64 results about "Embryoid body" patented technology

The invention provides preparation, sorting and application of LGE / CGE organoid and GABAergic neurons, and belongs to the field of brain organoid differentiation and GABAergic neurons. The preparation method of the LGE / CGE type organ comprises the following steps: S1, performing embryoid formation and initial culture on the 0th-4th day; s2, performing nerve induction and matrigel embedding on the 5th-11th day; s3, culturing the brain organoid on the 12th day to the 19th day; s4, dynamically culturing and screening the brain organoid on the 20th-35th day; and S5, carrying out differentiation and collection on LCOs of the LGE / CGE type organs after the 35th day. According to the method, the LGE / CGE organs containing the high-purity GABA energy neurons are obtained through differentiation from the brain organs for the first time, the high-purity LGE type GABA energy neurons and the high-purity CGE type GABA energy neurons are obtained through a specific sorting method, and the method has the deep application potential in the fields of disease model construction, medicine research and development and the like.

The invention relates to the technical field of cell differentiation, in particular to a method for directionally differentiating iPSC into iNK. Specifically, the method for preparing the CD34 + hematopoietic stem / progenitor cell and the iNK on the shaking table comprises the following steps: forming an embryoid (EB) by the iPSC on the shaking table, and then collecting a single cell supernatant to obtain the iNK. According to the method, high-proportion CD34 < + > hematopoietic stem / progenitor cells are obtained by adjusting the factor concentration and collecting the supernatant and are further induced and differentiated into the iNK, and more than 100 iNKs can be harvested before amplification of one iPSC according to the iNK harvested in batches.

The invention provides a method for promoting differentiation of induced pluripotent stem cells to trigerm layers and application of the method in differentiation potential detection, and relates to the technical field of biologication.The method comprises the steps that the induced pluripotent stem cells are inoculated into a culture medium containing 5-20 [mu] M of E-cadherin activator 1, shaking culture is conducted to form embryoid bodies, the embryoid bodies are added into a differentiation culture medium for differentiation culture, and the embryoid bodies are obtained. Obtaining an embryoid body for activating three-germ-layer differentiation potential; inoculating the embryoid body which activates the differentiation potential of the three-germ layer into a container coated with a cell adhesion matrix, adding a differentiation culture medium, continuing differentiation culture, and performing induced differentiation to form the three-germ layer cells. The technical problems that in the prior art, when iPSC is induced to be differentiated to three germ layers through an embryoid body path, the embryoid body forming capacity is poor, the embryoid body state is poor, the long-term maintaining capacity is poor, and the endoderm differentiation and climbing-out capacity is poor due to the fact that the embryoid body is poor in wall attaching capacity after plate rotation are solved.

The invention relates to the technical field of pepper anther tissue culture, and particularly discloses a method for improving pepper anther tissue culture embryoid induction rate and reducing pollution rate, and the method is characterized in that a culture medium containing GABA (gamma-aminobutyric acid) is adopted for culture. Specifically, the culture medium comprises an induction culture medium for inducing embryoid formation, an embryoid culture medium for culturing embryoid seedlings and a seedling growth culture medium for culturing seedlings to be mature. On the basis of a conventionally used culture medium formula, 50 mM of GABA is added into the culture medium in each stage, so that the embryoid induction rate can be remarkably improved, the pollution rate can be reduced, and finally the purpose of improving the culture success rate of the pepper anther is achieved. In the anther tissue culture process, compared with a conventional culture medium, the pollution rate of the culture medium added with 50 mM of GABA is reduced by 4.5-2.5 times, the embryoid induction rate is averagely improved by 1.6-2.2 times, and the survival haploid seedling is improved by 1.6-3.3 times.

The invention discloses a method for creating a new bolting-resistant radish germplasm by using a microspore culture technology, and belongs to the technical field of radish breeding. The method comprises the following steps: hybridizing a radish material R-16 and a bolting-resistant radish material LR-75, carrying out microspore culture, embryoid induction and regeneration on hybrid offspring plants to obtain double haplobionts, carrying out selfing, and carrying out character evaluation and screening on selfing offspring plants to obtain bolting-resistant double haplobionts with stable characters. Experimental results show that by selecting the parent material and optimizing the hormone concentration of the induction culture medium, the embryo emergence rate of the radish material is remarkably increased, and the homozygous bolting-resistant radish germplasm can be efficiently and quickly obtained. According to the method, a rich material foundation is laid for heterosis breeding of the bolting-resistant radishes, the breeding procedure is simplified, the breeding cost is reduced, the breeding process of new varieties of the bolting-resistant radishes is accelerated, and the method has great economic significance and wide application prospects.

The invention provides a 3D culture method for promoting stem cells to be differentiated into cardiac lineage cells. According to the method, four-stage time sequence induction is adopted, wherein in the stage I, BMP4, Activin A and bFGF are used for efficiently inducing the cells into mesoderm in an insulin-free culture medium; in the stage II, DKK1 is used for inhibiting WNT signals to lock the heart pedigree, and VEGF165 is added in the early stage to start cardiac endothelial differentiation; in the stage III, metabolic support is provided for an insulin-containing culture medium, and bilineage maturation is deepened; in the IV stage, bFGF is added to support long-term survival and function maintenance of the cells, and the whole process is carried out in a 3D embryoid body. According to the invention, the cheap RPMI1640 basal culture medium is used for replacing an expensive commercial culture medium, the high-purity heart micro-tissue which can spontaneously beat and contains cardiac muscle and cardiac endothelial cells can be obtained within about 10 days, and an economical and efficient cell source is provided for heart disease research, drug screening and regenerative medicine.

The application discloses a method for reducing vitrification of anther culture regenerated plants, and belongs to the technical field of plant tissue culture. The method comprises the following steps: inoculating anthers into a culture bottle containing a culture medium, covering a layer of breathable sealing film on the bottle mouth of the culture bottle, and tightening the breathable sealing film with an elastic band; then covering a layer of non-breathable sealing film on the outer layer of the breathable sealing film, and tightening the non-breathable sealing film with a rubber band; when embryoid is induced to be generated in 20-25 days, the covered non-breathable sealing film is removed, and only the breathable sealing film is left. The application has the following advantages: when the anthers are subjected to high-temperature treatment, the humidity in the culture bottle is not too low due to the covering of the non-breathable sealing film, and the survival of immature pollen is not affected; when embryoid is induced to be generated in 20-25 days, the non-breathable sealing film is removed, and only the breathable sealing film is left, so that the embryoid will not be vitrified due to excessive humidity; and the suitable humidity in the culture bottle can be realized only by covering or not covering the non-breathable sealing film, so that the workload of large-scale production of haploid plants is reduced.

A method of differentiating pluripotent stem cells into hemangioblasts comprising incubating the pluripotent stem cells in a first serum-free differentiation medium comprising bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor (VEGF) and stem cell factor (SCF) to induce differentiation of the pluripotent stem cells into hemangioblasts or hemangioblast-containing embryoid bodies is provided. The hemangioblasts or embryoid bodies may be cultured in a second differentiation medium comprising at least granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF) and interleukin-3 (IL-3) for a period of time sufficient to generate alveolar-like macrophages.

The invention provides a method for producing platelets through human iPSCs (induced pluripotent stem cells) in-vitro differentiation, aiming at the existing in-vitro induction method, a systematically optimized in-vitro culture scheme is adopted, and the method mainly comprises the following four aspects: (1) optimizing forming conditions of embryoid bodies (EB); (2) optimizing the composition of a basic cell culture medium; (3) establishing an induction scheme for replacing cell factors with small molecular compounds; and (4) adding a small-molecule inhibitor to promote maturation and differentiation of megakaryocytes. The efficiency is improved by a technology for promoting MKs maturation by adjusting the amount of initial EB cells and adding small molecules, the cost is reduced by a method for optimizing culture medium components and replacing cell factors with small molecules, and the differentiation time of megakaryocytes is shortened to 14 days; the differentiation efficiency of the MKs is remarkably improved by about 2.5 times (about 1.428 megakaryocytes are obtained by differentiating each iPSC, and about 0.57 megakaryocytes are obtained by differentiating each iPSC according to a basic scheme), and the final differentiation maturity is remarkably improved. Meanwhile, after the cell culture medium is optimized, the production cost is obviously reduced by 70.9%.

The application belongs to the field of stem cell biology and relates to a culture method of a 3D nerve immune organoid containing microglia cells, which comprises the following steps: S1, inducing induced pluripotent stem cells in ectoderm direction and mesoderm direction respectively to obtain nerve-like embryoid bodies and yolk sac-like embryoid bodies; S2, culturing the nerve-like embryoid bodies to make them differentiate into nerves to obtain nerve ring structures, re-digesting the nerve ring structures to obtain nerve progenitor cell single cells, and culturing the yolk sac-like embryoid bodies to make them generate macrophage progenitor cells; and S3, fusing the nerve progenitor cell single cells and the macrophage progenitor cells and continuing to culture to form the 3D nerve immune organoid containing microglia cells. The brain-like organoid of the application can realize the following functions: exploring the control of microglia cells on the proportion of progenitor cells in the development stage, the change of phagocytosis, the control on the number of mature neurons, the influence on cell proliferation and apoptosis, and simultaneously accepting external stimulation and making corresponding functional stress changes.

The invention provides a hormone inducible ZmEXPB6 promoter and application thereof in construction of a plant genetic transformation system, and belongs to the technical field of genetic engineering. The invention provides a hormone inducible ZmEXPB6 promoter. The nucleotide sequence of the hormone inducible ZmEXPB6 promoter is as shown in SEQ ID NO: 1. According to the embodiment of the invention, after a carrier containing the hormone-induced promoter and a target gene ZmWus2 gene is transferred into a corn immature embryo receptor, induced culture is carried out in a culture medium containing auxin 2 and 4-D. The result shows that the hormone-induced promoter drives the ZmWus2 gene to express, so that not only can a transgenic embryoid be quickly obtained and the conversion rate be increased, but also the yield of the corn immature embryo is increased. And compared with a constitutive promoter 35S promoter, the malformation rate of regenerated seedlings is obviously reduced. The hormone-induced promoter has a wide application prospect in the aspect of constructing a plant genetic transformation system.

A medium for direct differentiation of embryonic stem cell-derived mesenchymal stem cells, a method of preparing mesenchymal stem cells by using the same, mesenchymal stem cells prepared thereby, and a cell therapy product comprising the same mesenchymal stem cells. In a medium composition and in a method according to an embodiments, mesenchymal stem cells may be prepared at high yield within a short period of time. In addition, the method is simple in preparation procedure because of the absence of an embryoid body formation step that allows homogeneous cells to be prepared, thus advantageously providing a cell therapy product within a reduced period of time, compared to conventional methods.

The invention relates to the technical field of cytobiology and regenerative medicine, and discloses a composition and method for efficiently and directionally inducing iPSC (induced pluripotent stem cells) to be differentiated into functional microglial cells, and the method comprises the following steps: S1, digesting the induced pluripotent stem cells into single cells, resuspending the single cells by using an embryoid induction culture medium, inoculating the single cells into a low-adsorption culture plate, and culturing for 2-4 days to obtain a culture medium; forming an embryoid; s2, transferring the obtained embryoid into a matrigel-coated culture plate, adding a myeloid precursor cell induction culture medium for culturing, regularly changing the liquid during the period, and culturing for 2-3 weeks to obtain suspended myeloid precursor cells; s3, collecting the obtained myeloid precursor cells, centrifuging the myeloid precursor cells, re-suspending the myeloid precursor cells by using a microglial cell end induction culture medium, inoculating the myeloid precursor cells into a culture plate, adding the microglial cell end induction culture medium for culturing, regularly changing the medium during the culture period, and culturing for 10-14 days to obtain mature microglial cells. The invention solves the problems of long period and high cost of the existing scheme for differentiating the iPSC into the microglial cells.

The present invention provides a method for producing a natural killer cell from an iPS (Induced pluripotent stem) cell, comprising steps of: (i) contacting an iPS cell with a composition comprising a GSK-3 inhibitor and a ROCK inhibitor to obtain an embryoid body, (ii) contacting the embryoid body with a composition comprising a TGFβ receptor inhibitor to obtain a hematopoietic progenitor cell, (iii) culturing the hematopoietic progenitor cell to obtain a lymphocyte progenitor cell, and (iv) differentiating and expanding the lymphocyte progenitor cells to a natural killer cell.

This invention discloses a method for co-culturing and inducing stem cell differentiation into hematopoietic progenitor cells. The method includes the following steps: culturing pluripotent stem cells to obtain embryoid bodies; culturing the embryoid bodies for mesodermal differentiation to obtain mesodermal cells; culturing the mesodermal cells for hematopoietic endothelial differentiation to obtain hematopoietic endothelial cells; and co-culturing the hematopoietic endothelial cells with mesenchymal stem cells to obtain hematopoietic progenitor cells. Through optimization of the culture system, the differentiation efficiency and the number of hematopoietic progenitor cells obtained are significantly improved; by co-culturing mesenchymal stem cells with hematopoietic endothelial cells, the number of hematopoietic progenitor cells is further increased while maintaining high differentiation efficiency and uniform and stable expression of various markers.

The invention relates to the technical field of cell biology, and discloses a method for efficiently inducing hiPSCs to differentiate into mononuclear cells, which comprises the following steps: Step 1: culturing and amplifying human induced pluripotent stem cells; step 2: forming embryoid bodies by human-derived induced pluripotent stem cells: at the first stage, digesting hiPSCs to form cell agglomerates, inoculating the cell agglomerates to a cell culture dish, adding mTeSR Plus containing ROCK inhibitor Y-27632, and culturing for 0.5-1 day; in the second stage, the old culture medium is removed, EBM is added, half of the culture medium is changed every day, and culture is conducted for 4-6 days. Step 3: differentiation of the embryoid bodies into mononuclear cells: at the first stage, inoculating the embryoid bodies into a cell culture plate, adding MDM1, changing half of the liquid every 2-3 days, and culturing for 8-9 days; in the second stage, the old culture medium is removed, MDM2 is added, half of the medium is changed every 2-3 days, and culture is performed for 4-6 days; and a third stage: removing the old culture medium, adding MDM2, completely changing the medium every 2-3 days, and collecting the mononuclear cells. By adjusting EB culture conditions and inoculation density, the differentiation efficiency of the mononuclear cells is remarkably improved, the preparation time is shortened, and the production cost is reduced.

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.

The invention relates to a method and a culture medium for improving the embryoid induction rate of eggplant anther culture, and belongs to the technical field of plant tissue culture. The method comprises the following steps: (1) obtaining an embryoid; and (2) obtaining the regenerated haplobiont. The culture medium used in the method is an anther culture solid culture medium and a healthy seedling rooting solid culture medium. The method has the following advantages that the steps of the culture method are simplified, the operation is convenient, the embryoid induction rate of eggplant anther culture can be effectively improved, and particularly the problem that the culture efficiency of recalcitrant eggplant varieties with poor anther culture response is low is solved; according to the method, the embryo forming period is short, embryoid formation can be observed by naked eyes only in three weeks of anther inoculation, the eggplant haploid breeding process is remarkably accelerated, and the breeding efficiency is improved.

The application discloses a method for creating a DH line of eggplant by using anther culture and application thereof, and relates to the technical field of plant breeding. The application establishes a morphological index of a flower bud based on a calyx lobe separation state, replaces a time-consuming microscope examination procedure, and optimizes a combination of heat treatment and low-temperature pretreatment, so that the embryoid induction rate is significantly improved. Finally, through strict ploidy identification and self-pollination purification, a DH line with consistent traits and stable heredity can be obtained, an efficient pure line source is provided for eggplant hybrid breeding, and efficient conversion from a heterozygous genotype plant to a homozygous DH line is realized.

Human pluripotent stem cells (hPSCs), especially induced pluripotent stem cells (iPSCs) provide a promising starting material to produce mimetic innate immune cells such as natural killer (NK) cells and γδ T-cells for cancer immunotherapy. To facilitate consistent mass production, an overall manufacturing scheme to make mimetic innate immune cells from hPSCs was designed and demonstrated. Particularly, a robust protocol to differentiate hPSCs into NK cells or γδ T-cells through sequential hematopoietic differentiation on stromal cell line deficient in expressing M-CSF and lymphoid commitment on stromal cell line deficient in expressing M-CSF ectopically expressing DLL1 without employing CD34+ cell enrichment and spin embryoid body formation is established. Using this two-stage protocol, the generation of functional mimetic NK cells and functional mimetic γδ NKT-cells was demonstrated from hPSCs, including hESCs, peripheral blood cell-derived iPSCs (PBC-iPSCs). non-T cell-derived iPSCs or γδ T cell-derived iPSCs and the use of these mimetic innate immune cells in killing cancer cells.

The present disclosure relates to a method for induced differentiation of stem cells into hematopoietic progenitor cells. The method comprises the following steps: culturing pluripotent stem cells to obtain an embryoid body; performing mesoderm differentiation culture on the embryoid body to obtain mesoderm cells; performing hemogenic endothelium differentiation culture on the mesoderm cells to obtain hemogenic endothelial cells; and performing hematopoietic progenitor cell differentiation culture on the hemogenic endothelial cells to obtain hematopoietic progenitor cells. According to the present disclosure, the hematopoietic progenitor cells can be rapidly and efficiently prepared, and the prepared hematopoietic progenitor cells have an ability to stably differentiate into a plurality of different blood cells (including simultaneously having erythroid, myeloid, and lymphoid cells); by optimizing a culture system, the differentiation efficiency is significantly improved and the number of hematopoietic progenitor cells obtained is significantly increased.

The present invention is directed to methods of chemical reprogramming of a microspore plant cell using tyrosine kinase inhibitors. The tyrosine kinase inhibitors described facilitate microspore embryoid regeneration in maize, including maize varieties previously recalcitrant to microspore embryoid regeneration, and in a variety of other plant species.

The invention relates to the technical field of biological model construction, and discloses a construction method of a neurolyme disease model based on cerebrum orgonoids, which comprises the following steps: step 1, establishment and culture of the cerebrum orgonoids: inducing pluripotent stem cells (iPSCs) to be subjected to suspension culture to form embryoid bodies (EBs), inducing neuroepithelium, performing matrigel embedding to enable the neuroepithelium to be differentiated, and obtaining the cerebrum orgonoids; finally, culturing by using an orbit determination shaking table to make the brain organoid mature; and step 2, establishment of a neurolyme disease brain organ model: a pathogen Bb (Borreria burgdorferi) related to the neurolyme disease is introduced into the brain organ, a disease infection process is simulated, and the infection dose of the Bb is 1.0 * 10 < 5 > / each organ. According to the method, the whole maturation time of the brain organoid containing the microglial cells is successfully shortened by optimizing the culture conditions of the organoid, the structure and the function of the central nervous system can be more accurately simulated, and a more reliable experimental platform is provided for subsequently constructing a disease model and mining a key regulation mechanism.

A method for mesenchymal stem cell differentiation from human pluripotent stem cells is provided, particularly including the step of adaptation of embryoid bodies from 3-dimensional culture to 2-dimensional culture. The method enables rapid mesoderm and mesenchymal stem cell transformation by transferring from 3-dimensional culture to 2-dimensional culture. Also, the method relates to obtaining mesenchymal stem cell, which can be used in clinical applications, with this method.

The present disclosure relates to a method for preparing a cerebellar organoid, comprising: (A) a step of selective differentiation into hindbrain tissue, in which embryoid bodies formed from pluripotent stem cells are cultured in the presence of a hindbrain tissue induction medium including an endogenous WNT secretion inhibitor and a WNT signaling agonist; (B) a step of selective differentiation into dorsal metencephalic tissue, in which the embryoid bodies are cultured in the presence of a dorsal metencephalic tissue induction medium including a sonic hedgehog (SHh) signaling pathway inhibitor; and (C) a basal surface-apex surface polarization step of inducing basal surface-apex surface polarization, in which polarization of the embryoid bodies are induced in the presence of a basal surface-apex surface induction medium including an extracellular matrix-based hydrogel.

The invention relates to a culture medium and a method for preparing NK cells by inducing iPSC differentiation. According to the invention, a specific induced differentiation process and culture medium are designed, the whole differentiation process is divided into three key stages, the culture medium combined by different cell factors is specifically used, efficient induction and NK phenotype maturation are realized, high-proportion NK cells are obtained through 23-day differentiation, and iPSC suspension culture is applied to large-scale induced differentiation of the NK cells, so that the large-scale induced differentiation of the NK cells is realized. The uniformity and the induction efficiency of embryoid bodies (EB) are further improved, iPSC dynamic suspension differentiation is not limited by the bottom area of a container any more and is suitable for a bioreactor system, in addition, an amplification process based on a wave type bioreactor is designed, amplification of 200 times or above in 14 days can be achieved, the yield of 1011 or above levels of a single batch is finally achieved, and the method is suitable for large-scale production. A new thought and a new method are provided for large-scale preparation and application of the NK cells.

The invention relates to the technical field of biology, in particular to a construction method of a novel lung cancer brain transfer model. The method comprises the following steps: carrying out induction culture on stem cells to obtain an embryoid organ, and carrying out differentiation culture on the embryoid organ to obtain a midbrain ventral organ; the method comprises the following steps: shearing and digesting small cell lung cancer (SCLC) tissue blocks to obtain SCLC cells, and carrying out suspension culture to obtain small cell lung cancer (SCLC) organs; and placing the midbrain ventral organ and the small cell lung cancer organ in the same culture container, and carrying out co-culture to obtain the novel lung cancer brain transfer model. Compared with a human-derived tumor tissue xenograft technology with a low success rate, the novel lung cancer brain transfer model provided by the embodiment of the invention has high fusion efficiency and batch stability, and interference of other heterologous animal components can be reduced. In addition, the organoid fusion model established by using the technology of the invention is easier to observe, deliver drugs and sample.

The invention discloses a method for improving the regeneration efficiency of cucumber cotyledonary node embryoids by utilizing a cucumber gene CsRBOHD, a novel gene CsRBOHD for improving the regeneration efficiency of the cucumber cotyledonary node embryoids is identified, a homozygous mutant strain with the CsRBOHD gene knocked out is successfully obtained for the first time by utilizing a CRISPR / Cas9 gene editing technology, and the CsRBOHD gene knocked out homozygous mutant strain has the advantages that the CsRBOHD gene knocked out homozygous mutant strain is used for improving the regeneration efficiency of the cucumber cotyledonary node embryoids; and embryogenic callus culture and embryoid induction are carried out on the mutant seeds, and the result shows that the regeneration efficiency of the cucumber cotyledonary node embryoid is remarkably improved by the mutant strain. The method has important theoretical and practical application values for improving somatic embryogenesis efficiency and embryoid regeneration capacity of cucumbers.

The present invention relates to the technical field of cell culture, and specifically discloses a method for domesticating an adherent cell line into a fully suspended cell line. The adherent-dependent cells are inoculated on microcarriers and cell proliferation is carried out in DMEM medium containing 10% fetal bovine serum. After the cells proliferate to 10E6 cells / mL, the microcarriers are lysed, the cell clusters are centrifuged and collected. The cell clusters are inoculated in a medium containing 10% fetal bovine serum, 0.1% shear force protectant, and 2 g / L glucose and cultured at a rotation speed of 30 to 50 rpm for 3 to 4 days to enable the cells to form embryoid body clusters of 50 μm to 150 μm. The embryoid body clusters are mechanically pipetted to disperse them, and are domesticated for 18 to 22 days with DMEM medium containing different concentration gradients of low molecular weight dextran sulfate to obtain a successfully suspended and domesticated cell line. The method of the present invention improves the uniformity of cell embryoid bodies, makes the properties of the embryoid bodies more stable, and can efficiently obtain a suspended cell line.