitro culture doubling time is only 20 hours or so. The cells can be stably amplified by 50 generations.

The subject invention concerns novel and translatable materials and methods for expansion of stem cells, such as mesenchymal stem cells (MSC), that significantly improve translational success of the cells in the treatment of various conditions, such as stroke. The subject invention utilizes cell self-aggregation as a non-genetic means to enhance their therapeutic potency in a microcarrier bioreactor. The subject invention integrates a cell aggregation process in a scalable bioreactor system. In one embodiment of the method, thermally responsive microcarriers (TRMs) are utilized in conjunction with a bioreactor system. Cells are cultured in a container or vessel in the presence of the TRMs wherein cells adhere to the surface of the TRMs. Once cells are adhered to the TRMs they can be cultured at a suitable temperature for cell growth and expansion, e.g., at about 37° C. After a period of time sufficient for cell growth and expansion on the TRMs, the cell culture temperature is reduced so that the cells detach from the TRMs. The detached cells are allowed to form cell clusters that are then cultured under conditions such that the clusters aggregate to form 3D aggregates. The 3D aggregates can be collected and treated to dissociate the cells (e.g., using enzymatic treatment, such as trypsinization). Dissociated cells can then be used for transplantation in methods of treatment or for in vitro characterization and study.

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 provides a method for rapidly separating and extracting hUC-MSC (human Umbilical Cord mesenchymal stem cells). The method comprises the following steps: taking the freshly collected umbilical cord tissue of a healthy newborn baby, carrying out on-ice transportation on the freshly collected umbilical cord tissue in umbilical cord storage transportation liquid containing double antibodies, carrying out cleaning and disinfection by adopting 75% alcohol and normal saline, removing blood vessels, carrying out blunt dissection on wharton jelly, carrying out mechanical pulverization, treating the obtained product I by adopting red blood cell lysis buffer for 3 min, digesting the obtained product II by adopting IV collagenase, screening the obtained product III by adopting a 100-200-mesh sieve, carrying out suspension culture on the obtained product IV by adopting a serum-free medium, wherein the liquid is changed every 3-5 days, taking supernatant, detecting cell pollution, after the adherent rate in a plate reaches 30-70%, carrying out trypsinization, carrying out centrifugation, collecting cells, carrying out passage amplification, carrying out merging when the cell merging rate reaches 90% or above, collecting the cells, carrying out cryopreservation on the cells, and detecting the biological characteristics of hUC-MSC.

The invention provides a method for establishing a human nasopharyngeal carcinoma tumor stem cell line. The method comprises the following steps: (1) obtaining human nasopharyngeal carcinoma cells in the single cell state; (2) adding the human nasopharyngeal carcinoma cells in the single cell state, obtained in the step (1), to a serum-free stem cell culture medium containing epidermal growth factors and basic fibroblast growth factors to obtain single cell suspension; (3) adjusting the concentration of the single cell suspension in the step (2) to 900-1100 single cells/ml; (4) inoculating 1ml of the single cell suspension obtained in the step (3) into single pores on a cell culture six-pore plate and then adding the equivoluminal stem cell culture medium for culture for 72-96 hours; and (5) collecting the cells obtained in the step (4), carrying out trypsinization till the single cell and carrying out subculture. The method provided by the invention is simple and convenient to operate; and the obtained nasopharyngeal carcinoma stem cell line has obvious stem cell characteristics.

The common medical polymer material to be used as cell culture substrate is treated through laser scanning to form micron/nanometer or submicon/nanometer channels before sterilization. Anchorage dependent cell in condition after trypsinization is inoculated homogeneously to the surface of the substrate for culture. Owing to the regulation and control of the surface channels, cell will grow and orient in the channel direction. The present invention has simple technological process and easy operation, and is suitable for the basic research in cell biology, making of biological sensor and functional device, research of medical implant, tissue engineering, etc.

The invention discloses a method for induced differentiation of liver cells by using endometrium stem cells. The method comprises the following steps of: (1) carrying out isolated culture and amplification on the endometrium stem cells; and (2) carrying out in vitro induced differentiation: A, carrying out trypsinization on cells with good growth conditions of 2-4 generations, which are obtained in the step (1), ending the digestion by using a DMEM (dulbecco's modified eagle medium) culture medium containing fatal bovine serum of which the volume concentration is 9-11% when the cells, seen from a microscope, become single round cells, centrifuging, and cleaning the obtained cell deposit by using a PBS (phosphate buffered solution); B, inoculating the cell deposit which is obtained in the step A and cleaned by using the PBS to a six-hole plate to carry out induced culture; C, carrying out induced culture by selecting a system 1 adopting an induced culture medium I or a system 2 adopting an induced culture medium II; and D, weighing and changing the solution once every 2 to 3 and a half days, and observing the condition of the cells under an inverted microscope to obtain liver cell like cells with functions of the liver cells, wherein the induction time is 20-22 days.

The invention provides a bovine testicle cell line and an establishment method thereof. The establishment method comprises the following main steps: (1) primary culture, namely collecting a bovine testicle of a newborn calf in a sterile manner, cleaning adhesive tissues, separating by virtue of a trypsinization method so as to obtain bovine testicle cells with high vitality, culturing the separated bovine testicle cells with high vitality in a DMEM (Dulbecco modified eagle medium), and culturing at 37 DEG C under the condition of 5% CO2 to obtain primary bovine testicle cells; (2) transfection and screening, namely extracting and purifying eukaryotic expression plasmids pCI-neo-hTERT containing hTERT genes, leading the extracted and purified eukaryotic expression plasmids pCI-neo-hTERT to the primarily cultured bovine testicle cells in the step (1) to perform transfection by adopting a lipidosome transfection method; (3) screening and enlarged culture. The bovine testicle cells provided by the invention are easy in culture and relatively fast in growth speed, can be stably inherited, are high in motility rate and purity, and can be well preserved; the method provided by the invention is simple and convenient in operation and convenient in popularization and application. The cell line can be used for producing hog cholera vaccines, sheep poxes, ecthyma vaccines and the like.

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 discloses walnut protein polypeptide with ACE inhibiting activity and a preparation method and application thereof. The polypeptide is a mixture of compounds with amino acid sequences ofVal-Glu-Arg-Gly-Arg-Arg-Ile-Thr-Ser-Val and Phe-Val-Ile-Glu-Pro-Asn-Ile-Thr-Pro-Ala. According to the preparation method of the walnut protein polypeptide with the ACE inhibiting activity, firstly, waste walnut protein which is obtained after oil processing is conducted is subjected to degreasing treatment, then a continuous extraction method is adopted, pure water, a diluted salt solution, an ethyl alcohol solution and a diluted acid solution are sequentially used for extracting walnut glutelin-1 from degreased walnut protein, then the walnut glutelin-1 is used as a raw material, and technologies of limited trypsinization, sephadex chromatographic separation, reserve efficient liquid-phase chromatography purification and the like are adopted for preparing the walnut protein polypeptide with an ACE activity inhibition ratio over 92%. According to the walnut protein polypeptide with the ACE inhibiting activity and the preparation method and the application thereof, the waste can be turned into wealth, the walnut deep-processing and comprehensive utilization levels are raised, development of the walnut industry is promoted, good economic benefits are generated, and the obtained polypeptide can be used as an ACE inhibitor and can be used for preparing an antihypertensive drug.

The invention provides a human primary tumor cell separation and culture kit, consisting of a packing box, a cell culture plate with 6 pores, cleaning solution, trypsinization solution, collagenase-hyaluronidase digestive juice, a cell separating medium, selective tumor culture solution and an operating instruction, wherein sterile RPMI (Roswell Park Memorial Institute) 1640 cell culture solutioncontaining 400U/mL of penicillin and 400 mu g/mL of streptomycin serves as the cleaning solution. The human primary tumor cell separation and culture kit solves the problems that the conventional primary tumor cell culture process is complicated, the purity of obtained tumor cells are not high, and the tumor cells are easy to be contaminated by cells. According to the invention, the design is reasonable, the opportunity of germ contamination is less in the process of separation and culture of primary tumor cells in vitro, and only a few of centrifugal tubes and other common consumable items are needed, so that the kit is simple and convenient to use, the purity of the cultured tumor cells is higher, and the effect is good.

The invention discloses a non-trypsinization harvested cell culture method based on a temperature sensitive hydrogel material. The method includes first preparing a hydrogel solution by an emulsion polymerization method, wherein the hydrogel can maintain a solution state at the low temperature, and the hydrogel can be rapidly formed after approaching the physiological temperature of a human body.Based on the temperature sensitive hydrogel material, the method achieves non-enzymatic harvesting of cells by the following two schemes: 1) mixing a cell suspension and the hydrogel solution, placingthe mixture in a 37 DEG C cell incubator, adding a 37 DEG C medium for culture after the mixture forms the hydrogel rapidly and then releasing the cells by cooling; 2) first applying the hydrogel solution on the surface of a substrate, placing the hydrogel solution in the 37 o C cell incubator, adding the 37 DEG C cell suspension and the medium for culture after the hydrogel is formed and releasing the cells by cooling. According to the cell culture method, trypsinization is not required, a cell scraper is not required, cell surface proteins and markers can be well preserved to ensure high cell viability, and the method is a non-destructive cell culture method.

The invention discloses a method for restoring the dryness or proliferative ability of hair follicle-derived cells, comprising the following steps: cutting umbilical cord with removed arteries and veins, culturing by adding a low-sugar DMEM culture solution containing bovine serum and penicillin-streptomycin, and carrying out subculture by trypsinization when mesenchymal stem cell fusion reaches 70-80%; continuously culturing by replacing a medium without exosome serum when the cells are passaged to the 3rd to 5th generation and cell fusion reaches 60-80%; extracting by a ultracentrifugation method to obtain exosomes of mesenchymal stem cells; carrying out isolated culture on dermal papillae cells by an improved two-step enzymatic method; and culturing the obtained dermal papillae cells ina DEME basal medium, which contains PDGF, KGF, VEGF and exosomes of mesenchymal stem cells. The method of the invention can effectively restore the dryness of hair follicle-derived cells, and regulates the normal physiological metabolism of hair follicle cells by secreting various regulatory factors, thus solving the problem of alopecia from the perspective of hair follicle physiology.

The invention discloses a construction method of a human APP promoter-containing Luci cell line, and the specific method is as follows: construction of an expression vector containing a human APP promoter region-luciferase chimeric gene; cotransfection of constructed pGL3-APPP Luc-Puro plasmids and pRL-SV40 plasmids into a human embryonic kidney cell line HEK-293T cell line; laying and planting of transfected HEK-293T cells after trypsinization into a 10cm culture dish, puromycin pressurization screening to obtain five cell lines with genomes capable of stably integrating the human APP promoter region-luciferase chimeric gene, and detection of activity of firefly luciferase and renilla luciferase in the five cell lines to obtain the cell line containing the human APP promoter region-luciferase chimeric gene. The cell line prepared by the cell line construction method can be used for screening of new anti senile dementia compounds and therapeutic drugs for inhibiting the expression of human APP.

The invention belongs to the field of cell in-vitro culture, and particularly discloses a preparation method of human primary cartilage cells with high yield rate. The preparation method comprises the following steps of obtaining a cartilage tissue, and shearing the cartilage tissue by a pair of ophthalmology scissors; adding trypsinization, adsorbing supernatant, and discarding; adding II-type collagen digestion, staying overnight, filtering, adding into a culture medium, terminating digestion, centrifuging for 5min at the speed of 1000-1500rpm, obtaining precipitate of cartilage tissue, planting into a culture dish, and culturing, so as to obtain the human primary cartilage cells with high yield rate. The preparation method is mainly used for improving the shearing and centrifuging speeds of cartilage tissue blocks. The preparation method has the advantages that the centrifuging rotation speed is increased to 1000-1500rpm, the damage to the cartilage cells is avoided, and the yield rate of the cartilage cells is greatly improved; compared with the traditional cartilage cell preparation method, the yield rate of the cartilage cells is increased by about 3.55 times; after proofing by experiments, the cartilage cell has good cellular morphology.

The invention discloses a nose epithelial stem cell culture method which comprises the following steps: 1) laying nourishing layer cells into a culture dish; 2) separating primary cells of nose epithelial stem cells from nasal mucosa; 3) laying the primary cells of the nose epithelial stem cells on the nourishing layer cells, replacing a medium by a nose epithelial stem cell proliferation medium,and removing off residual tissue cells; 4) when the nose epithelial stem cells are proliferated to a density of 80%, carrying out trypsinization, and carrying out continuous passage amplification; 5)transferring the nose epithelial stem cells into a gas-liquid phase culture system and a PneumaCult-ALI differentiation medium, and carrying out differentiation culture. The invention further discloses a nose epithelial stem cell proliferation medium which comprises the following components: DMEM/F12, fetal calf serum, penicillin, streptomycin, insulin, an epidermal growth factor, hydrocortisone,3,3',5-triiodo-L-sodium adenoglycosine and an ROCK inhibitor. By adopting the method, a key application base is provided for transformation application of clinical transplanting of the nose epithelialstem cells.

The invention provides a mitochondria isolation method suitable for stem cells. The mitochondria isolation method includes the steps of firstly, marking and identifying the detection surfaces of mesenchymal stem cells, and identifying the differentiation ability of bones, cartilage and fat; secondly, performing trypsinization, and collecting cells in a centrifugal manner; resuspending, cleaning and centrifuging; thirdly, preparing mitochondria extract which includes lysate A and reaction termination liquid C; fourthly, precipitating the cells resuspended and cleaned by the lysate A, performing vortex treatment, standing, and repeating for several times; adding the reaction termination liquid C to stop the pyrolysis, and centrifuging to obtain white precipitate for the fifth step; fifthly, repeating the fourth step on the precipitate obtained by the first centrifuging in the fourth step; sixthly, placing the white precipitate obtained in the fourth step and the white precipitate obtained in the fifth step together, and centrifuging to obtain white precipitate, and using immediately or storing at 80 DEG C. The mitochondria isolation method has the advantages that mitochondria obtained by the method is good in activity and high in purity, and the method is suitable for isolating mesenchymal stem cells to obtain the mitochondria, is simple to operate and does not need grinding.

The invention relates to the field of biology. A serum-free suspension domestication method of Vero cells comprises the steps of step I, performing adherent culture on the Vero cells until the convergence rate reaches 80%, discarding supernatant, adding D-PBS free from calcium and magnesium ions, and performing washing; step II, discarding the D-PBS, adding pancreas enzymes, discarding trypsinization liquid, remaining a few pancreas enzymes which are enough to cover cells, performing incubation until the cells become round or performing incubation for definite time, and patting the bottom of aflask to dissociate cells; step III, performing re-suspension with a Vero-S suspension serum-free culture medium; step VI, discarding the supernatant, and performing re-suspension with Vero-S suspension serum-free culture medium; step V, diluting the density of the cells, performing inoculation to a disposable shake flask, and performing culture; step VI, when the density of the cells reaches 3*10<6> cells/ml, performing cell passage; and step VII, once the density of the cells can reach 3*10<6> cells/ml or above in 48h for continuous 10 generations or above and the motility rate of the cellsis not less than 95%, thinking that the cells are adapted to suspension culture.

The invention provides a method for promoting the survival of injured neurons by virtue of mitochondria transplantation. The method comprises the following steps: (1) carrying out trypsinization centrifuge on P1-P3-generation mesenchymal stem cells, and counting the number of the cells; (2) separating by virtue of a mitochondria separation agent to obtain mitochondria, and carrying out protein quantification and ATP content determination; and (3) preparing suspension from the mitochondria obtained in the step (2), and transplanting the suspension to a neuron injury region to contact with the neuron injury region. Compared with the transplantation of mesenchymal stem cells, the method has the advantages that the allogeneic immunogenicity problem is avoided, the implantation problem of chromosomes of allosome-derived stem cell nucleuses is avoided, and the regeneration of neuronal cells is promoted.

The invention discloses a culture method of lawsonia intracellularis. The culture method comprises the following steps of step 1, performing adherence culture to obtain pig intestinal tract cells; step 2, after cell adherence culture for 6-8h, inoculating cells with lawsonia intracellularis and cell culture fluid in the volume ratio being (0.01-1) to 1, performing placing in improved micro aerobicenvironment, and performing culture for 1-96h; and step 3, performing trypsinization on infected cells, collecting the infected cells, performing re-suspending on the infected cells with cell frozen-storage fluid, and performing transferring into a liquid nitrogen jar for storage. The invention further discloses an application of the lawsonia intracellularis cultured by the culture method. The lawsonia intracellularis is applied to research of lawsonia intracellularis separated culture and pathogenicity characteristics. The pig intestinal tract cells are used for culturing the lawsonia intracellularis, the culture in vitro environment in the body of a host is strictly simulated, besides, an improved micro aerobic culture box is used for culturing the lawsonia intracellularis, the operation is simple, the culture cycle is short, the cost is low, and the success rate of culturing the lawsonia intracellularis is greatly increased.

The invention discloses a preparation method and application of a CGA cross-linked sodium alga acid-gelatin blend membrane joint patch, and belongs to the field of medical technology. The preparationmethod of the joint patch comprises the following steps: after selection and trypsinization of chick embryo precartilage stem cells, resuspending a sodium alga acid solution and adding CGA in a cell suspension; after uniform blowing and beating, taking and adding a small amount of the cell suspension into a CaCl2 solution; after sodium alga acid is polymerized with the chick embryo precartilage stem cells to form a 3D compound with stable shape, changing a cartilage induction medium; after culture in vitro for 14 days, obtaining the CGA cross-linked sodium alga acid-gelatin blend membrane joint patch. Cartilage injuries on the surfaces of external condyles of thighbones of newly hatched chicken are taken as models, the joint patch is transplanted to the positions of the cartilage injuries,the restorative effect of the cartilage injuries are observed through observational means such as behavior, iconography and morphology, and possible mechanisms are developed by developing a molecularbiological method, so that experimental evidence is provided for comprehensively developing and using CGA and searching effective joint injury therapeutic methods.