109 results about "Feeder Cell" patented technology

The invention relates to the technical field of biology, in particular to a Treg cell amplification culture medium and a preparation method of Treg cells. According to the method for in-vitro amplification of the regulatory T cells (Treg) from peripheral blood, the amplification efficiency and purity of the Treg cells are improved, the culture cost is reduced, the culture period is shortened, and the problem of trophoblast cell residues in subsequent clinical application is solved.

The invention discloses a method for differentiating induced pluripotent stem cells into megakaryocytes and platelets, and relates to the technical field of induced pluripotent stem cell directional differentiation, and the method comprises the following steps: inoculating an iPSC single cell into a culture vessel, and carrying out adherent culture through a culture solution; after the cell adheres to the wall and before cloning is not formed, the liquid is changed into a first induction culture liquid, and the cell is induced and differentiated into a mesoderm cell; changing the solution into a second induction culture solution to induce and differentiate the mesoderm cells into hematopoietic endothelial cells; collecting the suspension cells, culturing the suspension cells in an amplification culture solution, and enriching megakaryocytes; inducing megakaryocyte polyploidy through a third induction culture solution; and culturing the polyploidy megakaryocyte through a fourth induction culture solution to promote platelet release. The method for inducing the pluripotent stem cells to differentiate into megakaryocytes and platelets is convenient and rapid, the megakaryocytes and platelets can be obtained through iPSC in a short time, and feeder cells, serum and additional gene editing are not needed; meanwhile, the yield is high.

Several embodiments disclosed herein relate to methods and compositions for enhanced expansion of NK cells in culture, through the repeated co-culturing of NK cells with feeder cells and the selective use of stimulatory interleukins. In several embodiments, the methods utilize one or more soluble interleukins as culture media supplements at one or more time points during expansion of the NK cell, or other immune cell, which results in a highly expanded and highly cytotoxic population of cells, for use in, for example allogeneic cellular immunotherapy.

The invention provides a construction method and application of a gene modified K562 cell line, and belongs to the technical field of biological medicines. According to the invention, two viruses CD86-4-1BBL and mbIL-21 are respectively subjected to gene transduction packaging by adopting a third-generation lentivirus four-plasmid packaging system, a K562 cell line is infected by the lentiviruses CD86-4-1BBL and mbIL-21, and a monoclonal cell line capable of stably expressing CD86, 4-1BBL and mbIL-21 proteins is obtained through screening. According to the method, cell lines with the cloning numbers of V5-E9, V10-A5, V5-D6 and V6-D7 are obtained through screening, the four cloning cell lines can serve as feeder cells for stimulating NK cell amplification after being amplified and subjected to irradiation treatment, NK cell amplification can be effectively promoted, the amplification multiple of the NK cells reaches up to 6000-8000, and large-scale preparation and production of the NK cells for treatment are met.

The present invention provides a method for producing retinal cells or a retinal tissue, comprising the following steps (1)-(3):(1) a first step of culturing human pluripotent stem cells in the absence of feeder cells and in a medium comprising a factor for maintaining undifferentiated state,(2) a second step of culturing the pluripotent stem cells obtained in the first step in suspension in the presence of a Sonic hedgehog signal transduction pathway activating substance to form a cell aggregate, and(3) a third step of culturing the aggregate obtained in the second step in suspension in the presence of a 1) a BMP signal transduction pathway activating substance to obtain an aggregate containing retinal cells or a retinal tissue.

Described herein are serum-free and feeder-free methods and compositions for the tunable differentiation and production of CD4+ (single positive) T cells and CD8+ (single positive) T cells, by adjusting T cell receptor (TCR) stimulation and notch activation (or notch signaling activation, stimulation or inducement). In some other aspects the invention provides a method and in vitro niche and cell culture media that results in enhancing CD4+ (SP) T cell generation, including naive mature CD4+ single positive T cells from double positive (DP) CD4+8+cells or CD8+(SP) T cells by TCR stimulation while having low or no notch signaling activation, stimulation or inducement. In yet other aspects, the invention provides a method, niche and culture media for producing cells derived from CD4+ (SP) T cells, including Treg cells.

This application provides a method, device, and storage medium for establishing a feeder link. The method includes: determining to provide a feeder service to a gateway station and broadcasting a system message about the feeder cell; and, after establishing a radio resource control (RRC) connection with the gateway station, configuring a feeder data radio bearer (DRB) for the gateway station. This method establishes a feeder link.

To provide a method for producing induced pluripotent stem cells (iPS cells) that can initialize somatic cells without using feeder cells or a substrate. For production of iPS cells, the following steps are carried out: I. introducing an initialization gene into a somatic cell; and II. performing initialization and amplification culture of the cell into which the gene has been introduced, in a liquid medium comprising at least one of a protein kinase Cβ (PKCβ) inhibitor and a WNT inhibitor under a suspension culture condition.

A method for generating or expanding a population of natural killer cells (NK cells) via using modified K562 feeder cells having expression of factors capable of increasing activation and proliferation of natural killer cells, wherein the factors capable of increasing activation and proliferation of NK cells include: (A) a co-stimulatory molecule comprises CD80 molecules, and 4-1BBL molecules; and (B) a cytokine, specifically membrane-bound IL-21 (mbIL-21).

The invention provides a method for obtaining GiNK cells through novel culture medium amplification, a large number of highly purified human natural killer (NK) cells are obtained by combining IL-15 active fragments, PI3K inhibitors, key receptors targeting the surfaces of the NK cells, antibodies enhancing activation of the NK cells and various cytokines, and the GiNK cells can be used for preparing the human natural killer cells. Expansion from human peripheral blood mononuclear cells (PBMCs) can be achieved without the use of feeder cells. Through culture for 2-3 weeks, the GiNK cells can be amplified by hundreds to thousands of times, and the proliferation efficiency, functional activity and anti-tumor effect of the GiNK cells are remarkably improved. The culture medium provided by the invention not only reduces the use concentration of cell factors, but also solves the problem of function depletion of GiNK cells in in-vitro culture through the synergistic effect of various additives, and provides important technical support for clinical application of the GiNK cells.

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.

Provided are methods for expanding tumor-infiltrating lymphocytes (TILs), which include co-culturing an initial cell population containing TILs with first feeder cells to obtain a first expanded cell population; and then co-culturing the first expanded cell population with second feeder cells to obtain an expanded TIL population. The methods can quickly produce a large number of TILs from a small tumor sample.

The present invention provides a method for producing neural cells or a neural tissue, including the following steps (1)-(3):(1) a first step of culturing pluripotent stem cells in the absence of feeder cells and in a medium containing 1) a TGFβ family signal transduction pathway inhibiting substance and / or a Sonic hedgehog signal transduction pathway activating substance, and 2) a factor for maintaining undifferentiated state,(2) a second step of culturing the cells obtained in the first step in suspension to form a cell aggregate, and(3) a third step of culturing the aggregate obtained in the second step in suspension in the presence or absence of a differentiation-inducing factor to obtain an aggregate containing neural cells or a neural tissue.

The present disclosure provides an engineered population of natural killer cells wherein the NK cells express at least one polypeptide selected from the group consisting of human leukocyte antigen E, human leukocyte antigen F, and human leukocyte antigen G. The NK cell comprises a genetically modified disruption of one or more copies of an endogenous beta-2 microglobulin and a genetically modified disruption of one or more copies of a human leukocyte antigen class II-related gene, the human leukocyte antigen class II-related gene is selected from a protein containing regulatory factor X-related ankyrin, a regulatory factor 5, a regulatory factor X-related protein and a class II trans-activator. Also provided are a method of producing human pluripotent stem cell-derived engineered natural killer cells without feeder cells, and a method of treating a disease or condition in a subject by administering the NK cells to the subject.

The present invention relates to the field of immune cell therapy, and specifically relates to a method for expanding NK cells and use thereof. The method comprises using OK-432 in combination with irradiated feeder cells, such as K562 cells, to expand and culture NK cells in vitro.

The present invention relates to a method for amplifying an invariant natural killer T (iNKT) population and an application thereof, the method comprising: providing a peripheral blood stem cell sample; culturing the peripheral blood stem cell sample in an amplification culture medium, wherein the amplification culture medium comprises interleukin 2, interleukin 15 and alpha-galactosylceramide; and adjusting the number of the cultured cells, and co-culturing the cultured cells and feeder cells in the amplification medium, the feeder cells being a dendritic cell and a peripheral blood mononuclear cell.

The present invention relates to the production and isolation of monoclonal antibodies, in particular (but not limited to) novel feeder cell lines for culturing B cells that produce monoclonal antibodies. The invention also relates to application of the feeder cell line in culturing B cells generating monoclonal antibodies and in separating the B cells from a cell culture medium. The invention also relates to a method for culturing and isolating B cells producing monoclonal antibodies and a method for isolating monoclonal antibodies.

The object of this invention is to provide a method of producing a nerve bundle including efficiently extending axons of neural cells. As a solution to accomplish this end, neural cells are cultivated in the presence of feeder cells including at least one type of cells selected from vascular component cells and perivascular cells.

The utility model discloses a feeder cell preparation operating table for a cataract mouse model, which comprises an operating table provided with a sliding hole and a mounting rod locked in the sliding hole through a nut, and further comprises a mounting piece elastically arranged on the mounting rod, the same spring piece is jointly installed at the center positions of the pair of rotating rods, one side of the spring piece is fixedly connected with the installation part, and the end of each rotating rod is sleeved with the same rubber belt so that the spring piece can release elastic potential energy to drive the pair of rotating rods to do circular motion around the rotating shaft to the bottom end to expand and tighten the rubber belt. Through cooperation of the mounting part, the mounting rod, the rotating rod, the spring piece and the rubber belt, the multi-purpose mouse cell extraction operation table is formed, the application range of the operation table is expanded, the operation accuracy is improved, meanwhile, injuries caused by mouse operation are reduced, the structural design is compact and reasonable, operation is convenient and fast, postoperative cleaning and disinfection are easy, and the operation table is worthy of popularization and application. And the practicability is high.

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.

A cell population comprising Corin- and / or Lrtm1-positive cells was produced by the following steps (1) and (2), from which Corin positive and / or Lrtm1 positive cells are collected using a substance that binds to Corin and / or a substance that binds to Lrtm1, and dopaminergic neuron progenitor cells are produced by performing suspension culture of the Corin positive and / or Lrtm1 positive cells in a culture solution containing one or more nutritional factors:(1) a step of performing adhesion culture of pluripotent stem cells in a medium for maintaining undifferentiated state containing a Sonic hedgehog (SHH) signal stimulant, and an undifferentiated state-maintaining factor in the absence of feeder cells but in the presence of an extracellular matrix, and(2) a step of culturing the cell population obtained in the step (1) in a culture solution containing one or more differentiation-inducing factors.

The invention belongs to the field of medical biology, and provides a method for in-vitro amplification of NK (Natural Killer) cells by utilizing extracellular vesicles of artificial antigen presenting cells. A feeder cell, namely an artificial antigen presenting cell, used in the invention carries four active molecules, namely CD48, anti-NKp46, mIL-15 and mIL-21. The structure of the exosome secreted by the exosome is complete, the particle size is 100-200 nanometers, and the exosome carries the four active molecules. The separated and purified exosome aAPC-Exo is effectively amplified in vitro to obtain high-purity NK cells, and the high-purity NK cells can be well proliferated in the later stage and have a relatively strong killing effect on tumor cells, so that a new methodology and material basis is provided for immune cell biological treatment.

The invention relates to a stem cell culture system purification method based on a double-helix inertial micro-fluidic chip. The double-helix inertial micro-fluidic chip comprises a base and five sequentially connected micro-pipelines fixed on the base, the five sequentially connected micro-pipelines are respectively an inlet micro-pipeline, a first helix micro-pipeline, an S-shaped micro-pipeline, a second helix micro-pipeline and an outlet micro-pipeline, and the first helix micro-pipeline and the second helix micro-pipeline are distributed in a staggered manner. The S-shaped micro-pipeline is positioned in the center of the double-spiral channel formed by the first spiral micro-pipeline and the second spiral micro-pipeline; the front end of the outlet micro-pipeline is divided into a first stage and a second stage, the width of the second stage is larger than that of the first stage, and the tail end of the outlet micro-pipeline is divided into a collection outlet micro-pipeline and a waste outlet micro-pipeline. According to the method, the stem cells are sorted and purified from a stem cell-feeder layer cell co-culture system in a high-flux and label-free manner.

The present invention relates to a growth medium, which does not include cholera toxin, for culturing cells, such as epithelial cells, in particular keratinocytes, use of the medium to grow cells, (for example epithelial cells, such as keratinocytes) and kits comprising the medium. Also provided is a method of generating human full thickness skin, fully human epidermis or fully human dermis involving culturing keratinocytes and feeder cells in said cell culture medium.

The method for producing a cell aggregate including glial progenitor cells according to the present invention comprises:(1) a step of subjecting pluripotent stem cells to suspension culture in an embryoid-body-forming culture medium containing one or more SMAD signaling inhibitors and one or more Wnt signaling activators in the absence of feeder cells for 5 days to 10 days, to form a cell aggregate;(2) a step of subjecting the cell aggregate obtained in (1) to suspension culture in an embryoid-body-forming culture medium containing retinoic acid;(3) a step of subjecting the cell aggregate obtained in (2) to suspension culture in an embryoid-body-forming culture medium or neuron-and-glia-proliferating culture medium containing retinoic acid and one or more SHH signaling activators; and(4) a step of subjecting the cell aggregate obtained in (3) to suspension culture in a neuron-and-glia-proliferating culture medium containing no retinoic acid and one or more SHH signaling activators.

A method for culturing tumor infiltrating lymphocytes (TIL), the method comprising co-culturing the expanded TIL with feeder cells after contacting the TIL with a T cell co-stimulatory molecule and / or a T cell growth factor for a period of time. In addition, further provided is a method for preventing and / or treating tumors by means of using the tumor infiltrating lymphocytes of the present application.

The present invention relates to a method for inducing and expanding natural killer cells derived from peripheral blood mononuclear cells, which comprises co-culturing, as feeder cells, irradiated Jurkat cells and irradiated Epstein-Barr virus transformed lymphocyte continuous line (EBV-LCL) cells in the presence of cytokines, along with peripheral blood mononuclear cells. According to the present invention, a large quantity of natural killer cells can be induced and proliferated from a small quantity of peripheral blood mononuclear cells even without the use of high-cost equipment or various kinds of expensive cytokines, thereby making it possible to significantly improve the efficiency and efficacy of the prevention and treatment of cancer using the natural killer cells.

The application provides a tumor infiltrating lymphocyte culture medium and application thereof. The culture medium adopted by the application is composed of a basic culture medium, a serum substitute, a low-concentration interleukin, a stimulating factor and an antibiotic and the like. The application discloses a method for preparing high stemness tumor infiltrating lymphocytes (TIL) in vitro under serum-free and feeder-free conditions. Through optimized two-step culture (primary culture for 7-14 days + expansion culture for 7-14 days) and activation stimulation, high stemness TIL products with a CD3+ active cell ratio of greater than or equal to 95% and a Tcm ratio of greater than or equal to 70% can be obtained, and the total expansion multiple reaches 500-1000 times. The method of the application avoids the safety risk brought by feeder cells, reduces IL-2 related side effects, shortens the preparation period to 14-28 days, significantly improves the stem cell characteristics and sustained killing ability of TIL cells, and meets the quality control requirements of clinical-grade cell treatment products.

Disclosed herein are method of producing NK cells that include one or more heterologous nucleic acids. The methods include culturing a population of isolated NK cells in the presence of one or more cytokines to produce a population of activated NK cells. The population of activated NK cells are transduced with a viral vector comprising the one or more heterologous nucleic acids, for example by contacting the activated NK cells with viral particles including the viral vector. The resulting transduced NK cells are then cultured in the presence of one or more cytokines, and optionally in the presence of irradiated feeder cells, to produce a population of expanded transduced NK cells. Also disclosed are methods of treating a subject with a disorder (such as a tumor or hyperproliferative disorder) by administering to the subject NK cells produced by the methods described herein.

The present application relates to a kind of tumor infiltrating lymphocyte (TIL) cell expansion culture process. Specifically, the present application provides a kind of TIL cell expansion method, comprising: in expansion culture medium, preliminary culture TIL cell is carried out fast expansion culture. In the TIL cell expansion method of the present application, it is not dependent on activation antibody CD3 antibody, high-dose IL-2 and feeder cell, that is, the TIL cell number hundreds of times expansion can be realized in short time, the demand of TIL cell quantity in TIL cell therapy clinically can be satisfied, and it has broad application prospect in the field of TIL cell treatment.