560 results about "In vitro incubation" patented technology

The invention provides an in-vitro culture method of T cells, and belongs to the technical field of genetic engineering.The culture method comprises the steps that immune cell serum-free culture media are added into peripheral blood mononuclear cells, and activated culture is conducted in the presence of cell factors to obtain activated T cells; the cell factor comprises an IL-2 monoclonal antibody and a CD3 monoclonal antibody, and further comprises two or three of an iCAM1 protein, a CCL21 protein and OK432. The T cells obtained through activation and multiplication culture are relatively stable in cell phenotype, relatively large in amplification multiple, relatively high in cell viability and relatively high in tumor killing efficiency. T cells are obtained after activation is performed for 24 hours and then multiplication culture is performed for 14 days, and the number of the cells is 6.2-8.9 * 10 < 9 >; the killing rate on Hela cells is 51.9 to 74.1 percent, and the killing rate on LOVO cells is 55.7 to 80.5 percent.

The invention discloses a marine fish myoblast in-vitro proliferation method. A basal culture medium is used as a background culture medium of a culture medium; the culture medium contains IGF2 protein with the final concentration of 20-200 ng / mL; the IGF2 protein is derived from plectropomus leopardus. The culture medium is used for culturing the passage cells to obtain the myoblasts proliferated in vitro of the plectropomus leopardus, the in-vitro culture of the myoblasts of the plectropomus leopardus is successfully realized, the number of the myoblasts reaches 30000, and the in-vitro proliferation rate reaches 87%. The invention provides raw materials for exploring the self function of myoblasts and gene regulation and control mechanism, provides preconditions for an in-vitro certification experiment for further exploring the influence of the myoblasts of marine fishes on the growth speed, and also lays a foundation for improved variety breeding of the marine fishes.

The present disclosure provides a preparation method and use of a hydrogel material for growth of blood vessel organoids (BVOs). The preparation method of the hydrogel material includes: (1) synthesis of gelatin methacryloyl (GelMA), and (2) synthesis of ns-GelMA-PEO. The hydrogel material may be used for in vitro cultivation of BVOs. The hydrogel material ns-GelMA-PEO prepared by the present disclosure may significantly improve the survival and budding abilities of BVOs. The ns-GelMA-PEO provides a novel solution for in vitro cultivation of BVOs, and may well support the growth and differentiation of organoids.

The invention belongs to the technical field of gene engineering, and relates to a construction method of a mouse model capable of rapidly degrading MSTN protein, which comprises the following steps: sequentially inoculating a 5'homologous arm, 3xEAAAK, an FKBP1A gene synonymous mutation sequence, 3xEAAAK, a red fluorescent protein mCherry sequence and a 3 'homologous arm on a Donor vector to obtain a Donor recombinant vector; designing gRNA based on a third exon of the Mstn gene, mixing the gRNA, Cas9 protein and a Donor recombinant vector, injecting the mixture into a mouse fertilized egg, and performing in-vitro culture and transplantation to obtain an F0-generation mouse; and hybridizing the F0-generation mouse with a wild type, or continuously breeding the filial generation to obtain the knock-in mouse model. According to the invention, a dTAG targeted protein degradation technology is utilized, the obtained homozygous knock-in mouse is subjected to dTAG administration, and rapid and reversible degradation and removal of MSTN protein can be realized.

The utility model relates to the technical field of liquid pressure control, and discloses a positive and negative pressure device for plant in-vitro culture, which comprises a gear pump, a liquid storage tank, a bubble remover, an electric control pressure regulating part, a liquid stirring part and a plant fixing part, and the electric control pressure regulating part comprises a first water flow regulating electromagnetic valve, a second water flow regulating electromagnetic valve and a hydraulic pressure gauge. The plant fixing part is provided with a flow guide pipe connecting pipe, a connecting pipe, a metal cable tie A and a metal cable tie B; according to the positive and negative pressure device for plant in-vitro culture, a nutrient solution is stirred through the liquid stirring part, the nutrient solution is conveyed through the gear pump, the water pressure in the flow guide pipe is controlled and adjusted through the electric control pressure adjusting part, and bubbles in the nutrient solution are filtered through the bubble remover. Therefore, the problems that an existing plant in-vitro culture device cannot culture plants with a nutrient solution under the same water pressure in the plants, the survival time of the plants is short, and leaves are prone to withering are solved.

The invention discloses an egg-carrying shrimp egg-removing liquid, an egg-removing device, an egg-removing method and a shrimp egg in-vitro culture method, and belongs to the technical field of aquaculture. The egg-carrying lobster egg-removing liquid is obtained by dissolving protease and sodium chloride in water and adding acid to adjust the pH value; the mass ratio of the protease to the sodium chloride to the water is (0.006-0.010): (0.006-0.009): 1. According to the invention, through the combined work of the egg-carrying shrimp egg-removing liquid, the egg-removing device and the fertilized egg in-vitro culture, the egg-removing and culture conditions are optimized. While the emergence rate of the fertilized eggs is guaranteed, intensive treatment of the shrimp eggs is carried out, and a new thought is provided for industrialization and industrialized breeding of the procambarus clarkii.

The invention provides an improved blastocyst culture solution as well as a preparation method and application thereof, and relates to the field of embryonic cell in-vitro culture. Based on a formula of a conventional blastocyst culture solution, colony stimulating factor-1 (CSF-1) and a transforming growth factor-alpha (TGF-alpha) are added, and the colony stimulating factor-1 and the transforming growth factor-alpha are dissolved in the conventional culture solution according to the concentration of 0.02-0.06 mmol / L and the concentration of 0.01-0.05 mmol / L; the obtained improved blastocyst culture solution can improve the mulberry embryo formation rate or blastocyst formation rate in in-vitro culture, promote the development of low-quality embryos and improve the mulberry embryo quality or blastocyst quality, and can be used for preparing medicines for promoting embryo implantation or subsequent growth and development.

The invention provides 3D tumor cell culture hydrogel as well as a preparation method and application thereof. The 3D tumor cell culture hydrogel is prepared from the following raw materials: polyvinylpyrrolidone and polyvinyl alcohol. The preparation method comprises the following steps: mixing a polyvinylpyrrolidone solution with a polyvinyl alcohol solution, adjusting the pH value, and freezing-unfreezing to obtain the 3D tumor cell culture hydrogel. The 3D tumor cell culture hydrogel provided by the invention does not need to be additionally added with a chemical cross-linking agent, and the material is high in biocompatibility and flexible in modulus adjustment, and can be well applied to a 3D in-vitro culture model of tumor cells.

The invention relates to isolation, purification, and in vitro cultivation of Symbiodinium algae symbiotic with soft corals. The deposit information for Symbiodinium sp. SY-1 is as follows: Depository Name: China General Microbiological Culture Collection Center (CGMCC); Depository Address: Institute of Microbiology, Chinese Academy of Sciences, Building 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China; Deposit Date: Jun. 7, 2023; Deposit Number: CGMCC No. 40681; Taxonomy: Symbiodinium sp.

The invention discloses application of a method for enhancing the treatment effect of salmonella bacteriophage, and relates to the technical field of microorganisms. According to the method, bacteriophage is added into salmonella for in-vitro culture, after continuous subculture, the salmonella is dropwise added to an SS plate containing the bacteriophage, and resistant bacteria of the bacteriophage are screened. The split bacteriophage is screened aiming at the resistant bacteria of the bacteriophage, and the split bacteriophage and the initial bacteriophage form a bacteriophage cocktail formula, so that the salmonellosis can be well prevented and treated, and the generation of the resistant bacteria is delayed. The method has the beneficial effects that compared with the existing method for screening the bacteriophage-resistant bacteria through a double-layer plate method, the method is more convenient, the operation is simple, and the screening efficiency of the bacteriophage-resistant bacteria is greatly improved. The bacteriophage cocktail contains the bacteriophage for cracking the resistant bacteria, so that the treatment effect of the bacteriophage is improved, and the practical application scene is quite high.

A method of evaluating disposition and / or effect of a candidate compound in an in vitro culture and / or suspension to predict in vivo disposition and / or effect of the candidate compound, which can include the steps of providing a cell culture and / or suspension; exposing a candidate compound to the culture and / or suspension; exposing the culture and / or suspension to a media providing an in vivo relevant extracellular environment, such as a media comprising a component (such as a protein and / or other component, such as lipoproteins, bile acids, and endogenous compounds such as bilirubin) at a physiologic concentration or a concentration having characteristics, such as binding characteristics, similar to the physiological concentration; wherein any combination of any of the exposing steps can occur in any order or simultaneously; and evaluating in vitro disposition and / or effect of the compound to predict in vivo disposition and / or effect of the candidate compound.

The invention relates to the field of cell culture, and discloses an NK cell culture method and application, and the NK cell culture method comprises the following steps: preparing a peripheral blood or umbilical cord blood sample, and extracting PBMC from the blood sample by using a Ficoll separating medium; using an NK cell sorting kit to separate CD3-CD56 + NK cells with flow cytometry detection purity greater than or equal to 95% from the PBMC; the obtained CD3-CD56 + NK cells are inoculated into an NK amplification culture medium for in-vitro culture, the period is 24 days, one or more traditional Chinese medicine polysaccharides, IL-2 and platelet lysates are added into the amplification culture medium, and thymoquinone is added on the 21st day of cell culture. The purified NK cells are long in proliferation time and high in proliferation multiple, and can be continuously cultured for 30 days; the traditional Chinese medicine polysaccharide promotes the proliferation efficiency and cell viability of NK cells and secretion of cell factors; thymoquinone can effectively inhibit T cell proliferation and activity within 72 h, and the purity of NK culture can be further maintained.

The invention discloses a preparation method of cells with LTo-like functions, which is characterized in that adipose tissue-derived stem cells are treated by LTo differentiation induction signal molecules to obtain cells with LTo-like phenotypes, secretion of cytokines and chemotactic factors of the cells is up-regulated, and the cells have recruitment and support capabilities on lymphocytes in vivo and in vitro. The problems that LTo cells are rare and difficult to obtain are solved, and the adipose-derived stem cells are higher in efficacy, easy to obtain and culture in vitro, capable of being massively amplified, low in cost, efficient and safe.

The invention discloses a rapid and efficient extraction method of extracellular vesicles rich in mitochondria. The extraction method comprises the following steps: (1) culturing mitochondrial donor cells in vitro; (2) treating the mitochondrial donor cells with cytochalasin D; (3) collecting the cells; (4) oscillating a cell suspension; according to the method disclosed by the invention, by combining cytochalasin D treatment with a subsequent gradient centrifugal separation step, a sample of extracellular vesicles rich in mitochondria can be efficiently extracted, meanwhile, the defects existing in a traditional mitochondrial transplantation technology are overcome, and a new thought and method are provided for clinical application of mitochondrial transplantation.

The invention relates to the technical field of in-vitro embryo culture, in particular to an in-vitro culture method and culture system of a mouse embryo. The method comprises the following steps: dropwise adding hydrogel onto a PDMS substrate, putting at least one, at least two, at least three, at least five or more embryos into hydrogel liquid drops, curing, and then adding a culture solution for embryo culture; wherein the Young modulus of the cured hydrogel liquid drops is 10 to 100 Pa. The in-vitro culture system comprises a PDMS (Polydimethylsiloxane) substrate and hydrogel liquid drops arranged on the PDMS substrate, and the hydrogel liquid drops are used for coating the mouse embryo; wherein the Young modulus of the cured hydrogel liquid drops is 10 to 100 Pa. When the method and the system are used for in-vitro embryo culture of mice, the embryos can normally develop and have a relatively high development rate within the in-vitro culture time of 9 days during in-vitro embryo culture.

The invention discloses an establishment method of an oat germ tip callus induction and efficient regeneration system based on small peptides. The establishment method comprises the following steps: firstly, culturing pretreated oat seeds in a 1 / 2 MS culture medium; when the seeds germinate for a certain length, taking the tips of the germs, putting the tips of the germs into an induction culture medium added with small peptides for induction, then transferring into a differentiation and proliferation culture medium added with small peptides for continuous culture, and then transferring into a rooting culture medium for culture to obtain differentiated seedlings with a certain root length; and finally, transplanting seedlings, hardening the seedlings, and culturing with vermiculite to obtain complete regenerated plants. According to the method, the oat germ is selected as the explant for plant tissue regeneration culture, materials are low in cost and easy to obtain, operation is easy, by introducing the small peptide capable of regulating and controlling the response and regeneration capacity of the local wound of the plant, the callus induction capacity of the apex of the oat germ in in-vitro culture is enhanced, the regeneration condition of the callus is optimized, and the tissue regeneration capacity of the apex of the oat germ is improved. Therefore, an efficient and stable oat regeneration system is constructed, and an important basis is provided for gene editing and oat germplasm innovation.

The invention provides oligonucleotide and method for marking cells, the nucleotide sequence of the oligonucleotide is shown as SEQ ID NO.1, the oligonucleotide sequentially comprises a primer 1, a bar code, a primer 2, a Poly A tail and a tail adding signal from the 5'end to the 3 'end, the nucleotide sequences of the primer 1 and the primer 2 are sequentially shown as SEQ ID NO.2-SEQ ID NO.3, and the bar code is composed of 10 random basic groups. According to the present invention, the primer 1 and the primer 2 in the oligonucleotide for labeling the cells do not match the sequence in the human genome, such that the library expression condition can be simply and rapidly verified through the PCR, the cells are labeled in batch during the in-vitro culture so as to provide the unique RNA bar code for each cell, such that the cells can be tracked, and the detection result is accurate. In addition, a single cell transcriptome sequencing technology is particularly optimized, so that the single cell transcriptome can be accurately detected in subsequent single cell sequencing.

The invention discloses an application of creatine in improving the development rate of an in-vitro blastocyst of a sheep oocyte. The concentration of the creatine is 0.5 mmol / L. The creatine is added into an in-vitro maturation system and an in-vitro embryo culture system. The key metabolite creatine is analyzed and screened based on metabonomics of sheep oocytes, early embryos, follicular fluid and fallopian tube fluid. On the basis, the development regulation effect of the creatine on the oocytes and early embryos is researched, the in-vitro blastocyst development rate of the sheep oocytes can be remarkably increased by adding the creatine into an in-vitro maturation system and an in-vitro embryo culture system of the sheep oocytes, and it is proved that the creatine can serve as an additive of the in-vitro culture system; and the creatine is a key metabolite contained in the development microenvironment and is friendly to oocyte and embryo development.

The invention discloses a method for promoting development of preantral follicles of a mouse and application of the method. The establishment of the follicle in-vitro culture system provides an ideal research model for the research of sodium butyrate; a mouse precavity follicle in-vitro 3D culture system is utilized, and the promoting effect of sodium butyrate on follicle development is fully revealed; the application of the sodium butyrate in promoting the development of the preantral follicles is disclosed for the first time, the ectogenesis rate of the preantral follicles of mice is improved, a theoretical basis is provided for improving the reproductive performance of animals by the sodium butyrate, and a technical support is provided for the application of the sodium butyrate in animal production.

It comprises a receptacle for the culture of plant material, a cap for closing an opening to said receptacle, and means for allowing the entry and / or exit of gas from said receptacle, and is characterized in that it comprises a container for receiving a liquid culture medium inside the receptacle, and support means for supporting the plant material on said reception container, wherein said container is attached to the lid of the receptacle in a position suitable for use, said lid including at least one medium inlet and / or exit conduit that connects with the container to be able to fill and / or empty said container through said conduit.

The invention discloses a three-dimensional biological printing meniscus model for simulating the mechanical heterogeneity of a natural meniscus and a preparation method of the three-dimensional biological printing meniscus model. The model comprises a structural PCL fiber frame (red region fiber circumferential arrangement and white region radial arrangement, and simulates the trend of natural collagen fiber), regional specific hydrogel (the red region contains GelMA, HAMA and SilMA, and the white region contains GelMA and HAMA), and partitioned cell components (the red region contains BMSC, HUVEC and CTGF, and the white region contains BMSC and TGF-beta3), the PCL frame and the hydrogel cooperate to form a mechanical gradient, and the compression modulus from the red region to the white region is simulated to continuously change. The preparation method comprises the following steps: designing a three-dimensional model, printing a PCL framework, preparing hydrogel and a cell suspension, preparing bio-ink, filling and printing by multiple nozzles, curing and culturing in vitro to obtain a target model. Compared with the prior art, the method has the advantages that the key problem of mechanical mismatch of an existing support is effectively solved, the model better fits a natural meniscus in mechanical property and biological function, and a more adaptive in-vitro model support is provided for individualized meniscus repair and transplantation substitution research.

The invention provides genetically engineered fibroblasts, a preparation method thereof, a culture medium for proliferating and / or differentiating B cells and application, and belongs to the technical field of genetic engineering. The genetically engineered fibroblast contains a CD40L gene, an IL-2 gene, an IL-4 gene, an IL-21 gene and a BAFF gene, can express CD40L, IL-2, IL-21, IL-4 and BAFF, can promote B cells to be differentiated into plasma cells, supports proliferation of the B cells, and can prolong the in-vitro survival time of the B cells, so that long-term in-vitro culture and efficient amplification of the low-density B cells are realized. Experimental results show that the genetically engineered fibroblasts constructed by the invention can support in-vitro culture of B cells for more than 16 days, and in-vitro survival of the B cells is effectively maintained. In addition, the cell line can also significantly promote B cells to differentiate into plasma cells and secrete high-level antibodies.

The invention provides an immune cell culture medium and a culture method thereof, and the culture medium significantly improves the amplification efficiency and functional activity of immune cells by optimizing the combination of cytokines, small chemical molecules and bioactive polypeptides. According to the invention, cell factors such as IL-2, IL-7 and IL-15 are integrated, and a specific antibody and a specific peptide fragment derived from a tumor-associated antigen are combined, so that a culture environment is obviously optimized. The culture medium disclosed by the invention shows an excellent effect in amplification and function enhancement of various immune cells, and is suitable for in-vitro culture and function research. Particularly, a new technical support and a breakthrough scheme are provided for immune cell treatment in the aspects of enhancing the killing activity of immune cells and maintaining the phenotypic stability.

The utility model provides a cell culture box for cell in-vitro culture, which relates to the technical field of cell culture boxes and comprises a cell culture box, a control panel is arranged at the top of the cell culture box, a sealing door is rotatably connected to the outer surface of the cell culture box, a heating plate and a guide rail are fixedly mounted on the inner surface of the cell culture box, and the control panel is arranged on the control panel. According to the tray fixing device, through the arrangement of the wedge-shaped block, the fixing groove, the limiting rod and the first spring, when the tray moves on the guide rail, accurate positioning and stable clamping connection can be achieved, so that the tray is effectively fixed, and through the arrangement of the pull ring, the clamping block, the fixing frame and the rotating block, the tray can be effectively fixed. The unlocking or fixing of the tray can be easily realized during the operation of a user, and the operation convenience and safety of the device are improved.

The application provides a culture medium and a culture method of ovarian cancer primary cells. The culture medium comprises an MST1 / 2 kinase inhibitor, sodium pyruvate, forskolin, epidermal growth factor, gastrin, fibroblast growth factor 7, nicotinamide, SB431542, and fetal bovine serum. Compared with the existing culture method, the in-vitro culture using the culture medium of the application has higher amplification efficiency; the culture of the ovarian cancer primary cells using the culture medium can maintain the morphological structure and pathological characteristics of the primary tissue, and improve the success rate and survival rate of the ovarian cancer primary cell culture.

This invention discloses an antibody that specifically recognizes Toxoplasma gondii surface antigens and its applications. The invention utilizes hybridoma technology to establish a hybridoma cell line by fusing Balb / C mouse spleen cells with SP2 / 0 cells. Monoclonal antibodies were prepared from these cells using in vitro culture and in vivo ascites induction methods, yielding the monoclonal antibody SH12-S7. This invention provides clinical and research institutions with an antibody that specifically recognizes Toxoplasma gondii surface antigens. It can be used for ELISA assays to detect clinical samples, for immunohistochemical assays to diagnose pathological specimens, and provides a convenient research tool for toxoplasmosis research departments. It has broad application value in the diagnosis and detection of definitive host infections and provides technical support for epidemiological investigations and evaluation of control effects of Toxoplasma gondii.

The invention relates to a composite microbial inoculant and an evaluation method for the antibacterial effect thereof. The method optimizes the in-vitro culture mode of the microbial agent compounded by aerobic bacteria and anaerobic bacteria by simulating the intestinal environment of probiotics in an animal body, and adopts a facultative anaerobic culture method for the microbial compound agent, namely, the outer layer of a sealing film of a triangular flask is wrapped with four layers of preservative films, and the neck of the triangular flask is tightened by a rubber band. The facultative anaerobic environment of aerobic bacillus and facultative anaerobic enterococcus faecalis is considered in the microbial composite inoculant, in the subsequent oxford cup bacteriostasis evaluation experiment, the experiment result with the accurate bacteriostasis effect and good parallelism can be obtained, and the action effect of bacillus and enterococcus faecalis composite bacteria can be efficiently and scientifically evaluated.

The invention discloses construction of a Guangdong small-ear spotted pig spermatogonial stem cell in-vitro culture system and screening of surface markers, and belongs to the technical field of cell culture.The technical scheme includes that testis are broken firstly, then enzyme digestion, density gradient centrifugal purification and differential adherent purification are conducted, and purified spermatogonial stem cells are obtained; inoculating the obtained spermatogonial stem cells to feeder layer cells and culturing in a culture medium; the culture medium is a DMEM culture medium added with FBS and growth factors; the mass fraction of the FBS in the DMEM culture medium is 8-12%, an enzyme digestion method, a density gradient centrifugation method and a differential adhesion method are combined for application, the Guangdong small-ear spotted pig spermatogonia stem cells with certain purity are obtained, cells enriched and purified in vitro can proliferate massively, and an in-vitro culture system of the Guangdong small-ear spotted pig spermatogonia stem cells is established.

Production and maintenance of hematopoietic stem cells in in vitro culture systems has proven to be elusive. Disclosed herein are hematopoietic stem cell compositions that originate from organoids such as liver organoids. These organoids also comprise a rare type of immune cell that is not yet fully elucidated due to the difficulty in isolating said immune cell from biological samples. Also disclosed herein are methods of producing said hematopoietic stem cells and immune cells from organoids, as well as methods of expanding hematopoietic stem cells from other sources using these organoids.

The invention discloses a culture method of in-vitro uniform monolayer cells of prawns, and belongs to the technical field of biology. The method comprises the following steps: (1) obtaining lymphatic tissues of a last-stage prawn at the early stage of shelling, completely stripping an outer membrane to obtain internal tissues, and cutting the internal tissues into small tissue blocks; (2) cleaning the small tissue blocks, inoculating the small tissue blocks to a culture plate coated with a gelatin solution, adding a complete culture solution into an inoculation system at intervals within 12 hours after inoculation for culture, replacing the complete culture solution 24-48 hours after inoculation, and inducing tissue cells to continuously migrate to form an in-vitro cell layer; (3) blowing and beating the cell culture to obtain a cell suspension, and centrifuging to obtain a cell monolayer; and (4) suspending the cell monolayer by adopting a complete culture solution, then inoculating the cell monolayer into a culture plate coated with a gelatin solution, and culturing to obtain the in-vitro uniform monolayer cells of the prawns. According to the method, the prawn monolayer cells which are long in in-vitro survival time, uniform and stable and are cultured in vitro can be obtained, and an important tool is provided for related virology research.