479 results about "In vitro incubation" patented technology

The invention discloses preparation and application of exosome secreted by human derived blood or mesenchymal stem cell. The preparation process of exosome comprises: extracting mesenchymal stem cells from health-human body blood or a stem cell culture supernatant, performing in-vitro culture and amplification, and utilizing means such as low-temperature separation, purification and the like to prepare high-purity high-bioactivity exosome. The blood or mesenchymal stem cell secreted exosome is added into medicines, health-care products and cosmetics according to ratios for developing of products with bioactivity, is capable of effectively regulating and controlling body cell signal transduction, activating cell regeneration, enhancing health cell proliferation and differentiation capacity, further regulating physiologic restoration or removing damaged, pathologic and aged cells of a body, fundamentally changing body physiologic functions, and helping to generate anti-tumor treatment effect by adjusting cellular immunity. The blood or mesenchymal stem cell secreted exosome is applicable to fields of medical science, health care and beauty treatment, and has wide application prospect.

The invention discloses a three-dimensional porous chitosan/gelatine microsphere, a preparation method thereof and an application thereof in liver cell culture. The preparation method thereof comprises the following steps of: rapidly freezing mixture into spheres by dropping liquid nitrogen by virtue of a high-pressure pulse microcapsule moulding instrument after chitosan and gelatine B are mixed, carrying out secondary freeze-drying after microspheres subjected to primary freeze-drying are crosslinked and cured with saturated tripolyphosphate-85% ethanol solution, fully hydrating the microspheres obtained by the secondary freeze-drying, adding cross linking agent carbodiimide/N-hydroxy succinyl, modifying the microspheres with gelatine A, removing unreacted carbodiimide/N-hydroxy succinyl and gelatine A by washing after water bath and reaction in dark place are carried out, and carrying out tertiary freeze-drying, thus the three-dimensional porous chitosan/gelatine microspheres with the diameter of 300-800Mum and the surface aperture of 50-200mu m are obtained, and the surface of each microsphere is connected with the aperture of the interior of the microsphere. The three-dimensional porous chitosan/gelatine microspheres obtained by the invention can be used for in vitro culture of high-density and high-activity liver cells.

The invention provides an assembling type cell-derived extracellular matrix membrane (ECM) compound bone repairing material as well as a preparation method and application thereof. The bone repairingmaterial is prepared by the following steps: completely covering a three-dimensional degradable biological scaffold with a sheet-shaped acellular matrix membrane under sterile conditions and then freezing the three-dimensional degradable biological scaffold for 24 to 48h under the condition of -20 to -80 DEG C; then carrying out freeze drying treatment to obtain the ECM membrane compound bone repairing material, wherein the sheet-shaped acellular matrix membrane is a cell-derived extracellular matrix which is generated by in-vitro culture of cells, is subjected to decellularization treatment and has certain bone induction activity, and the three-dimensional degradable biological scaffold is a biological scaffold which is prepared from natural polymers, has a three-dimensional porous microscopic structure and can be completely degraded and absorbed in human bodies. The assembling type cell-derived extracellular matrix membrane compound bone repairing material has a good bone induction capability; furthermore, the bone regeneration capability of the compound material is strengthened; a construction method is simple and feasible and the material can be stored for a long time and has awide clinical application potential.

The invention relates to a method for in vitro incubation of fertilized eggs of macrobrachium nipponense, which is characterized in that the method comprises the steps of selection of parent shrimps, mating and oviposition, treatment of incubation water, egg stripping, egg washing, incubation, daily management and the like. The method for the in vitro incubation of the fertilized eggs of the macrobrachium nipponense solves the problem of biological characteristics that the fertilized eggs depend on parent bodies to be incubated, lays a foundation for genetic operations of the macrobrachium nipponense such as gene transfer, nuclear transplantation and the like, fills the research gap of the field, and has important practical application value.

The present invention provides isolated cells that comprise, integrated into the genome of the cell, a transcription-competent immunodeficiency virus or a transcription-competent immunodeficiency virus-based retroviral vector. Under basal in vitro culture conditions, the immunodeficiency virus is latent, and the expression of the latent immunodeficiency virus can be reactivated. The invention further provides methods of making a subject cell. The invention further provides screening methods for identifying agents that activate a latent immunodeficiency virus; and screening method for identifying agents that block reactivation of latent immunodeficiency virus expression in response to T cell activation signals. The invention further provides agents identified in the subject screening assays. The invention further provides methods of treating an immunodeficiency virus infection.

The invention provides a stem cell culture medium and a method for culturing endometrium stem cells by using the stem cell culture medium. The method comprises the following steps: separately collecting menstrual blood and endometrium tissues, respectively culturing the menstrual blood and endometrium tissues in the stem cell culture medium provided by the invention to respectively obtain menstrual blood adherent cells and endometrium adherent cells, culturing the menstrual blood adherent cells and endometrium adherent cells in a cell culture bottle, collecting the adherent cells by trypsinization, inoculating the adherent cells in a cell coculture dish, and culturing the adherent cells in the stem cell culture medium provided by the invention. The stem cell culture medium has the advantages of simple components, fewer added components and lower cost. After more than 20 generations of in-vitro culture, the cells can not easily have the phenomenon of aging or degeneration, and can maintain the activity and stem property of the stem cells for a long time. The stem cell culture method is simple and effective, the cell proliferation efficiency is high, and the in-vitro culture doubling time is only 20 hours or so. The cells can be stably amplified by 50 generations.

Disclosed is a method of mass producing potato seedlings, comprising collecting growing points of seed potatoes and culturing the growing points in a liquid or solid medium; introducing in vitro plantlets obtained from the culture of the growing points to solid culture; and removing the in vitro plantlets from the solid culture, and planting through stem cutting and acclimatizing the in vitro plantlets in deep flow culture, in which a nutrient solution is circulating. When in vitro-cultured plantlets are planted in DSCAC through stem cutting, and sprouts from the terminal and lateral buds of the plantlets are hardened by gradually increasing light levels from the dark condition, the plantlets have healthy dark-green leaves and a high accumulation of carbohydrates, thereby obtaining potato seedlings having long stems that appear robust but not overgrown, and elastic and short nodes. Upon planting in hydroponic facilities, such potato seedlings have high adaptability to the external environment and thus rapidly, uniformly generate roots in a short time. The rapid root anchoring prevents planted seedlings from withering, leading to death, growing poorly, and the like. The direct planting of in vitro plantlets through stem cutting without a separate acclimatization process shortens the overall production period of potato seedlings by omitting the acclimatization process.

Disclosed is a method for detecting CYP450 enzymatic activity by means of micro-liver tissue incubation in vitro, which is characterized in that: a liver trace puncturing method used in clinic takes micro-liver tissues of 0.1 to 0.2g, or kills a mouse to take out the liver in an animal experiment, and builds a micro-liver tissue in vitro incubation system, then uses a one-probe medicine to perform the liquid phase chromatography tandem mass spectrometry for detecting probe substrates and concentration of corresponding metabolites, and obtains the activity of P450 enzyme according to metabolite ratios thereof. The method has the advantages of: 1. micro scale: only micro-liver tissues of 0.1 to 0.2g are needed for detecting cytochrome P450enzyme activity, and can be used for animal experiment and checking clinic micro-liver tissue liver drug enzyme activity; 2. time saving and convenience: costly ultracentrifugation equipment used in the conventional method is saved, costs and time for detection are greatly saved, and liver enzyme metabolic condition simulation is better; and 3. establishing indexes for evaluating the enzyme metabolic activity: metabolite ratios.

The invention discloses a cultural method for inducing a human embryonic stem cell to directionally differentiate into a corneal limbal stem cell, which comprises the following steps that firstly, a DMEM (Dulbecco's Modified Eagle Medium)/F12 conditioned medium is adopted to culture a human primary corneal limbal stem cell to prepare a corneal limbal stem cell conditioned medium, and then the corneal limbal stem cell conditioned medium is utilized and combined with IV type collagen culture in vitro to induce the human embryonic stem cell to directionally differentiate into the corneal limbal stem cell. According to the corneal limbal stem cell obtained by utilizing the cultural method, through light microscope observation in vitro, electron microscope observation, real-time quantitative polymerase chain reaction, immunofluorescence, flow cytometry, cloning efficiency determination and the like, the induced cell has a similar shape and phenotype with a normal corneal limbal stem cell, has good differentiation and proliferation capacity in vitro, can be transferred in vitro for more than four generations and can be used as a seed cell for preparing a corneal graft.

The invention discloses a human-derived stem cell bioactive substance, its preparation and application. The method comprises: first extracting healthy human-derived stem cells, which are then subjected to in vitro culture and amplification, collecting stem cells of specific phases, and utilizing modern ultralow temperature as well as other biological technologies to prepare a stem cell bioactive substance group composition. Mainly coming from mesenchymal stem cells and hematopoietic stem cells, the stem cell substance group can be developed into products with a cytobiological activity function. When applied on human bodies, the products can activate body stem cells, repair or substitute damaged, pathological and aging cells, adjust the metabolic function of body cells, enhance body immunity, and delay aging, etc. The human-derived stem cell bioactive substance group composition in the invention boasts substantial application prospects in medicine, healthcare and beauty maintaining fields.

The invention discloses multipotent stem cells carrying human adult premature senility syndrome gene mutations, mesenchymal stem cells and a preparation method thereof. The method comprises the following steps: (1) taking human multipotent stem cells cultured in vitro, and mutating human adult premature senility syndrome genes WRN in the human multipotent stem cells to enable the WRN to lose functions, so as to obtain the human multipotent stem cells with the function-lost WRN; and (2) carrying out induced directional differentiation on the human multipotent stem cells with the function-lost WRN to obtain the mesenchymal stem cells with the function-lost WRN. The mesenchymal stem cells derived from the multipotent stem cells provided by the invention can be used for establishing a medicine screening platform for screening a WS treatment medicine, and providing more clues for delaying a natural senility process.

The present invention relates to a cell construct cultured in vitro characterized in that it comprises (i) animal cells cultured in vitro in conditions ensuring formation of a three-dimensional tissue structure and (ii) an endogenous extracellular matrix in which said cells are imprisoned, and in that it comprises, among said cells, cells having a mineralization and/or ossification capacity. The invention is particularly useful in the fields of grafts and implantology (tissue reconstruction or repair), as well as in the pharmaceutical industry, for research on tissue and analysis of the toxic or beneficial profile of molecules capable of being used in pharmaceutics development and/or pharmaceutical compositions.

Disclosed are processes for producing stably transformed fertile wheat a system of transforming wheat via Agrobacterium. This invention provides methods transforming a variety of explants, such as freshly isolated or pre-cultured immature embryos, embryogenic callus and suspension cells. Also disclosed are methods for recovering transgenic plants after transformation within a short period of time, if the explants are regenerable at the time of transformation. Thus the frequency of somaclonal variation associated with prolonged in vitro culture period is significantly reduced. The transformation frequency using this system is comparable to or better than published methods using other systems, such as microprojectile bombardment.

The invention relates to a rat liver cancer model with different liver matrix hardness backgrounds and a preparation method of the rat liver cancer model. The method for preparing the rat liver cancer model with different liver matrix backgrounds is characterized by specifically comprising the following steps of: after grouping the rats, injecting pure carbon tetrachloride to an abdominal region in a hypodermic manner; injecting a carbon tetrachloride-olive oil solution to the abdominal region in the hypodermic manner; detecting through a texture analyzer to form the rat model with different liver matrix hardness; taking liver cancer cells which are in-vitro cultured and grown well and injecting the liver cancer cells to the rat to form hypodermic liver cancer cells; in-situ planting the formed liver cancer tumor source to the rat model with different liver matrix hardness to obtain the rat liver cancer model with different liver matrix hardness backgrounds. The rat liver cancer model with different liver matrix hardness backgrounds can be used for well displaying liver cancer malignant pathological characteristics under the liver matrix hardness interference, simulating and reproducing pathological characteristics of clinical liver matrix hardening background liver cancer and solving the problems that an ideal experiment system, an animal model and the like are lacked in reaching the development of the liver cancer due to the liver matrix hardness changes.

The invention relates to a construction method of a human amniotic mesenchymal stem cell bank. The construction method comprises the following steps: taking a human amnion for detection, flushing and washing the human amnion with a phosphate buffered solution, then smashing the human amnion, diluting with the phosphate buffered solution, digesting with trypsin, digesting with collagenase IV and deoxyribonuclease I, and filtering to obtain a single-cell suspension; by adding human serum albumin, transferring, insulin and sodium selenite into a DMEM/F12 basal culture medium with the ratio of VDMEM to VF12 being 1 to 1, placing human amnion mesenchymal stem cells in an incubator under the serum-free condition, and then performing liquid exchange and culture transfer; subjecting the mesenchymal stem cells obtained through in vitro culture and proliferation to liquid nitrogen refrigeration, and preserving the cells according to the gender of newborn infants, the ABO/Rh type and the HLA type; establishing cell information files, so that the human amniotic mesenchymal stem cell bank is constructed. The method has the characteristics of no other animal derivation, wide source range and no ethic limitation. With adoption of the method, the human amniotic mesenchymal stem cells can be provided for cell therapy and other application.

The invention belongs to the field of use of a stem cell conditioned medium in medicinal cosmetics, and especially relates to an application of the conditioned medium in skin ageing resistance. Researches find that a large amount of complex active substances secreted by stem cells are stored in a medium in the in vivo culture process of mesenchymal stem cells, and is called as the stem cell conditioned medium, and an obtaining method of the medium comprises the following steps: carrying out digestive passage on p2 generation mesenchymal stem cells by using 0.05% pancreatin, inoculating 2*10<6> into a 150mm culture dish, and culturing to obtain p3 generation cells; and sucking the obtained culture supernatant when 80% of the p3 generation cells fuse, washing by using PBS twice, adding a new serum-free medium, putting the obtained cells in a 37DEG C 5% CO2 incubator, culturing for 48h, and collecting the obtained culture supernatant to obtain the stem cell conditioned medium. The stem cell conditioned medium obtained in the invention can supplement skin cell metabolism promotion substances lacked in skins and also can induce new collagen synthesis in order to maintain elasticity of skins and reduce wrinkles.

The invention discloses a composite microcapsule model for use in in-vitro cell coculture. The model is a sphere-in-sphere composite microcapsule model consisting of an inner sodium alginate gel microsphere and an outer sodium alginate gel microsphere, which is formed by using natural polysaccharide sodium alginate as a substrate material, dripping the polysaccharide sodium alginate into multivalent cation solution and performing gelatinization treatment twice, wherein the two gel microspheres may contain two different kinds of cells. The model can be used for the study on in-vitro coculture of various cells and overcomes the drawback that the interaction between cells of the same kinds and the interaction between cells of different kinds cannot be controlled, the drawback that different kinds of cells are difficult to separate in late-stage study and the drawback that cell dependency exists of the conventional coculture model; and the model can simulate cell-cell interaction, cell-extracellular matrix interaction and cell-soluble factor interaction and provide theoretical and technical support for in-vitro construction of a tumor model, optimization of in-vitro culture mode of cells and tissue engineering construction of various tissue and organ analogues or equivalents.

The invention provides a separation method for mouse female germline stem cells in the technical field of transgenosis engineering. The separation method comprises the following steps: step one, collecting a mouse ovary, and adopting a two-step enzymic digestion method for preparing germline stem cell suspension; and step two, adding mvh first antibody and then second antibody magnetic bead in the cell suspension and re-suspending the cells, thus separating the female germline stem cells. The invention also provides a culture in vitro method for the mouse female germline stem cells, which comprises the following steps: transferring the female germline cells on an STO cell culturing layer, adding female germline stem cell culture solution, and conducting primary culture and subculture on the female germline stem cells, thus obtaining purified and stable germline stem cells. In the method, the mouse female fermline stem cells are separated and cultured in vitro successfully at first time, the methods are simple, the cost is low, the requirements on experiment conditions are low, and the operability is strong.

The invention relates to an in vitro culture kit for T-lymphocyte cells, which fundamentally solves the problems of existing T-lymphocyte cell in vitro culture methods that the used materials and culture time are inconsistent, and the culture periods, quality and quantity of effective cells are different. The technical key points are as follows: the content comprises a coating bottle for inducting the differentiation of the T-lymphocyte cells; a CD3 monoclonal antibody, gamma- interferon and interleukin-1 alpha are pre-coated in the coating bottle; the component of a cell factor A, a cell factor B and a cell factor A for stimulating the division and proliferation of the T-lymphocyte cells is 200000 units of interleukin-2, and the component of the cell factor B is 2 million units of interleukin-2. According to the in vitro culture kit for the T-lymphocyte cells, the composite reagent and materials required for the in vitro culture of the T-lymphocyte cells are combined together in a standard way, all the operation is standardized and modularized, so that not only is the working efficiency improved, but also the cell quality and yield difference caused by the operation of different people is reduced.

The invention discloses an in vitro maturation culture method of denuded oocytes of mice. The method comprises the following steps: (1) performing in vitro maturation culture on the denuded oocytes; (2) performing parthenogenetic activation on matured denuded oocytes; (3) performing in vitro fertilization on the matured denuded oocytes. According to the method, an environment for artificially simulating natural maturation of oocytes can be created, so that the denuded oocytes can efficiently maturate and grow in vitro and can be subjected to in vitro fertilization and embryonic development as same as normally growing oocytes after in vitro maturation culture. The practice proves that the in vitro maturation culture efficiency of the denuded oocytes of the system is high, the system is mature and stable, and the embryonic development quality of the denuded oocytes is high after in vitro natural fertilization, so that the application and popularization of the denuded oocytes in reproductive health of human beings and genetic breeding of modern livestock can be greatly facilitated. In addition, the operation of obtaining blastulae from the denuded oocytes is simplified and the denuded oocytes serving as ovum sources can be conveniently put into actual production.

The invention provides a method for improving the blastocyst rate of in-vitro embryos of animals. According to the method, the developmental capacity of a parthenogenetic embryo and an in-vitro fertilization embryo can be remarkably improved by adding type I interferon into an embryonic development culture solution for the in-vitro culture of the embryos; compared with the blastocyst rate obtained under the culture of the conventional culture solution, the blastocyst rate is improved by 7 to 13 percent; and the blastocyst hatching rate is also remarkably higher than the blastocyst hatching rate under the culture of the conventional culture solution. By the method, the early development capacity of in-vitro embryos of cattle and sheep is improved, so that the in-vitro production efficiency of embryos of the cattle and sheep is improved, and high-quality embryos are provided for the scientific research and production field in which the embryos of the cattle and sheep are taken as materials.

The invention discloses a swine in-vitro embryo culture solution. The swine in-vitro embryo culture solution disclosed by the invention is obtained through adding ligustrazine, with the final concentration of 0.05-5.00 microgram/milliliter, into an embryo culture solution NCSU-23. A traditional Chinese medicine monomer component, namely ligustrazine, is added into the swine in-vitro embryo culture solution disclosed by the invention; shown by experiments, through carrying out in-vitro culture on the swine parthenogenetic activated embryos and the swine in-vitro fertilized embryos by using the culture solution, the development efficiency of the embryos and the number of the cell masses and total cells in blastulae can be effectively increased. The swine in-vitro embryo culture solution has the advantages that a foundation is laid for the development of other biotechnologies, medical research and animal husbandry, and meanwhile, a certain theoretical basis for clarifying the development mechanism of early swine embryos is provided.