41 results about "Embryoid body" patented technology

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 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.

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.

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.

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.

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 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.

Disclosed are various embodiments for growing, culturing, monitoring, and analyzing embryoid bodies, fused embryoid bodies, spheroids, organoids, or other multi-cellular bodies using a system of microplates. Different types of microplates are designed to be used during the various stages of growing and culturing of cells to form embryoid bodies, fused embryoid bodies, spheroids, organoids, or other multi-cellular bodies. The different microplates are designed to mate with one another to allow for the transfer of cells from wells in one plate to wells in the other plate. An assay plate includes an array of perfusable units that include a supply well that is in fluid communication with a culture well to allow for an exchange of fluid.

The invention discloses a culture medium and a method for obtaining an induced cortical-like neuron precursor cell cluster from iPSCs (induced pluripotent stem cells), and belongs to the technical field of biology. The culture medium comprises a neural stem cell induction liquid I and a neural stem cell induction liquid II, a single-layer adherent induction method is adopted, and iPSCs are efficiently differentiated into an induced cortical neuron precursor cell cluster through optimized inoculation density, matrix treatment and a staged induction process. The neural stem cells comprise neural stem cells, transition-state nerve cells and differentiated mature nerve cells (the cell type composition is highly similar to that of tissue-derived neural stem cells). The method overcomes the problems of complex induction process and high cell heterogeneity in a traditional embryoid body method, the induction efficiency can reach 100%, single cell sequencing analysis shows that all cells are nerve cells, the purity is 100%, and non-target cells are not detected. A uniform, stable and efficient cell tool is provided for brain development research, nervous system disease modeling and cell therapy.

Provided herein is a method of producing an embryoid model including: co-culturing one or more first cells and one or more second cells in serum-free stem cell culture medium, thereby generating an embryoid structure, wherein: the first cells include induced pluripotent stem cells or embryonic stem cells expressing GATA binding protein 6 (GATA6) or a variation or derivative thereof; and the second cells include non-engineered stem cells; and culturing the embryoid structure in cell culture medium, thereby generating a heterogenous tissue that has embryonic and extraembryonic components.

The invention provides a preparation method of macrophages derived from pluripotent stem cells. Dynamic culture is combined in the process that an embryoid body is sequentially differentiated into mesoderm progenitor cells, hematopoietic stem cells and myeloid cells, a specific culture medium is used in the process that the myeloid cells are differentiated into macrophages, finally induced and differentiated macrophages are mainly M2 type (accounting for nearly 70%), and tissue regeneration markers are highly expressed on the surfaces of the macrophages. The cartilage regeneration can be promoted; the suspension cells can be collected at a fixed time in a myeloid cell maturation process. According to the invention, the problems of low differentiation efficiency, low purity, long period, immature cell function, dependence on animal-derived components, difficulty in large-scale production and the like in the process of differentiating the pluripotent stem cells into the macrophages are solved.

Methods are provided for automated embedding of embryoid bodies in a hydrogel, media exchange, culturing organoids, and monitoring the development of organoids such as brain organoids. Methods for automated testing of compounds and toxicity effects are also provided.

This invention provides a method for producing NK cells from undifferentiated stem cells without using feeder cells in serum-free culture medium. [Solution] A method for producing NK cells without using feeder cells, comprising culturing undifferentiated stem cells in a serum-free medium containing BMP4 and a GSK-3 inhibitor to form embryoid bodies, culturing them in a serum-free medium containing VEGF, SCF, and a TGF-β inhibitor, culturing them in a serum-free medium without a TGF-β inhibitor containing VEGF and SCF to differentiate them into hematopoietic progenitor cells, culturing them in a serum-free medium containing IL-7, SCF, Flt-3L, IL-15, and IL-3, culturing them in a serum-free medium without an IL-3 containing IL-7, SCF, Flt-3L, and IL-15 to differentiate them into NK progenitor cells, and culturing them in a serum-free medium containing IL-2, IL-7, SCF, Flt-3L, IL-15, and a serum substitute to differentiate them into mature NK cells.

The invention relates to a method of generating an in vitro endometrium assembloid comprising growing endometrial stromal cells inside a hydrogel matrix and layering cells and / or fragments from endometrial epithelial organoids on top, thereby generating a three-dimensional endometrium assembloid comprising a stromal compartment, an outer luminal epithelial layer and a functional secretory glandular endometrium. The invention further relates to an in vitro endometrium assembloid produced according to the methods defined herein. Also provided are methods for determining embryo implantation ability and / or window of receptibility to embryo implantation and for screening for embryo implantation-modulating agents comprising, providing the in vitro endometrium assembloid defined herein. The invention further relates to the use of three sequential human embryo media (HEM) for culturing human stem cell based embryo-like (blastoids) or human blastocysts, and to the compositions of the three media.

Provided herein are multilineage cardiovascular organoids and methods of generating the same. In some aspects, provided herein is a system comprising a plurality of multilineage cardiovascular organoids derived from embryoid bodies. The embryoid bodies can be aggregated from pluripotent stem cells of varying genotypes. The multilineage cardiovascular organoids and systems described herein may be used for screening of agents, such as anti-cancer agents, for cardiotoxicity.

The invention provides an improved Spin-EB culture method for promoting in vitro differentiation of human iPSCs (induced pluripotent stem cells) to produce platelets, the iPSCs are inoculated into a differentiation culture system with a high initial inoculation quantity for in vitro culture and induced to form embryoid bodies (EB), the differentiation culture system consists of a basic culture medium and an additive factor, and the additive factor is a platelet lysis buffer. Experiments prove that the HPL can completely replace human albumin in a platelet culture solution produced by in-vitro differentiation of human iPSCs, the cost is reduced, the HPL is a culture medium component capable of more effectively promoting differentiation of the iPSCs to hematopoietic cells, and the hematopoietic cells can be further differentiated into MKs and platelets. Therefore, the invention provides a new way for producing platelets through in vitro differentiation of human iPSCs.

The invention belongs to the field of stem cell biology, and relates to a culture method of a 3D neural immune organoid containing microglial cells, which comprises the following steps: S1, respectively inducing induced pluripotent stem cells in an ectodermal direction and a mesodermal direction to obtain a neural embryoid and a yolk sac embryoid; s2, culturing the neural embryoid to perform neural differentiation to obtain a neural garland structure, and re-digesting the neural garland structure to obtain a neural progenitor cell single cell; culturing the yolk sac embryoid to enable the yolk sac embryoid to generate macrophage progenitor cells; and S3, fusing the neural progenitor cell single cell and the macrophage progenitor cell, and continuously culturing to form the 3D neural immune organoid containing the microglial cells. The brain-like organ provided by the invention has the functions of exploring the control of microglial cells on the progenitor cell proportion, the change of phagocytic function, the control on the number of mature neurons and the influence on cell proliferation and apoptosis in the development stage, and meanwhile, the brain-like organ can accept external stimulation and make stress change of corresponding functions.

Provided herein are methods of producing induced embryo feeder cells and embryoids.