84 results about "Feeder Layer Cells" patented technology

The invention belongs to the field of medical biology engineering, and particularly relates to a method for effectively multiplying gamma delta T cells by stimulating peripheral blood in vitro and application of the method. The method comprises the step of using feeder cells, an OKT3 (ornithine ketoacid transaminase) antibody, interleukin-2 and zoledronic acid. The feeder cells are formed by specifically inserting CD64, CD86 and CD137L genes in a target site of a genome of the feeder cells. After the zoledronic acid and the nterleukin-2 are used for increasing the proportion of the gamma delta T cells of the peripheral blood, protein products of genes, the OKT3 antibody and the interleukin-2 act in a combined manner, and the gamma delta T cells can be stimulated so that a large amount of gamma delta T cells can be multiplied. The multiplied gamma delta T cells can be used for killing tumor cells which are pretreated by the zoledronic acid, or the tumor cells can be directly killed by modifying and expressing chimeric antigen receptors (CAR) via a genetic engineering means. The gamma delta T cells which are obtained by the method have complete anti-tumor cytotoxicity, and can kill solid tumor cells and non-solid tumor cells.

The invention discloses an expansion method of various lymphocyte subpopulations and application of the expansion method to preparation of an adoptive immunotherapy medicine for cancers. The expansion method comprises the following steps: S1, adding IFN gamma and zoledronic acid into a culture medium of PBMC collected in the blood of a tumor patient, and re-adding an OKT3 antibody and recombinant human interleukin-2 after culture to stimulate PBMC so as to finish preliminary expansion; S2, after finish of preliminary expansion, mixing immune lymphocytes with feeder layer cells, and after adding zoledronic acid, an OKT3 antibody and recombinant human interleukin-2, continually performing expansion. By adoption of the expansion method, after twice expansion, the sum of the immune cells can be 10,000 to 80,000 times by expansion, the expanded immune cells include alpha beta T cells, gamma delta T cells, NKT cells and NK cells, and tumor cells can be directly killed, or a cellular immunotherapy drug is prepared to kill the tumor cells.

The invention provides a culture system which contains defined components and efficiently acquires induced pluripotent stem cells (iPS cells) from somatic cells. The culture medium additive provided by the invention contains vitamin C, vitamin B12, insulin, glycogen synthase kinase-3 inhibitor, receptor tyrosine kinase and antioxidant. The culture medium additive provided by the invention can also contain a substituted serum cell growth promoter. The invention also provides a complete medium acquired by inducing pluripotent stem cells, which is prepared from one or more of basic culture medium, serum and substituted serum additive and the culture medium additive. The culture system provided by the invention does not have serum, has no animal source pollution, contains defined chemical components, efficiently acquires iPS cells, can maintain cell growth and proliferation in the process of conversion from somatic cells to iPS cells under the condition that feeder cells do not exist, simultaneously accelerates the induction course of the iPS cells obviously, and greatly improves the efficiency of inducing the somatic cells into the iPS cells.

The present invention provides stem cell feeder layer cell lines that contain are readily triggered to differentiation. The expression vector encodes the senescence-triggering factors (STFs) consisting of Cip / Kip, INK4A, Cy protein or ankyrin-binding protein motifs. Each expression vector also contains an inducible transcription regulation element for conditional expression of the STFs.

The invention discloses a method for encapsulated osteoblasts to support and regulate the in-vitro amplification of cord blood hematopoieticstem / progenitorcells (He) in the hypoxia condition, belonging to the fields of biotechnology and tissue TSPCS engineering. The method is characterized in that the human osteoblasts are embedded by gelatin-alginate-chitosan (GAC) microcapsule as matrix feeder cells and co-cultured with the cord blood HSPCs in the 5% hypoxia environment to obtain the HSPCs. The invention adopts GAC microcapsule to separate the human osteoblasts from the cord blood HSPCs to avoid the cell contamination and immunological rejection. Furthermore, the pore passages of the microcapsule are capable of causing hemopoietic growth factors secreted by the osteoblasts in the microcapsule to expand to the habitat of the HSPCs and play a role of stimulating the amplification of the hematopoietic stem / progenitor cells. Moreover, the material of the microcapsule has the slow release function to the expansion of the growth factors to increase the concentration gradient of the growth factors on the surface of the microcapsule, which is helpful for the long-term function of the growth factor. In addition, the microencapsulated osteoblasts form the 3D environment, which not only causes the culture system to contain more osteoblasts, but also causes the culture system to have bigger HSPCs-OBs action area compared with the co-culture pattern of the osteoblasts cultured by adherence. Besides, in the 5% hypoxia environment, the co-culture system is capable of better simulating the hematopoietic microenvironment iche of human bone marrow the osteoblasts have significant effects on the characteristic maintenance and number amplification of stem cells of the HSPCs.

Undifferentiated primordial stem cells are manipulated to permit their long term growth in defined media lacking serum and feeder layer cells by shifting the apoptotic balance of the cells, through increasing the activity of Bcl-2 family anti-apoptotic proteins or decreasing the activity of Bcl-2 family pro-apoptotic proteins. In some embodiments of the invention, the Bcl family protein is Bcl-2. The ES cells sustain the characteristics of undifferentiated, pluripotent stem cells during long-term serum- and feeder layer cell-free conditions, including the ability to be expanded in vitro, but maintain their potential to differentiate into mature cell types.

The invention relates to a mesenchymal stem cell of marrow, umbilical cord or bleeding of the umbilicus and purpose in the field of cell transplantation, stem cell culture, expansion and oriented differentiation. The mesenchymal stem cell for expressing a related gene of a neurotrophin family is prepared by the following steps of extracting a mesenchymal stem cell from the marrow, the umbilical cord or the bleeding of the umbilicus; recombining the related gene of the neurotrophin family to an AAV (Adeno-Associated Virus) used as a carrier; preparing the AAV expressing the related gene of the neurotrophin family; and infecting separated and purified mesenchymal stem cell through AAV particles to obtain the mesenchymal stem cell expressing the related gene of the neurotrophin family. The invention is used as a culture layer cell to provide a manually controlled microenvironment for stem cell culture, expansion and oriented differentiation and promote the proliferation and the oriented differentiation of the stem cells so as to provide a new curing measure for human diseases.

The invention provides a test method for induced differentiating human embryo stem cells to retina pigment epithelial cells, which comprises the following steps that: A) 0.25% pancreatic enzyme and 0.1mg / mL collagenase iv are mixed into phosphate buffered saline (PBS) solution containing 1mM calcium chloride (CaCl2) and 20% KSR, the solution is used for digesting embryo stem (ES) clones, so the ES becomes an ES cell group with 5 to 10 cells; B) the ES clone is incubated in a culture dish which is wrapped by gelatin in a short time so as to remove the feeder layer cells; and C) the ES cell group is cultured in a continuous differential culture medium with the concentration of 6.7*102 cell groups per liters in a non-adhesive bacteria culture dish. The test method has the advantages such as simple and convenient method, good repeatability, high successful rate, good omnisexuality, rich and economical source and the like.

The invention belongs to the field of induced differentiation of blood cells, and provides a method for inducing and differentiating human multipotent stem cells into aged blood cells. The method comprises the following steps of A, inducing and differentiating the human multipotent stem cells into hematopoietic progenitor cells CD31 and CD34; B, inducing and differentiating the hematopoietic progenitor cells CD31 and CD34 into human aged cells. The method has the advantages that by adopting the two-step inducing type of single-layer cell culture and suspension cell culture, the inducing procedure is simplified, the inducting time is shortened, the operation is simple, the efficiency is high, and the stability is realized; by adopting the inducing and differentiating system without feed layer cells, the induced blood cell has biological safety guarantee, the requirements of clinical conversion and clinical experiment can be met, and the medical conversion value is high.

The invention relates to a serum-free medium of embryonic stem cells, and its application. The medium comprises a micro-molecular inhibitor CHIR99021, a micro-molecular inhibitor XAV939, an additive B27 / N2 and a basic medium DMEM / F12. The serum-free medium of embryonic stem cells, which contains no animal source substances and has a determined composition, is obtained by using the micro-molecular inhibitor CHIR99021, the micro-molecular inhibitor XAV939 and the B27 / N2 additive having a clear composition to substitute serum and feeder cells in a traditional embryo hepatocyte medium. Experiments prove that the medium can maintain the self updating of human embryonic stem cells, and maintains the undifferentiated state and totipotency of the embryonic stem cells; and the medium has a clear composition, and proteins and other components contained in the medium are from human recombinant proteins or are obtained through chemical synthesis, so there is no embryonic stem cell pollution, thereby no corresponding immune response is excited, and no immune immunological rejection is initiated.

The invention relates to the technical field of stem cells, and especially relates to culture media and applications thereof, and differentiation methods of induced pluripotent stem cells into pancreatic islets. In the provided differentiation methods, a differentiation process from iPS stem cells to pancreatic progenitor cells needs 14 days (2 weeks); and the process mainly includes following four steps:directional endoderm - archenteric canal -foregut / hindgut - pancreatic progenitor cells. The differentiation process from the pancreatic progenitor cells to functional islets needs about 31 days (1 month); and the process is mainly divided into following three steps: endocrine progenitor cells - early islets - islets in late functional maturity. Each of the above steps needs specific induction culture media; and the culture media can be formed by adding corresponding inducing factors into basic media, and the corresponding culture media need to be daily replaced for culture. Feeder layer cells are not needed during the whole induction of the method; and the adopted culture media have no animal-derived components, so that the risks of alloimmunization can be reduced.

The invention relates to a method for differentiating an induced pluripotent stem cell into a hepatocyte through directed induction, and the hepatocyte thereof. The method is characterized in that different media are added in different stages of directed induction differentiation to carry out directed induction differentiation of the induced pluripotent stem cell. The change of the form of the cell can be momentarily observed in the operating process to ensure normal implementation of induction, so a required liver precursor cell can be fast and efficiently obtained through induction. The method for differentiating the induced pluripotent stem cell into the hepatocyte through directed induction adopts a culture system of feeder-free cells, the hepatocyte can be obtained only through adding an induction medium in a corresponding stage, and a detection result shows that the purity is high. The method for differentiating the induced pluripotent stem cell into the hepatocyte through directed induction has the advantages of short induction period, high efficiency, stable performances and mature functions of the obtained hepatocyte, and no matrix cell pollution.

Dopamine-producing neural precursor cells are produced by: producing a cell population containing Corin and / or Lrtm1 positive cells via steps (1) and (2) as will be shown below; collecting the Corin and / or Lrtm1 positive cells from the cell population thus obtained by using a substance that binds to Corin and / or a substance that binds to Lrtm1; and suspension-culturing the Corin and / or Lrtm1 positive cells in a liquid culture medium that contains one or more neurotrophic factors: (1) a step for adhesion-culturing pluripotent stem cells in the absence of any feeder cells in an undifferentiatedstate-maintaining culture medium that contains a sonic hedgehog (SHH) signal stimulant and an undifferentiated state-maintaining factor in the presence of an extracellular matrix; and (2) a step for culturing the cell population obtained in step (1) in a liquid culture medium that contains one or more differentiation inducing factors.

In the field of biological technology, a stem cell culture method is provided. The method includes preparing an amniotic epithelial cell feeder layer that is not treated to lose the division ability; and seeding the stem cells onto the amniotic epithelial cell feeder layer, and culturing in a culture medium. The stem cell culture method according to the present invention does not require the treatment of the feeder layer cells to lose the division ability, and is thus simple and safe, thereby effectively solving the problem of contamination caused by animal-derived ingredients in culture of human stem cells at present, greatly reducing the culture cost of the stem cells, and providing a safe, effective, and inexpensive stem cell culture method for the industrialization of the stem cells in the future.

The invention relates to a method for preparing breast cancer bone metastasis organs, which comprises the following steps: mixing breast cancer bone metastasis cells, feeder layer cells, a temperature-sensitive hydrogel and a first culture medium, and culturing to obtain breast cancer bone metastasis organs; wherein the feeder layer cells comprise bone marrow mesenchymal stem cells without a proliferation function. By means of the technical scheme, the bone marrow mesenchymal stem cells without the proliferation function are used as feeder layer cells for breast cancer bone metastasis organ culture, and the success rate of breast cancer bone metastasis organ culture through the method is high.

The invention provides a method for preparing NK by using freeze-dried feeder layer cells. In the NK preparing method, engineering cells expressing cytokines and membrane proteins are constructed, andthe engineering cells expressing the cytokines and membrane proteins are freeze-dried. According to the method, after freeze-drying of feeder layer cells, the stimulation effect of the engineering cells as feeder layer cells is retained, the costs for cell inactivation and preservation process are also reduced, the costs for laboratories and clinical use are reduced, and the use convenience is improved. Compared with an existing feeder layer using technology, NK cells quantity for clinical treatment can be provided within 12-21 days, the cell viability is high, and the cells have high killingeffect on K562 and A549; The NK medium method has obvious advantages in proliferation ratio and quantity compared with a factor method and a special NK culture medium method; the cell quantity and purity obtained through amplification after stem cells are induced and split up into NK are poor compared with the method.

The invention relates to a method for directionally inducing and differentiating induced pluripotent stem cells into vascular endothelium-like cells. According to the method, a culture system without feeder cells is adopted, the vascular endothelium-like cells can be obtained only through expressing FLI1 related genes and activating a protein kinase C pathway, and a test shows that the purity is relatively high. Through the adoption of the method for directionally inducing and differentiating induced pluripotent stem cells into vascular endothelium-like cells, the induction period is short, the efficiency is high, the obtained vascular endothelium-like cells are stable in performance, and the problem of matrix cell pollution is avoided.

The invention discloses a method for induced differentiation of inner ear hair cells by virtue of human mesenchymal stem cells. The method comprises the steps of separating bone marrow MSCs (mesenchymal stem cells) from bone marrow of human as a source, and inducing the bone marrow MSCs by combining factor combination with feed layer cells so as to differentiate the mesenchymal stem cells. According the method, the human mesenchymal stem cells can be induced to be differentiated into the inner ear hair cells, and the cells obtained by differentiation express specific protein of the hair cells and have certain hair cell electrophysiological functions.

The invention discloses a cultivation method for activating human memory B cells into plasma cells in vitro. According to the method, irradiated 3T3-CD40L cells serve as feeder layer cells, IMDM of 10% FBS serves as basis culture solution, IL-2 and IL-21 serve as growth factors, and the memory B cells are induced in vitro to activate into the plasma cells and secrete antibodies. The cultivation method is simple to operate, low in cost, fewer in needed memory B cells and high in activation efficiency and activation quality.

The invention provides a tomato transformation method of agrobacterium tumefaciens mediation. Cotyledon explants (the seedling age is 8 days) are precultured for one day in an MSB5 culture medium with 2.0 mg / L of zeatin and 0.1mg / L of IAA, and are then infected by EHA105 agrobacterium for 20 minutes; the concentration of OD600 is 0.2 to 0.6; after the culture is performed for 3 days on a co-culture medium, the co-culture is performed for one day on a culture medium of 500mg / L of cephalosporin; the screening is performed for 6 to 8 weeks on the culture medium containing 100mg / L of kanamycin and 500mg / L of carbenicillin. The method has the advantages that the conversion efficiency is high; feeder layer cells, acetosyringone and bleeding sap are not needed; the conversion efficiency reaches 20 percent or higher; simplicity and high repeatability are realized.

Undifferentiated primordial stem cells are manipulated to permit their long term growth in defined media lacking serum and feeder layer cells by shifting the apoptotic balance of the cells, through increasing the activity of Bcl-2 family anti-apoptotic proteins or decreasing the activity of Bcl-2 family pro-apoptotic proteins. In some embodiments of the invention, the Bcl family protein is Bcl-2. The ES cells sustain the characteristics of undifferentiated, pluripotent stem cells during long-term serum- and feeder layer cell-free conditions, including the ability to be expanded in vitro, but maintain their potential to differentiate into mature cell types.

A stem cell culture system is disclosed. The system comprises culture-layer cells, a cell sieve and a three-dimension medium in order from bottom to top. The three-dimension medium comprises a three-dimension support prepared from Matrigel and a complete medium. The complete medium comprises a DMEM medium, FCS, beta-mercaptoethanol, LIF, glutamine and insulin. The system is advantageous in that the system adopts the Matrigel and growth factors secreted from culture cells to stimulate an extracellular matrix wherein the stem cells in vivo are in, thus allowing an in-vitro living environment to be similar to an in-vivo living environment and ensuring proliferation and a normal form of the cells; stem cell proliferation is promoted through combined action of the culture-layer cells, the beta-mercaptoethanol and the insulin; stem cell differentiation during proliferation is inhibited through synergistic effects of LIF; and effects of capability of stimulating the in-vivo cytoplasmic matrix, high stem cell activity, safety and convenient separation are achieved.

The invention discloses a method for culturing a single porcine embryo in vitro, and belongs to the field of agriculture-animal husbandry and veterinary medicine. The method particularly involves thatfeeder layer cell micro-droplets are adopted to be co-cultured with a single embryo so as to obtain a better development result; the development process of single embryo culture is consistent with the development process of traditionally used multi-embryo culture, and the single embryo attached with a transparent belt or removing a transparent belt can obtain a porcine parthenogenetic developmentblastosphere in 7 days. The method for culturing the single porcine embryo in vitro has the advantages that the provided feeder layer cell micro-droplets preparation is simple, a reliable original material for studying the development of a single embryo can be provided according to the provided method, and the needs of tracking, observing and further researching the development process of the single porcine embryo can be fully met; the provided single embryo culture method can be widely applied to single embryo culture of various species.

The invention discloses a serum-free culture medium for hematopoietic stem cell in vitro expansion culture and a preparation method thereof. The culture medium for in vitro expansion of the hematopoietic stem cell is free from introducing exogenous gene expression, and the genome stability of the original stem cell is unchanged, the tumorigenic risk is avoided, and the stem cell transplantation safety problem caused by feeder layer cell and other exogenous cell mixing is not involved; through the combined application of various cytokines, a proper culture condition and properexpansion time are selected to overcome the defect that the expansion number is insufficient, the number is effectively expanded, and the quality is maintained at the same time, namely, the effect of maintaining hematopoietic reconstitution capacity through the expansion of the early hematopoietic progenitor cell and reservation of the HSC is achieved.

The invention relates to a feeder-free and serum-free human induced pluripotent stem cell isolation culture method. By the feeder-free and serum-free human induced pluripotent stem cell isolation culture method, feeder cells and serum are not needed, a culture medium has clear components, operation is simple, human iPS cells can be efficiently and stably isolated, self-renewal and pluripotency are maintained, and a good experimental foundation is laid for final establishment of a clinical application-level human iPS cell isolation culture system.

The invention provides a stem cell culture perfusion device which comprises a perfusion bottle, a feeder layer cell culture unit, a cell perfusion tank and a waste liquid bottle, wherein the opening of the to-be-cultured cell perfusion bottle is provided with a first filter head and an input tube; the input tube is provided with a first controller capable of displaying pH value and controlling temperature; the tail end of the input tube is immersed in the feeder layer cell culture unit; a first connecting tube, which is connected through the feeder layer cell culture unit of which the substrate is provided with a semi-permeable membrane, is provided with a second controller and a first peristaltic pump; both ends of the cell perfusion tank are respectively connected with a second connecting tube and a third connecting tube; the other end of the second connecting tube is connected with the first peristaltic pump; the other end of the third connecting tube is connected with the a second peristaltic pump; the opening of the waste liquid bottle is provided with an output tube and a second filter head; and the other end of the output tube is connected with the second peristaltic pump. Under the actions of the first controller and second controller, the temperature and pH value variations can be observed, the rate of the peristaltic pumps can be controlled, and error values caused by manual observation and recording can be avoided; and the equipment output of the cell perfusion tank can enhance the stem cell culture rate.

The invention relates to the field of cell biology technologies and application, and particularly relates to a stomach cancer tumor primary organ culture method. The method comprises the steps of obtaining feeder layer cells and stomach cancer tumor primary cells by adopting an enzyme digestion method and cell culture method; adding magnetic suspension particles to the feeder layer cells and the stomach cancer tumor primary cells respectively, and obtaining the magnetic particle containing feeder layer cells and stomach cancer tumor primary cells by incubation-digestion; inoculating the magnetic particle containing feeder layer cells and stomach cancer tumor primary cells to a stomach cancer tumor primary organ culture medium, executing culture under the magnetic driving conditions to obtain stomach cancer tumor primary organs. By adopting a magnetic suspension culture system, the feeder layer cells and stomach cancer tumor primary cells are co-cultured, the mutual effect of tumor microenvironment and the cells is better simulated, and the new in-vitro efficacy testing method is provided for the drug research and development industry.

The invention discloses a method for preparing feeder layer cells. The method provided by the invention comprises the following steps: (1) treating the isolated fibroblasts with 10 μg / ml mitomycin C, collecting the culture supernatant and adherent cells respectively; (2) treating the isolated fibroblasts The cells are treated with the culture supernatant collected in the previous step, and the adherent cells are collected; between the step (1) and the step (2), the following steps of 0 to 2 times are also included: the isolated fibroblast The cells are treated with the culture supernatant collected in the previous step, and the culture supernatant and adherent cells are collected respectively; the adherent cells obtained in each of the above steps are feeder layer cells. The method for preparing feeder layer cells provided by the invention has simple flow, economical and reliable effect. Human embryonic stem cell culture experiments show that the feeder layer cells prepared by the method can be passed on for a long time, and can effectively support the growth of human embryonic stem cells and keep them in an undifferentiated state.

The invention discloses a fetus liver matrix cell strain for expressing alkaline fibroblastic growth factors. The fetus liver matrix cell strain of the invention is a recombinant fetus liver matrix cell strain carried with alkaline fibroblastic growth factor genes. By experimental verification, recombinant fetus liver matrix cells of the invention can highly efficiently produce secretory alkaline fibroblastic growth factors, and embryonic stem cell proliferation of human beings and embryonic stem cell drying can be maintained under the condition of no exogenous alkaline fibroblastic growth factors by taking the cells as feeder layer cells for in vitro culture of embryonic stem cells of human beings. The recombinant fetus liver matrix cells of the invention and a construction method thereof provide appropriate microenvironment for researches in the aspects of construction of a low-cost (reduction of introduction of exogenous alkaline fibroblastic growth factors), safe and highly efficient (capable of replacing odontogenic zooblast as the feeder layer cells) in vitro culture system of the embryonic stem cells of human beings and development and differentiation of hematopoietic cells, and the invention has wide application prospect.

The invention provides new application of rabbit fetal fibroblast, and particularly relates to a method for preparing a ZEN (Zearalenone) monoclonal antibody by utilizing the rabbit fetal fibroblast.The method is characterized in that mouse hybridoma is cultured by using rabbit fetal fibroblast as feeder cells of murine hybridoma, the murine feeder cells can be completely replaced, growth of monoclonal hybridoma can be promoted, and a monoclonal antibody with high valence, good stability and strong specificity can be generated; the hybridoma bred by different animal cells as the feeder cellsis used for preparing an antibody, and very high valence and very high specificity are obtained; meanwhile, the rabbit fetal fibroblast can be revived by many times for use after being cryopreserved,animal tissue isolating culture is not required to be carried out every time. The method disclosed by the invention is simple, the cost is low, the monoclonal antibody which is used for fast, sensitive, economic and efficient ZEN immunological detection can be prepared, and a wide application prospect and huge market value are obtained.