25 results about "Embryoid body" patented technology

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 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 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 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 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.

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 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.

The present invention provides a method for producing mesodermal lineage cells from an embryoid body, the method comprising: (1) a step for inducing an embryoid body into a differentiated embryoid body in which differentiation into a mesodermal cell lineage is promoted; and (2) a step for determining the solidity of the induced differentiated embryoid body.

This invention provides a method for inducing pluripotent stem cells to differentiate into ovarian granulosa cells and its applications, comprising: constructing an NR5A1-iPSC engineered cell line expressing the NR5A1 gene, the expression of which is regulated by a tetracycline-inducible element; inducing differentiation using a three-stage differentiation culture system: in the first stage, embryoid bodies are induced to form using a culture medium containing Activin-A, BMP4, GSK-3β inhibitors, and Rock inhibitors; in the second stage, primary granulosa cells are induced to form using a culture medium containing BMP4, GSK-3β inhibitors, bFGF, and tetracycline; and in the third stage, mature granulosa cells are induced using a culture medium containing BMP4, bFGF, and tetracycline. This invention also provides a culture medium combination for this method and the granulosa cells obtained therefrom. The granulosa cells prepared by this method can be used to promote in vitro maturation of oocytes, construct in vitro models of ovarian-related diseases, and for drug screening.

The invention provides culture platforms, cell media, and methods of differentiating pluripotent cells into hematopoietic cells. The invention further provides pluripotent stem cell-derived hematopoietic cells generated using the culture platforms and methods disclosed herein, which enable feed-free, monolayer culturing and in the absence of EB formation. Specifically, pluripotent stem cell-derived hematopoietic cell of this invention include, and not limited to, iHSC, definitive hemogenic endothelium, hematopoietic multipotent progenitors, T cell progenitors, NK cell progenitors, T cells, and NK cells.

The invention provides a towel gourd radiation induced embryoid seedling culture medium and a towel gourd double haploid creation method, and belongs to the technical field of breeding. The invention provides a loofah radiation mutagenesis embryoid seedling culture medium. The loofah radiation mutagenesis embryoid seedling culture medium comprises 4.74 g / L of an MS culture medium, 30 g / L of sucrose, 0.04 mg / L of thidiazuron, 0.225 mg / L of 6-benzylamino adenine and 7 g / L of agar. The radiation induced embryoid seedling culture medium for the towel gourd is used for culturing the radiation induced embryoid of the towel gourd, so that the radiation induced embryoid seedling of the towel gourd can be promoted, and the grown plant is identified as the double haploid of the towel gourd. Technical support is provided for creation of double haploids of towel gourds, high-quality parents are provided for cultivation of new varieties of towel gourds, and the creation process of the new varieties of towel gourds is accelerated.

The invention relates to a method and a culture medium for directly obtaining haplobionts through tomato 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 advantages that the steps of the culture method are simplified, the operation is convenient, and the haplobiont is obtained by directly inducing the embryoid through anther in-vitro culture; as the method adopted by the invention does not need a callus stage, the tedious process of callus induction, transfer to a differential medium and induction of a regenerated plant is omitted; according to the method, ploidy mixing caused by the fact that diploid somatic cells such as anther walls and filaments participate in callus formation is completely eradicated from the development path, it is ensured that all obtained plants are haploids from pollen, and the purity and obtaining efficiency of haploid materials are remarkably improved.

The present disclosure relates to a method for selective differentiation from pluripotent stem cells to hindbrain tissue, the method comprising 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 containing endogenous WNT secretion inhibitors and WNT signaling agonists.