16 results about "Primary culture" patented technology

The application discloses a construction method and application of an ovary cell line of Quasipaa spinosa. The method comprises the following steps: selecting metamorphosis period larva ovary tissue, disinfecting and obtaining the ovary tissue, and then using collagenase I digestion solution to digest the ovary tissue at 26 DEG C for 1 hour; after the tissue block adheres to the wall, using a low-osmotic-pressure complete culture medium to carry out primary culture at 25-27 DEG C, wherein the culture medium is 60% diluted DMEM / F12 culture medium containing 10%-15% fetal bovine serum; when the cell confluence degree is higher than 70%, using trypsin-EDTA to digest the cells for 1-2 minutes to subculture, and the subculture culture medium contains 30% cell conditional culture medium; and finally, the cells are cryopreserved through programmed cooling. The ovary epithelial cell line of Quasipaa spinosa is successfully established for the first time, the survival rate of the cells after recovery is high, and the karyotype is stable. The constructed cell line can be used for amphibian reproductive biology research, compound screening and in-vitro conservation of germplasm resources.

The application discloses a tissue culture and rapid propagation method for Yangmei of Alocasia macrorrhiza and a special combined culture medium thereof, and the method comprises the steps of sterilizing an explant, carrying out primary culture and subculture and proliferation culture in stages, wherein the primary culture is performed by first inoculating a stem segment into an induction culture medium containing 6-BA 0.8-1.2 mg / L, IBA 0.05-0.15 mg / L and PVP-40 to induce germination, and then transferring the stem segment into an elongation culture medium containing 6-BA 0.1-0.5 mg / L, NAA 0.01-0.05 mg / L and PVP-40 to promote elongation; the new shoots are then transferred into a subculture medium containing 6-BA 1.0-1.8 mg / L, NAA 0.1-0.2 mg / L, GA3 0.5-1.5 mg / L and PVP-40 to proliferate. The application effectively solves the technical problems that Yangmei is prone to brown and death, and it is difficult to simultaneously achieve budding and high growth by means of two-stage hormone stepwise regulation and cooperation with a brown prevention agent, and realizes efficient and large-scale breeding of high-quality seedlings.

The application relates to a method for separating and primary culturing of monopterus albus liver cells. The method comprises the following steps: obtaining monopterus albus liver tissue, perfusing the liver tissue with HBSS buffer solution without calcium and magnesium until blood is completely drained; removing the capsule, blood clots and connective tissue after rinsing, and cutting the liver tissue; sequentially using 0.02% EDTA solution, 0.25% trypsin solution and 50 U / mL collagenase IV working solution to perform three-step digestion treatment on the liver tissue fragments; obtaining a cell suspension by blowing and filtering after the digestion is completed, and then performing gradient centrifugal purification of the cells at rotating speeds of 600 rpm, 250 rpm and 80 rpm in sequence; and finally resuspending the cells in DMEM / F12 culture medium containing 10% FBS and inoculating and culturing the cells. The monopterus albus primary liver cells obtained by the method have the advantages of large quantity, high activity and high purity, and provide a reliable in-vitro model for monopterus albus related research.

This invention belongs to the field of biology and discloses a method for co-culturing enteric glial cells (EGCs) and intestinal organoids. Primary cultured and purified enteric glial cells are digested and seeded onto the membrane of a Transwell chamber. Intestinal organoids are placed in individual Matrigel drops in a co-culture plate. The Transwell chambers are placed in the co-culture plate, and growth medium is added to ensure free exchange of the growth medium through the Transwell membrane. The EGC-organoid co-culture system obtained based on this method provides a model for studying diarrhea and intestinal health in large mammals. It also lays the research foundation for developing innovative intervention strategies targeting the enteric nervous system and provides a research model for further exploring the regulatory roles of nutrients or related genes in intestinal health.

The present application relates to the technical field of aquatic animal cell culture, and particularly relates to a rapid enrichment and separation method of Plectrypops leopardus gonadal somatic cells. The method comprises the following steps: selecting healthy Plectrypops leopardus for anesthesia; taking gonadal tissue, and after aseptic treatment, cutting, centrifugal washing and filtration, inoculating in a culture medium for primary culture to obtain gonadal cells migrated from the tissue block, and after trypsin-EDTA digestion and centrifugal washing, using 1xPBS containing polysorbate for treatment; after blocking, antibody incubation and washing, verifying the labeling effect by a fluorescence microscope to realize specific enrichment of gonadal somatic cells; after filtration and purification of the enriched fluorescent labeled cells, placing the cells under a fluorescence microscope, accurately picking fluorescent positive cells under a fluorescence field, and transferring the cells to a culture dish containing complete culture medium, inoculating the separated fluorescent positive cells in optimized complete culture medium for purification culture, so that the cells adhere to the wall and realize stable subculture.

The application provides a tumor infiltrating lymphocyte culture medium and application thereof. The culture medium adopted by the application is composed of a basic culture medium, a serum substitute, a low-concentration interleukin, a stimulating factor and an antibiotic and the like. The application discloses a method for preparing high stemness tumor infiltrating lymphocytes (TIL) in vitro under serum-free and feeder-free conditions. Through optimized two-step culture (primary culture for 7-14 days + expansion culture for 7-14 days) and activation stimulation, high stemness TIL products with a CD3+ active cell ratio of greater than or equal to 95% and a Tcm ratio of greater than or equal to 70% can be obtained, and the total expansion multiple reaches 500-1000 times. The method of the application avoids the safety risk brought by feeder cells, reduces IL-2 related side effects, shortens the preparation period to 14-28 days, significantly improves the stem cell characteristics and sustained killing ability of TIL cells, and meets the quality control requirements of clinical-grade cell treatment products.

The present application relates to the technical field of plant tissue culture, in particular to a kind of golden bean tissue culture rapid propagation method and application;Select non-lignified young stem section as explant, clean and then use 75% alcohol, saturated bleaching powder solution, 0.05% mercury successively disinfection;The explant after disinfection is primary culture for 25-35 days, the single bud of the cluster buds of primary culture is cut and subculture for 40-45 days, and the single bud of the cluster buds obtained by subculture is cut and rooted for 40-45 days.The disinfection contamination rate of the present application can be controlled within 40%, the disinfection mortality rate is controlled within 10%, the success rate of establishing sterile system in laboratory can be effectively improved, the primary induction rate is more than 70%, the proliferation coefficient is more than 4, and the rooting rate is more than 95%.

The application discloses a kind of Vibration Mountain beard toad hind leg / toe tissue cells and its quick acquisition method and application.The method comprises: the body surface disinfection of Vibration Mountain beard individual, cut and take hind leg or toe tissue, and put back after wound disinfection;After tissue is soaked in 75% alcohol and washed with PBS containing penicillin, streptomycin and amphotericin B, it is temporarily stored in 4 ℃ temporary storage solution;After tissue is cut, it is sequentially digested with trypsin and type I collagenase;In the optimized low permeability medium, primary culture is carried out at 25-27 ℃;When cell confluence reaches 60%-70%, it is passaged using trypsin-EDTA digestion;Freezing and recovery.The application realizes the non-lethal cell of Vibration Mountain beard for the first time Quick acquisition, cell first time migration time is only 3 days, grow into monolayer only 12-17 days, pollution rate is reduced to 5.6%, recovery survival rate reaches 76.19%, and provides efficient and reliable technical support for endangered amphibian germplasm resource preservation and biological research.

FIELD: neurology; laboratory diagnostics.SUBSTANCE: used for the differential diagnosis of Parkinson's disease not associated with mutations in the GBA1 gene, and multiple system atrophy. The patient's peripheral venous blood is collected, from which mononuclear cells are isolated by gradient centrifugation, followed by their differentiation into a primary culture of macrophages in the presence of the macrophage colony-stimulating growth factor M-CSF to ensure proliferation and differentiation of monocytes into mature macrophages. The obtained blood macrophage cells are applied to 903 filter cards at a concentration of 2×106 cells / ml. Whatman 903 Sample Collection Cards can be used as 903 filter cards. The activity of lysosomal enzymes is determined: glucocerebrosidase GCase, alpha-galactosidase GLA, acid sphingomyelinase ASMase, galactosylceramidase GALC by high-performance liquid chromatography in combination with tandem mass spectrometry. The value of the canonical linear discriminant function CLDF is calculated using the stated formula. If the value of CLDF is ≥ 43.44, the patient is diagnosed with multiple system atrophy. If the value of CLDF is < 43.44, Parkinson's disease is diagnosed in patients who do not have a mutation in the GBA1 gene.EFFECT: method enables reliable and accurate differential diagnosis of Parkinson's disease and multiple system atrophy by assessing the activity of lysosomal enzymes.2 cl, 2 dwg, 2 ex

The application discloses a Plectropomus leopardus spleen cell line and application thereof. Plectropomus leopardus The spleen cell line PLS has a preservation number of GDMCC No: 68131. The PLS is obtained through primary culture and multiple subculture, has a fast cell growth speed, has been stably subcultured for more than 60 generations, has good activity after cryopreservation, and lays a foundation for Plectropomus leopardus gene germplasm resource preservation. Meanwhile, the PLS is sensitive to fish nervous necrosis virus and iridovirus, can be applied to exogenous gene function research, fish virus isolation, virus infection pathogenesis, virus host interaction and virus in-vitro infection model, and provides a high-efficiency amplification system for Plectropomus leopardus virus vaccine preparation.

This invention relates to the field of edible fungi spawn production technology, and more particularly to a yellow-lineage morel mushroom containing two mating type genes. Morchella sp. This invention discloses a pure strain of morel mushrooms, its preparation method, strain products containing the pure strain, and its use in the cultivation of *Morchella asiatica*. The genome of this pure strain contains both mating type genes MAT1-1-1 and MAT1-2-1. The advantage of this invention is that by placing multiple spores in a liquid culture medium for co-germination, the primary hyphae carrying different mating type genes will naturally fuse, forming visible antagonistic lines at the colony interface. Hyphae are picked from the antagonistic line region, and combined with PCR screening of the mating type genes, to efficiently obtain double-mating type strains. Furthermore, during the primary culture stage, covering the material surface with sterilized soil and controlling the temperature at 18-24℃ can induce the hyphae to expand in a directional, parallel manner from disordered twisting, and promote the differentiation of sclerotium-related structures at the casing soil-bottle wall interface, thereby significantly improving the quality of the primary culture.

This invention discloses a chicken embryo fibroblast, its preparation method, and its application, relating to the field of biopharmaceutical technology. The preparation method of the chicken embryo fibroblast includes the following steps: S1, providing chicken embryos and obtaining the muscle tissue of the chicken embryos; S2, mixing the muscle tissue with a plant-derived compound digestion solution, and sequentially performing digestion, purification, and primary culture treatment using a serum-free special culture medium to obtain primary chicken embryo fibroblasts; wherein, the plant-derived compound digestion solution includes bromelain and papain. This invention, using a plant-derived compound digestion solution and a serum-free special culture medium, significantly improves the batch-to-batch stability, cell yield, and cell purity of the cells, reducing the risk of exogenous viral contamination. Therefore, the prepared chicken embryo fibroblasts exhibit high batch-to-batch stability and good reproducibility in viral culture.

This invention relates to a rooting culture medium and tissue culture method for Rhizoma Cypripedium, belonging to the field of plant culture technology. The tissue culture method includes the following steps: obtaining explants, sterilizing them, transplanting them to a seedling culture medium for primary culture, subculturing, and then transplanting the subcultured explants to a rooting culture medium for rooting culture to obtain Rhizoma Cypripedium seedlings; the seedling culture medium and rooting culture medium are sugar-free media. This method enables the artificial culture of Rhizoma Cypripedium, with high rooting rate, high seedling establishment rate, and high survival rate of transplanted Rhizoma Cypripedium seedlings.

This application relates to the field of plant cultivation technology, and in particular to a method for establishing a high-frequency regeneration system for *Gnaphalium affine*. The method includes: S1, explant preparation and disinfection: *Gnaphalium affine* seeds are disinfected and inoculated onto a primary culture medium under aseptic conditions to obtain sterile seedlings; S2, shoot induction: the terminal buds of the sterile seedlings obtained in step S1 are cut as explants and inoculated into a first induction culture medium to induce the generation of shoot clusters; S3, shoot proliferation: the shoot clusters obtained in step S2 are divided and transferred to a second induction culture medium to promote the proliferation and growth of the shoot clusters; S4, rooting culture: the robust shoot clusters obtained in step S3 are transferred to a rooting culture medium to induce the formation of adventitious roots; S5, hardening and transplanting: the tissue culture seedlings with complete root development from step S4 are hardened and then transplanted into a cultivation substrate to obtain regenerated *Gnaphalium affine* plants. This application establishes a rapid propagation system for *Gnaphalium affine*.

The present application belongs to the technical field of forest tree breeding, and provides a micro-cutting method of heteromorphic Jiaoye Jinhua, comprising the following steps: inoculating the heteromorphic Jiaoye Jinhua terminal bud into a primary culture medium and culturing for 25-35 days to obtain culture 1; placing the culture 1 into a subculture medium and culturing for 55-65 days to obtain a cluster bud; after the cluster bud is seedling-raised for 5-9 days, the cluster bud is divided into single plants to obtain single buds; the single buds are cut into a rooting substrate, rooting induction is performed for 28-32 days, and rooted seedlings are obtained; and the rooted seedlings are transplanted into a mixed substrate for daily management. The method has the advantages of simple operation, controllable cost, short breeding cycle, high rooting rate and high survival rate after transplanting, and provides stable and efficient technical support for large-scale production, population protection and resource utilization of the heteromorphic Jiaoye Jinhua, and has significant application value and popularization prospect.

The application discloses a tumor cell primary culture device, which comprises a cell culture bin, a transfer mechanism and a plurality of cell culture bottles. The cell culture bin comprises a culture bin main body, which is internally provided with a plurality of gas chambers which are arranged at intervals along a horizontal preset direction. Any two adjacent gas chambers are provided with an isolation chamber. Each gas chamber is provided with a first air inlet pipeline, a second air inlet pipeline and a pressure relief pipeline. The isolation chamber is provided with a sealed state, a first communication state and a second communication state. The transfer mechanism is arranged outside the culture bin main body and comprises a moving frame, a moving part and an electromagnet. The moving frame is movable along the horizontal preset direction. The moving part is connected to the moving frame and is movable in the longitudinal direction. The electromagnet is connected to the moving part. One side of the cell culture bottle is provided with a first metal sheet. The moving part can adsorb the cell culture bottle in the gas chamber through the electromagnetic force between the electromagnet and the first metal sheet.