31 results about "Feeder Layer Cells" patented technology

The invention discloses construction of a Guangdong small-ear spotted pig spermatogonial stem cell in-vitro culture system and screening of surface markers, and belongs to the technical field of cell culture.The technical scheme includes that testis are broken firstly, then enzyme digestion, density gradient centrifugal purification and differential adherent purification are conducted, and purified spermatogonial stem cells are obtained; inoculating the obtained spermatogonial stem cells to feeder layer cells and culturing in a culture medium; the culture medium is a DMEM culture medium added with FBS and growth factors; the mass fraction of the FBS in the DMEM culture medium is 8-12%, an enzyme digestion method, a density gradient centrifugation method and a differential adhesion method are combined for application, the Guangdong small-ear spotted pig spermatogonia stem cells with certain purity are obtained, cells enriched and purified in vitro can proliferate massively, and an in-vitro culture system of the Guangdong small-ear spotted pig spermatogonia stem cells is established.

The present invention belongs to the field of insect cell culture technology and discloses a method for preparing an Sf9 cell feeder layer. The method mainly comprises two steps: step S1 is subculture of Sf9 cells, in which Sf9 cells are cultured in a suitable culture medium to a suitable cell confluence; step S2 is mitomycin C treatment of the Sf9 cells, in which a specific concentration of mitomycin C is used to treat the subcultured Sf9 cells at a specific time and temperature to inhibit their cell division ability, followed by washing to remove mitomycin C and replacing with fresh culture medium. The feeder layer cells obtained by the present invention can effectively inhibit their own proliferation while maintaining good cell activity and structural integrity. They can survive stably and provide necessary support for the co-culture system. They can be used for the cultivation of insect symbiotic bacteria and other difficult-to-culture insect cells. The method is simple to operate, has mild conditions, and good reproducibility. The prepared feeder layer has a stable effect, providing a powerful tool for related research fields.

The invention provides a preparation method of a monoclonal antibody of coccidian oocyst protein, and belongs to the technical field of antibodies. The method comprises the following steps: carrying out flow sorting on single antigen-specific B lymphocytes, carrying out stimulated differentiation culture in a specific culture medium by taking EL4B5 as feeder layer cells, cracking obtained plasma cells, carrying out PCR (Polymerase Chain Reaction) amplification to obtain a variable region sequence of a monoclonal antibody, and carrying out recombinant expression to obtain the coccidian oocyst protein monoclonal antibody. The monoclonal antibody of the coccidian oocyst protein provided by the invention comprises at least one of coccidian monoclonal antibodies 6B10, 5C8 and 5D5. According to the present invention, the antigen affinity verification is performed on the monoclonal antibodies obtained through the recombinant expression, and the results show that the three monoclonal antibodies have good antigen affinity and good detection specificity, such that the effective tool is provided for the subsequent diagnosis and detection of the porcine coccidiosis and the porcine coccidiosis infection diseases.

The application discloses a method for inducing NK cells based on a porous material. The method for inducing NK cells provided by the application is based on a device containing a mesh culture tank and porous gelatin sponge and a culture medium, and can efficiently induce iPSCs-NK. The method solves the problems that current differentiation culture technologies of iPSC-NK rely on feeder layer cells of animal origin, the differentiation efficiency is low, and the purity is insufficient and needs to be further sorted. The method has the potential for large-scale industrial production.

The invention discloses a sheep female stem cell separation and in-vitro culture method, and relates to the technical field of biologication.According to the technical scheme, the sheep female stem cell separation and in-vitro culture method is characterized in that 0.25% pancreatin is adopted for digestion for 1 hour at the room temperature, mechanical cutting operation is combined, intercellular connexin can be effectively decomposed, and the survival rate of the sheep female stem cells is increased; the ovarian granular cells are quickly released. Medical alcohol and a culture medium containing double antibodies are used for cleaning ovaries for many times, so that the probability of bacterial and fungal contamination is remarkably reduced. A DPBS buffer solution preheated at 37 DEG C and a DMEM / F12 culture medium containing 10% of FBS are used in the whole process, and stress damage to cells caused by temperature shock is avoided. Complete granular cells and tissue fragments are efficiently separated through filtration and centrifugation, and the purity of a cell suspension is improved. The method is developed on the basis of ovaries of sheep, does not depend on feeder layer cells or a complex three-dimensional culture system, can be operated only by conventional laboratory equipment, and is suitable for research scenes with limited resources.

The invention discloses a high-activity NK cell as well as an in-vitro amplification method and application thereof. The method comprises the following steps: S1, selecting a basic culture medium; s2, other culture medium components are added, wherein the following components are added according to the ratio: 5-10 [mu] g / mL of lactobacillus, 30-50 [mu] M of ergothioneine, 80-100 [mu] g / mL of fucoidan polysaccharide, 2-3 [mu] g / mL of holothurin, 3-5 [mu] M of astaxanthin, 150-200 IU / mL of IL-2150, 20 ng / mL of IL-1515, 10 ng / mL of IL-218, 2 mmol / L of stable L-glutamine, 1 mmol / L of human pyruvic acid and 5% v / v of human serum albumin; s3, carrying out feeder layer cell treatment and co-culture, mixing inactivated K562 cells and purified NK cells according to a ratio of 1: 10-1: 20, inoculating to a culture container, and removing feeder layer K562 cells after 10 days; and S4, regulating and controlling culture conditions, maintaining the constant temperature of 37.0 + / -0.5 DEG C, controlling the pH value to 7.2-7.4, culturing in a 2%-5% low-oxygen environment, and culturing for 14-21 days to obtain the high-activity NK cells. According to the in-vitro amplification method of the high-activity NK cells, disclosed by the invention, the core problems of difficult in-vitro proliferation and low activity of the NK cells in the prior art are effectively solved by optimizing the synergistic matching of natural active substances, key cell factors and nutritional supplements in the basic culture medium.

The present invention relates to the application of a dynamically cross-linked hydrogel. Specifically, the dynamically cross-linked hydrogel is formed by mixing gelatin and a cross-linking group-modified cyclodextrin into a hydrogel prepolymer. After embryonic stem cells or immune cells are mixed in, a cross-linking reaction occurs under the action of a cross-linking initiator or ultraviolet light to produce the dynamically cross-linked hydrogel. The hydrogel maintains the stemness of embryonic stem cells in a basal culture medium without the need for feeder cells or differentiation inhibitors, is simple to operate, and is suitable for large-scale culture. The immune cell-loaded hydrogel can be directly used for injection and filling in vivo.

The invention discloses a preparation method of feeder layer cells and application of the feeder layer cells in primary tumor cell culture, and belongs to the technical field of biology. The method comprises the following steps: digesting tumor tissues by using collagenase and hyaluronidase, resuspending cells by using a culture medium, passing through a cell sieve, inoculating into a culture vessel for culture, obtaining tumor-related fibroblasts through differential digestion, repeating the steps of culture and differential digestion, adding the obtained fibroblasts into a culture medium containing 25-35mu g / ml mitomycin C, and treating for 3-5 hours, thereby obtaining the tumor-related fibroblasts. And cleaning the cells with a fresh culture medium for 1-5 times to obtain the feeder layer cells. The operation process is simple, compared with general trophoblast cells 3T3-J2 (Kerafast company), the trophoblast cells 3T3-J2 are low in price and easy to obtain, the problem that the culture period is limited is solved, the prepared feeder layer cells can be directly applied to primary tumor cell culture, and the cost of primary tumor cell culture is further reduced.

The invention discloses a suspension culture cell monoclonal screening culture method. The method comprises the following steps: selecting cells under suspension culture conditions and inducing the cells to recover adherence; separating the single cells developed into obvious clones, and carrying out radioactive ray irradiation on the remaining cells, so that the remaining cells lose the multiplication capacity and become feeder layer cells; culturing the separated monoclonal cells, recording the culture time and culture environment, establishing a high-density cell bank, and establishing a growth time prediction formula for the multiple monoclonal cells according to the culture time; and calculating the specific time for culturing the required number of cells according to the growth time prediction formula established in the step S3. According to the method, the remaining cells are subjected to radioactive ray irradiation and are converted into feeder layer cells with important effects, so that the waste of cell resources is avoided, the flexible utilization of the cell resources is realized, and the treatment cost of the remaining cells is also remarkably reduced.

Provided are a cow milk-derived stem cell, and an isolation and culture method therefor. The method comprises the following steps: (1) taking fresh cow milk, and isolating cells therefrom; (2) coating a culture dish with a matrix gel in advance, discarding the coating solution, then washing the culture dish with a washing solution, and discarding the washing solution; (3) inoculating the cells treated in the step (1) on the coated culture dish, and culturing the cells with a stem cell culture medium; and (4) when the cell confluence reaches 80% or more, digesting the cells with a digestion enzyme, terminating the digestion, isolating the cells, resuspending the cells using a CTFR culture solution, then inoculating the cells on a feeder layer for culture, promoting colony formation, and performing subculture to obtain cow milk-derived stem cells. The cow milk-derived stem cell has OCT4, SOX2, NANOG and SSEA4 markers.

The present application provides a method and application for enhancing Treg cell function, which belongs to the field of cell engineering technology. In this method, hUC-MSCs are irradiated with gamma rays to form feeder layer cells, and then the feeder layer cells are co-cultured with Treg cells to obtain Treg cells with enhanced cell function. The co-culture of feeder layer cells and Treg cells can enable Treg cells to enhance their ability to inhibit the activation and proliferation of immune cells, and can be used as universal cells in clinical research on autoimmune diseases, graft-versus-host disease, etc.

The invention relates to a culture medium for obtaining pig pluripotent stem cells and an induction culture method of the pig pluripotent stem cells. The method comprises the following steps: transferring transcription factors for inducing reprogramming into fetal fibroblasts of pigs, then inoculating the obtained cells on feeder layer cells of a culture plate, culturing for 1 day, then continuously culturing the cells by using an iCD3 culture medium for 9-14 days, changing the liquid by using the iCD3 culture medium every other day or two days, and culturing the cells for 5-7 days to obtain compact epithelial cell morphology; culturing the cells obtained in the step 1 by using an LACID culture medium until stem cell-like clones appear; the method comprises the following steps: cloning and subculturing original cells by using a mechanical separation method, and establishing piPSCs cells; and carrying out passage on the obtained piPSCs cell line, and culturing after passage. The piPSCs culture medium disclosed by the invention has relatively high multiplication capacity, one generation can be amplified every 17.1 hours, a gene editing experiment can be quickly carried out, and the cultured piPSCs have good development and differentiation capacity.

The present disclosure relates to methods and compositions for reprogramming cells to a pluripotent state. In particular, it relates to an integration- and feeder cell-free method for reprogramming primary human fibroblast cells to induced pluripotent stem cells (iPSCs).

The invention discloses a method for high-density amplification of feeder layer cells and application thereof. According to the method, the time for transferring the feeder layer cells to the high-density culture bottle in the feeder layer cell resuscitation and multiplication culture stage process is defined, and the cell concentration and the liquid changing and supplementing mode of feeder layer cell high-density multiplication culture are controlled. According to the method for high-density amplification of the feeder layer cells, on the premise that the quality of the feeder layer cells is guaranteed, the consumption of a culture medium used for producing the same number of feeder layer cells is reduced by 50%, the operation steps are remarkably simplified, and the inter-batch stability is higher.

This invention relates to a modified feeder cell and a method for culturing natural killer (NK) cells, aiming to enhance the proliferation capacity and killing activity of NK cells by using a specific feeder cell.

The present invention relates to a method for inducing the differentiation of corneal epithelial cells from pluripotent stem cells. More specifically, the present invention relates to a method for autonomously differentiating pluripotent stem cells, such as human iPS cells, into ectodermal cell lineage in a serum-free medium without using feeder cells and inducing the differentiation of the resultant ocular surface ectodermal lineage cells into corneal epithelial cells.

Provided are an HLA-E modified feeder cell and a method for culturing natural killer cells (NK cells). By means of culturing NK cells using the HLA-E-modified feeder cell, NK cells having a low expression of inhibitory receptor NKG2A and a high expression of activating receptors, such as NKG2C and NKp44, can be obtained, thereby significantly enhancing the killing activity of NK cells against target cells, particularly those with a high HLA-E expression.

The invention relates to the technical field of biology, and discloses a culture method of autologous peripheral blood NK immune cells, which comprises the following steps: in a first stage, carrying out selective activation culture in a culture medium containing an activated cytokine, an SIRT1 activator and an NAD + precursor; in the second stage, transferring into a culture medium containing amplification type cytokines, tricarboxylic acid cycle intermediate products and fatty acid-albumin compounds for metabolic reprogramming and amplification culture; and a third stage of performing pulse-type treatment on the cells by using a culture medium containing a function-enhanced cytokine and a histone deacetylase inhibitor so as to enhance the effector function. Through three-stage sequential regulation and control, under the condition of no feeder layer cells, the amplification multiple, purity, cytotoxic killing activity and cytokine secretion ability of the NK cells are remarkably improved, and an NK cell product with both quantity and quality is obtained.

The invention belongs to the technical field of insect cell culture, and discloses a preparation method of an Sf9 cell feeder layer, which mainly comprises the following two steps: S1, subculturing Sf9 cells: culturing the Sf9 cells in a suitable culture medium to a suitable cell confluence degree; and S2, mitomycin C treatment of the Sf9 cells: treating the subcultured Sf9 cells at specific time and temperature by using mitomycin C with specific concentration to inhibit the cell division ability of the Sf9 cells, then washing to remove the mitomycin C, and replacing a fresh culture medium. The obtained feeder layer cells can effectively inhibit self proliferation, maintain good cell activity and structural integrity, can stably survive and provide necessary support for a co-culture system, and can be used for culture of insect symbiotic bacteria and other insect cells difficult to culture; the method is easy and convenient to operate, mild in condition and good in repeatability, the prepared feeding layer is stable in effect, and a powerful tool is provided for the related research field.

The invention provides a method for culturing PBMC (peripheral blood mononuclear cells) by taking red blood cells as feeder layer cells, and belongs to the technical field of immune cell culture. The method comprises the following steps: adding red blood cells into a culture container, and fixing by using a fixing reagent; adding the PBMC and then adding the complete culture medium; and finally adding at least one cell factor. According to the method, the erythrocytes are used as feeder layer cells for in-vitro culture of the PBMC, the proliferation pore ratio of the PBMC is high, and the method has a wide application prospect.

The invention provides a line establishment method of primordial germ cells from chick embryo blood. The line establishment method comprises the following steps: obtaining fertilized eggs incubated to a 14-17HH period; sucking chick embryo blood from the dorsal aorta by adopting a micro-injection needle with a specific size and specific suction pressure; carrying out centrifugal treatment, and resuspending the cells by adopting a PGCs complete culture solution added with an antifungal drug; carrying out subculture by adopting a PGCs complete culture solution until the PGCs monoclonal cell line is successfully established; according to the line establishment method, a traditional enzyme digestion separation mode is not adopted, dependence on feeder layer cells is avoided, and the undifferentiated state of the PGCs can be maintained directly through culture medium components of a specific formula by adopting the chick embryo blood obtained through specific operation; compared with the prior art, the operation is more efficient and simpler; more importantly, the technical scheme can be used for quickly and successfully establishing the line, is good in repeatability and high in line establishment success rate, and can be popularized and applied to in-vitro line establishment of the PGCs and subsequent biological design breeding and model animal production.

Cardiovascular diseases represent the leading cause of mortality and accounts for approximately one-third of deaths globally each year. During the last decade, tremendous efforts have been made to take advantage of advances in regenerative cardiology using the approaches of adult or pluripotent stem cells. However, the current protocols do not recapitulate cardiac-related architectures. Thus, there remains a need in the art for further methods for producing cardiac organoids actually mimicking a cardiac tissue, starting from pluripotent stem cells. Notably, there remains a need in the art for producing cardiac organoids comprising cardiac muscle cells and endothelial cells, including both atrium-like and ventricular-like chambers. Such cardiac organoids that reliably mimic a cardiac tissue would notably allow testing for the physiological effects of known drug substances as well as candidate molecules, particularly in embodiments wherein the said cardiac organoids are produced by starting from cells originating from patients affected with a cardiac disease or disorder such as a cardiac disease or disorder of genetic origin. It is now provided a novel matrix-free and feeder-free method of generating mammal iPSC-derived cardiac organoids, which method includes both steps of dynamic culture conditions and static culture conditions. As it is experimentally shown, cardiac organoids obtained with the matrix-free and feeder-free method disclosed herein are relevant models for testing potentially active substances on cardiac organoids obtained from iPSCs derived from subjects affected with a cardiac disease or disorder.

The invention discloses a line establishment method of porcine formation state pluripotent embryonic stem cells, and belongs to the technical field of cell biology. The invention aims to obtain a stable passage pig formation pluripotent embryonic stem cell line. The invention provides a line building method of a pig formation state pluripotent embryonic stem cell, which comprises the following steps of: inoculating a pig parthenogenetic activated blastocyst, a pig in-vitro fertilized blastocyst or a pig in-vivo blastocyst into a pFESCM culture medium on which feeder layer cells are paved in advance, replacing the culture medium once every two days until the diameter of a growth halo reaches 100-500 microns, blowing away the cells into a single cell or a small block mass with 3-4 cells, and culturing the small block mass in the form of the pig formation state pluripotent embryonic stem cell. The method comprises the following steps: inoculating the porcine embryonic stem cells into a pFESCM culture medium which is pre-paved with feeder layer cells, continuously culturing for 2-4 days to obtain first-generation porcine formational pluripotent embryonic stem cells, and carrying out passage to eight generations to obtain a stably inherited cell line. The prepared embryonic stem cells are used for breeding, gene editing, animal cloning, medical models, drug development carriers and induced production of reproductive gametes.

The present invention belongs to the field of microbial culture technology and discloses a method for culturing insect symbiotic bacteria. The method comprises the following steps: Step S1, preparing an insect cell feeder layer; Step S2, collecting insect eggs; Step S3, disinfecting the insect eggs; Step S4, crushing the insect eggs and inoculating and culturing the symbiotic bacteria; and Step S5, isolating and expanding the insect symbiotic bacteria. The present invention utilizes an insect cell feeder layer to cultivate insect symbiotic bacteria, and utilizes cytokines or other unknown components secreted by the feeder layer cells to promote the growth and reproduction of symbiotic bacteria that are difficult to culture directly.

The invention belongs to the technical field of microorganism culture, and discloses an insect symbiotic bacterium culture method which comprises the following steps: S1, preparing an insect cell feeding layer; s2, insect eggs are collected; step S3, disinfection of insect eggs; s4, crushing insect eggs and performing inoculated culture on symbiotic bacteria; and S5, separation and amplification culture of the insect symbiotic bacteria. The insect cell feeding layer is used for culturing the insect symbiotic bacteria, and cell factors or other unknown components secreted by cells in the feeding layer are used for promoting growth and reproduction of the symbiotic bacteria which are difficult to directly culture.

The invention discloses a culture medium and a culture method for selectively amplifying NK (Natural Killer) cells. The culture medium for selectively amplifying the NK cells comprises a KBM581 basal culture medium and an additive, and the additive comprises the following components in concentration: 500IU / mL of IL-2, 25ng / mL of IL21, 20ng / mL of IL15, 1ug / mL of OK432, 10% of human platelet lysate and 0.05 ug / mL-20ug / mL of dexamethasone sodium phosphate injection. According to the culture method for selectively amplifying the NK cells, the culture medium for selectively amplifying the NK cells is adopted, coating, cell sorting and feeder layer cells are not needed, and operation is easy. After the culture medium and the culture method for selectively amplifying the NK cells are used for culturing for 2 weeks, the amplification multiple of the NK cells can reach 200 times, the proportion of the NK cells reaches 90% or above, the viability of the NK cells reaches 95% or above, and the NK cells are stable in batches and can meet clinical requirements.

Provided are methods of producing avian embryonic stem cells. In certain embodiments, the methods comprise culturing epiblast cells obtained from a stage IX-XI avian embryo in a cell culture medium present in a feeder-free cell culture container. Also provided are methods of producing two-dimension cultures, spheroids, or organoids from the avian embryonic stem cells produced according to the methods. For example, provided are methods of producing avian telencephalic organoids from the avian embryonic stem cells. Also provided are methods of using the avian embryonic stem cells, spheroids and organoids produced according to the methods of the present disclosure. For example, provided are screening methods comprising contacting the avian embryonic stem cells, spheroids or organoids with a test agent, and assessing for an effect of the test agent on the avian embryonic stem cells, spheroids or organoids.

The invention relates to the technical field of cell culture, in particular to a method for reprogramming somatic cells into pluripotent stem cells in a non-integrated mode. According to the non-integrated reprogramming method provided by the invention, only any combination (preferably at least comprising OCT4 and SOX2) of five transcription factors such as OCT4, SOX2, SV40LT, has-miRNA-302 / 367 and JDP2 is used, and human somatic cells can be reprogrammed into iPSC. Due to the fact that a reprogramming tool cannot be inserted into a genome, after the cells are reprogrammed into the iPSC, the reprogramming tool (plasmid, mRNA or non-conformity virus) of the iPSC can be gradually lost in the subsequent passage process. A transcription factor KLF4 or MYC family gene (such as c-Myc, L-Myc or n-Myc) is not used in the reprogramming process; a p53 inhibitor is not used; feeder layer cells are not used; small molecular compounds such as a GSK-3alpha / GSK-3beta inhibitor, a TGF beta inhibitor, an MEK inhibitor and a histone deacetylase inhibitor do not need to be used in the reprogramming culture medium. The reprogramming method is safe, simple, convenient and easy to use.

The present disclosure relates to methods and compositions for reprogramming cells to a pluripotent state. In particular, it relates to an integration- and feeder cell-free method for reprogramming primary human fibroblast cells to induced pluripotent stem cells (iPSCs).