148results about "Coculture" patented technology

The invention relates to a cell collection method capable of simultaneously collecting three cells in a co-culture model, and belongs to the technical field of biology. The invention provides a cell collection method capable of simultaneously collecting three cells in a co-culture model, and the cell collection method comprises the following steps: after a three-cell co-culture model is constructed, taking out a Transwell chamber, retaining cells in the lower chamber, and collecting the cells in the lower chamber; respectively digesting the cells on the two sides of the Transwell cell membrane by using a trypsin solution with the concentration of 0.5 g / 100mL so as to respectively collect the cells on the two sides of the Transwell cell membrane. According to the cell collection method disclosed by the invention, the three cells in the three-cell co-culture model are simultaneously collected in a manner of digesting the cells on the two sides of the Transwell membrane step by step by using pancreatin, so that not only is the cell and consumable cost saved, but also the experimental synchronism of the three cells is ensured, and convenience is provided for optimizing the experimental process.

The present invention belongs to the field of biomedical engineering technology and relates to a bone organoid, its construction method, and its use. The bone organoid is obtained by three-dimensionally culturing a cell mixture comprising stem cells and macrophages. The bone organoid and its construction method of the present invention enable rapid preparation of bone organoids with minimal differences from natural bone, enabling regulation of cell fate and better bone formation and repair.

The invention relates to the technical field of cell biology, in particular to a construction method and application of intestinal organs for children with Crohn's disease, the intestinal organs for children with Crohn's disease are successfully constructed by utilizing intestinal tissues from children with Crohn's disease, and the intestinal organs have various characteristic cells of the intestinal tissues; besides, immune cells are extracted from peripheral blood of a child patient homologous with intestinal tissues, a co-culture system is established with the organ-like model, the intestinal immune microenvironment of the child patient suffering from the Crohn's disease can be reproduced, and the system provides a new model for related mechanism exploration and drug screening of the child Crohn's disease.

The invention discloses a bone organ chip loaded with a microelectrode array and a use method, and relates to the technical field of organ chips, the bone organ chip is made of a polydimethylsiloxane material, and the bone organ chip comprises a three-layer structure: a top layer, a middle layer and a bottom layer; the middle layer comprises two blood vessel micro-channels, two osteoblast micro-channels and a neuron micro-channel, the channels are separated through columns and micro-columns, and cell interaction in a bone micro-environment is simulated; a microelectrode array is arranged on the top layer and is used for monitoring electrical activity of neurons in real time; the bottom layer is provided with a pneumatic valve which can control opening and closing of the micro-channel. According to the invention, nerve and blood vessel channels are introduced, a nerve-blood vessel-immunoregulation bone microenvironment is constructed, physiological processes such as osteogenesis-blood vessel formation coupling and the like are embodied, intervals of different sizes can enhance intercellular crosstalk and allow blood vessels and neuron axons to selectively pass through, so that the chip can simulate a complex microenvironment of bone tissues, and the microenvironment of the bone tissues can be simulated; and an efficient and accurate in-vitro model is provided for bone disease mechanism research and drug development.

The invention provides a culture system for simulating neutrophile granulocyte extracellular trap net intervention oligodendrocyte in vitro, and belongs to the technical field of cell and function detection. The invention establishes a stable and repeatable research system by screening and determining the optimal condition for in-vitro co-culture of the neutrophil extracellular trap net and the oligodendroglia. Based on the condition, the experiment can observe the obvious change of the oligodendroglia after the intervention, and the result shows that the condition can accurately simulate the endoplasmic reticulum stress regulation effect of the neutrophile granulocyte extracellular trap net on the oligodendroglia, thereby providing a necessary experimental basis for the subsequent mechanism research.

The invention relates to a retina nerve and blood vessel unit in-vitro three-culture model and a preparation method thereof, and belongs to the technical field of biology. The invention provides an in-vitro three-culture model of a retina nerve and blood vessel unit. The in-vitro three-culture model is obtained by carrying out in-vitro Transwell indirect co-culture on retina microvascular endothelial cells, retina ganglion cells and retina Muller cells. The in-vitro three-culture model of the retina nerve and blood vessel unit is based on a three-cell indirect co-culture method, retina microvascular endothelial cells, retina ganglion cells and retina Muller cells are simultaneously incorporated by utilizing Transwell, and the three cells are in indirect contact. The in-vitro three-culture model of the retina nerve and blood vessel unit can be used for more objectively simulating pathophysiological states of various cells of the retina nerve and blood vessel unit, so that common pathophysiological changes of mutual dependence and interaction of the three cells under in-vitro conditions can be observed.

The invention provides a stem cell exosome for overexpressing A beta degrading enzyme as well as a preparation method and application of the stem cell exosome. Specifically, the invention provides an application of the stem cell exosome overexpressing A beta degrading enzyme in preparation of drugs, and the drugs are used for at least one of the following: prevention, improvement or treatment of cognitive impairment related diseases; neuronal cell damage is improved; wherein the stem cells for overexpressing the A beta degrading enzyme are prepared through lentivirus transfection. According to the stem cell exosome overexpressing the A beta degrading enzyme, the content of the A beta degrading enzyme is obviously higher than that of the exosome prepared by a traditional method. Therefore, when the compound is prepared into a medicine for treating or preventing cognitive impairment related diseases, the medicine can be efficiently conveyed to the brain, and the concentration of the medicine in the brain is remarkably improved, so that the curative effect is enhanced, the dosage of the medicine can be reduced, the side effect is reduced, and the prognosis of brain diseases is effectively improved.

The application discloses a kind of hepatic organoids containing mesenchyme induction method, belong to organoid culture technical field.The method includes the following steps: (1) human embryonic stem cell WIBR3 pretreatment;(2) utilize human embryonic stem cell WIBR3 and induce liver organoids and mesenchymal cells;(3) mesenchymal cells and liver organoid cells are co-cultured, i.e. obtain the hepatic organoids containing mesenchyme.There is beneficial effect: the application changes the culture mode and method of the hepatic organoids containing mesenchyme.The hepatic organoids containing mesenchyme induction method proposed in the application is simple, and mesenchymal cells and other liver cells have the same genetic background source, suitable for the safety evaluation of drug and other chemical substances, liver fibrosis, non-alcoholic hepatitis modeling and pharmacodynamic evaluation.

The invention discloses a preparation method and application of a conditioned culture solution for improving the development rate of bovine in-vitro embryos. The preparation method comprises the following steps: culturing immortalized bovine endometrial epithelial cells by using a DMEM / F12 cell culture solution containing 10% FBS, collecting the cell culture solution in the culture process, and filtering to obtain an immortalized bovine endometrial epithelial cell conditional culture solution, namely the conditional culture solution capable of improving the development rate of the bovine in-vitro embryos. The prepared conditioned culture solution can simulate a liquid environment of an in-vivo environment, is used for culturing in-vitro embryos such as parthenogenetic activated embryos and in-vitro fertilized embryos, improves the cleavage rate and blastocyst rate of the in-vitro embryos and improves the development rate of the in-vitro embryos of cattle.

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

The invention belongs to the technical field of biomedical engineering and tissue engineering, and particularly relates to an acellular matrix modified bionic micro-fluidic organ chip constructed by 3D printing composite near-field direct writing as well as a preparation method and application of the bionic micro-fluidic organ chip. According to the bionic micro-fluidic organ chip prepared by the invention, the microstructure and physiological similarity of a model are remarkably improved, the problems of insufficient bionic property, single cell and the like at present are solved, and the preparation method is simple, is suitable for batch production and solves the problem of high production cost. The invention provides a new technical direction for the construction of the bionic organ model.

The invention discloses a preparation method of a cochlear organ chip integrated with a blood labyrinth barrier, and belongs to the field of biomedical engineering. The preparation method of the simulated blood labyrinth barrier comprises the following steps: constructing an endothelial cell culture cavity and a pericyte culture cavity, and separating the endothelial cell culture cavity and the pericyte culture cavity through a simulated basement membrane; and respectively inoculating percutaneous cells and endothelial cells into a percutaneous cell culture cavity and an endothelial cell culture cavity, and culturing to obtain the simulated blood lost barrier. The chip can effectively simulate the structure and the function of the BLB, and the barrier integrity of the BLB is evaluated through TEER (trans-epithelial electrical resistance) measurement and an apparent permeability coefficient. The invention also relates to application of the chip in NIHL drug screening, in particular to drug evaluation for oxidative stress induced inner ear organ injury. Through the platform, the protection effect of the candidate drugs on the TBHP-induced oxidative stress injury of the inner ear organs can be evaluated, and a new tool and thought are provided for developing novel NIHL treatment drugs.

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

The invention belongs to the technical field of organ chips, provides an in-vitro brain lymphatic system chip construction method and a brain lymphatic system chip, and makes up the blank of brain lymphatic system in-vitro model construction. The method comprises the following steps: firstly, carrying out flushing treatment, disinfection and sterilization treatment, rinsing treatment and cell culture on a micro-fluidic channel in the chip; then, in the treated micro-fluidic channel, blood vessel channel cell inoculation and brain parenchyma channel cell inoculation are carried out; and finally, in the blood vessel channel, perfusion is carried out once at a preset flow speed every preset duration, shear force stimulation simulating pulse type blood flow is provided, basic formation of in-vitro cerebral vessels is achieved after continuous perfusion, perfusion is carried out once at a preset flow speed every preset duration, shear force stimulation simulating pulse type blood flow is provided, and in-vitro cerebral vessels are formed. Intermittent liquid flow simulating arterial fluctuation is achieved.

The invention relates to an embryo model and a construction method thereof, and belongs to the technical field of stem cells. The invention provides an embryo model. The embryo model is obtained by spontaneously assembling # imgabs0 # induced pluripotent stem cells and induced hypoderm stem cells, the # imgabs1 # induced pluripotent stem cells and the induced hypoderm stem cells are both obtained by reprogramming somatic cells. According to the invention, stem cells of two different pedigree generated by somatic cell reprogramming are utilized to spontaneously assemble and construct an embryo model for simulating a human perioriginal period. The embryo model better reproduces the interaction and communication mechanism among the multi-lineage cells in the human embryo development process by combining the cells of different lineages; under the embryo model, pedigree tracking and gene regulation network research can be performed on cells of different pedigree through a gene editing tool, so that a molecular regulation mechanism of human early embryonic development is deeply understood.

Provided is a method for comprehensive evaluation of the biological potency of mesenchymal stem cells, comprising: simulating in vitro a scenario where mesenchymal stem cells enter an inflammatory site in vivo, detecting expression levels of PD-L1, IL-6, HGF, and IDO1 in the mesenchymal stem cells, and detecting inhibition rates of proliferation of immune cells and secretion of inflammatory factor TNF-α, so as to evaluate the immunomodulatory function and tissue regeneration function of the mesenchymal stem cells.

The invention relates to the technical field of tumor model construction, and discloses a chick embryo allantoic membrane model (CAM)-based liver cancer tumor planting improvement method, which comprises the following steps: S1, preparing a tumor cell suspension; s2, carrying out nude mouse subcutaneous transplantation; s3, nude mouse subcutaneous tumor treatment; and S4, performing CAM planting. According to the chick embryo allantoic membrane model (CAM)-based liver cancer tumor planting improvement method, liver cancer cells are firstly cultured subcutaneously in a nude mouse to form tumor tissues, then the tumor tissues are cut into pieces and mixed with matrigel and a DMEM culture medium, the tumor tissues are planted in a chick embryo allantoic membrane, the growth state of the tumor tissues is optimized in the nude mouse subcutaneous environment, a bionic microenvironment is provided by a mixed solution, the tumor formation rate is increased, and the survival rate of the chick embryo allantoic membrane model (CAM) is increased. A silica gel ring is fixed at a bleeding point of the chick embryo allantoic membrane, tissue suspension is dropped, a preservative film with holes is covered for culture, the silica gel ring prevents the suspension from leaking out, the preservative film guarantees gas and nutrition exchange, the contact between tumor tissues and chick embryo allantoic membrane blood vessels is promoted, the tumor is larger, and observation and experimental operation are facilitated.

The present disclosure provides methods and compositions for differentiating pluripotent stem cells and derived hematopoietic lineage cells, including hematopoietic endothelial cells, hematopoietic progenitor cells, and natural killer cells. The efficiency of differentiation of hematopoietic endothelial cells, hematopoietic progenitor cells, and natural killer cells can be improved by using the methods and compositions described herein.

The invention provides a preparation method of efficient activated NK cells, and belongs to the technical field of NK cell culture, and the preparation method comprises the following steps: S1, centrifuging whole blood, removing upper plasma to obtain a cell part, and diluting with a reagent I to obtain a cell diluent; s2, adding the cell diluent obtained in the step S1 into a centrifugal tube filled with a lymphocyte separating medium, centrifuging, sucking a middle mononuclear cell layer, and cleaning with a reagent II for multiple times to obtain target cells; s3, inoculating the target cells obtained in the S2 into a cell culture dish or a cell culture bottle, adding a lymphocyte basal culture medium and IL-2, activating the NK cells according to a traditional culture method, adding stem cell source exosomes on the seventh day of activated culture of the NK cells, adding the stem cell source exosomes once every three days later, continuously culturing to the 12th-17th day, and collecting to obtain the activated NK cells. The tumor killing effect is enhanced, and meanwhile the immune function of NK cells is adjusted.

The present invention relates to systems, compositions and methods for multicellular bioprinting vascularized tissue constructs comprising a hierarchical vascular network. The methods employ a dual bio-ink strategy involving fibrous microgel matrix bio-inks with adjustable hardness, self-healing rheology, and microporosity to support capillary self-assembly, and sacrificial bio-inks that define a large pourable channel after removal. The system integrates top-down printing and bottom-up cell self-organization within a programmable bio-printing platform. The key aspect of the invention is to engineer the physical topography of the matrix to actively guide vascular morphogenesis beyond conventional permissive stents. The integrated platform enables the manufacture of complex bionic vascular networks with enhanced mass delivery, cellular viability, and in vivo integration potential. The invention represents an important progress in the aspect of extendable production of functional human tissues, and has application in regenerative medicine, disease modeling and drug discovery.

A method for culturing a human hepatic parenchymal cell, the method comprising culturing the human hepatic parenchymal cell in a culture medium comprising a culture supernatant of a human hepatic nonparenchymal cell, a Wnt signaling promoter and a cell-division-promoting growth factor.

The invention discloses an edible beefsteak scaffold based on vegetable protein and a construction method thereof, and belongs to the fields of tissue engineering, material science, food science and biology. The edible beefsteak scaffold is a 3D printing scaffold based on vegetable protein, combined with gellan gum and sodium alginate, and is a cell culture meat bionic beefsteak composed of a bovine fibroblast and bovine muscle satellite cell co-culture system dynamically constructed by using a rotary culture system. The selected materials are all edible protein and polysaccharide, have high biocompatibility and safety, and are wide in source; the mode of dynamically constructing the bionic beefsteak can effectively reduce the formation of local necrotic areas, and the bionic beefsteak has excellent physical characteristics, has texture structures and flavor characteristics similar to those of real beefsteak, is sterile in surface, and can significantly reduce food sanitation and safety problems. The scheme provided by the invention has remarkable advantages in the aspects of food safety, texture regulation and control, sensory simulation and the like, and provides important technical support for the field of cell culture meat.

The present invention discloses a microfluidic chip and its construction method and application. A microfluidic chip of the present invention includes a polydimethylsiloxane layer and a base layer, the polydimethylsiloxane layer contains three or more microchannels; the microchannels are connected by a plurality of parallel microgrooves; the microchannels are provided with channels connected to the outside world; the base layer is at least one of a glass base, a polydimethylsiloxane base or a polystyrene base. The preparation method of the microfluidic chip of the present invention is simple and can achieve co-culture of tumor nerve cells. The pancreatic cancer-neural-stromal cell microenvironment model constructed using the microfluidic chip of the present invention can achieve the co-construction of the pancreatic cancer stromal microenvironment and the neural microenvironment, and can also perform qualitative and quantitative evaluation of nanomedicines targeting the neural microenvironment and the stromal microenvironment, which plays an important role in the screening of drugs related to the treatment of pancreatic cancer.

The invention discloses a chimeric antigen receptor exosome targeting a CD8T cell, a CAR-T cell and a construction method and application of the chimeric antigen receptor exosome, and relates to the technical field of biomedicine, and the construction method comprises the following steps: co-transfecting three plasmids, namely BRD-PTK-CD8 < + >-Lamp2b, pcDNA3.1-CD63-L7Ae and pcDNA3.1-N6 (SP)-Nb60-T20-C / D box, into a 293F cell, incubating, and purifying to obtain the exosome capable of targeting the CD8T cell. The exosome can deliver CAR-mRNA to T cells and convert CD8 + cells into CAR-T cells, and blood of an infected person does not need to be drawn; cAR molecules aiming at in-vivo malignant cells can be prepared in vivo; chemotherapy and radiotherapy are not needed, and side effects are avoided; and universality is achieved.

The invention discloses a novel ALS disease model and application thereof in screening ALS treatment drugs, and belongs to the technical field of disease models and drug screening. According to the method, induced pluripotent stem cells derived from a sporadic ALS patient are directionally induced into spinal cord organs, the spinal cord organs and immune cells derived from the same donor form a co-culture system, the co-culture system is infected with viruses, and the ALS disease model is successfully constructed. According to the method, ALS diseases are simulated on the three-dimensional level, ALS disease models of all stages (including the initial stage of the diseases) are obtained, and the pathological features of all the disease stages of ALS (especially 90% or above of sporadic ALS) can be accurately reflected; the problem that key disease-promoting factors in the early stage of the disease cannot be obtained due to complete disorder of downstream signal channels in the end stage of the disease after definite diagnosis caused by slow definite diagnosis of hidden disease onset of the ALS is solved.

Compositions and methods for stimulating the proliferation and cytotoxicity of γδ T cells are described. Therapeutic compositions and methods using the expanded and stimulated γδ T cells are described.

The invention discloses a preparation method and application of tumor exosome loaded gamma delta T cells. The tumor exosome-loaded gamma delta T cells are researched to be used for treating lung cancer, after the gamma delta T cells and the tumor cell exosome-loaded gamma delta T cells are co-cultured with lung cancer tumor cells respectively, a large number of tumor cell exosome-loaded gamma delta T cells surround the tumor cells, the tumor cells are attacked, and the tumor cell exosome-loaded gamma delta T cells are used for treating lung cancer. And the gamma delta T cells do not attack surrounding tumor cells. The tumor cell exosome loaded gamma delta T cells are intravenously injected into multiple lung cancer patients, compared with the prior art, the proportion of CD3 + and CD4 + positive cells is remarkably increased after 4 cycles and 6 months after treatment, the proportion of CD8 + positive cells is remarkably reduced, the ratio of CD4 + / CD8 + is remarkably increased after 6 months, and the number of NK cells is remarkably increased after 4 cycles after treatment, so that the tumor cell exosome loaded gamma delta T cells can be used for treating the lung cancer patients. The tumor exosome loaded gamma delta T cell has a good anti-tumor effect.

The invention provides a construction method and application of a sepsis model based on a lung and kidney organ chip. The method comprises the following steps: S1, carrying out ultraviolet sterilization on an organ chip device; s2, modifying a transwell plug-in unit; s3, inoculating human lung microvascular endothelial cells and human umbilical vein endothelial cells; S4, inoculating human alveolar epithelial cells and human renal tubular epithelial cells; s5, transwell inserts are arranged in two holes in the middle of the organ chip device respectively, a mixed culture medium is added into liquid adding holes in the two sides of the organ chip device, and the lung chip and the kidney chip are communicated through a lower channel of the organ chip device; s6, dynamically culturing the lung and kidney organ chip; and S7, sucking up the culture medium in the constructed lung and kidney organ chip, adding the LPS-containing mixed culture medium into a lower channel, and respectively adding the LPS-containing mixed culture medium into the human alveolar epithelial cell side and the human renal tubular epithelial cell side of the lung and kidney organ chip and the transwell plug-in hole for culturing for 2 days. According to the invention, the lung and kidney organ chip is applied to the sepsis model for the first time, and the constructed sepsis model is more suitable for basic research and drug development of sepsis.

The invention relates to bio-ink, microspheres for bio-ink, and preparation methods of the bio-ink and the microspheres. The bio-ink comprises: a microsphere comprising a methacrylated gelatin and a first conductive polymer, the microsphere optionally comprising a first cell; a hydrogel comprising a methacrylated gelatin and a second conductive polymer, the hydrogel optionally comprising second cells, the microspheres having pores. The bio-ink provided by the invention has good biocompatibility, conductivity, rheological property and mechanical property, and can promote interaction among biological materials.

The invention discloses a method for co-immunoculture of patient-derived tumor cell antigen peptide loaded by PBMC (peripheral blood mononuclear cells) and autologous DC (dendritic cells) and application of the method. The method comprises the following steps: extracting tumor-associated antigen peptide from a patient-derived tumor organ (PDO), and performing IFN-gamma pretreatment and freeze-thaw cycle purification; separating CD14 < + > mononuclear cells from the PBMC, inducing mature DCs (Dendritic Cells) by adopting a two-stage culture method, and loading antigens; and co-culturing the PBMC and the antigen-loaded DC, and detecting IFN-gamma expression after culturing for 48 hours. According to the method, the sensitivity and consistency of immunoreaction are remarkably improved, the experimental period is shortened, and the method is suitable for individualized drug screening and tumor immunity research.