79results about How to "Stable proliferation" patented technology

The invention relates to the field of bioengineering technology, and in particular to a method for in-vitro induction of cholangiocyte-like transformation of primary hepatocytes and for long-term culture, amplification and differentiation and an application of the method. The invention provides a hepatocyte cholangiocyte-like transformation medium determined by chemical ingredients and / or a system which is composed of a hepatocyte mature medium and is applicable to long-term stable culture, amplification and differentiation of the primary hepatocytes; the invention also provides the method for in-vitro induction of cholangiocyte-like transformation of the primary hepatocytes and for long-term culture, amplification and differentiation; and with the application of the method, the cholangiocyte-like hepatocyte conversion of the primary hepatocytes can be induced in vitro, so that the obtained hepatocytes have characteristics of biliary epithelial cells and hepatic precursor cells, and the hepatocytes are applicable to long-term stable culture and amplification. The breedable cholangiocyte-like hepatocytes and hepatocytes, which are mature in differentiation, prepared by the invention are applicable to such aspects as toxicologic and pharmacological evaluation of compounds and drugs, researches and diagnosis & treatment of hepatitis viruses, treatment by hepatocyte transplantation, preparation of bioartificial liver and the like.

The invention disclose a technique for the tissue culturing and breeding of the red-welcome cuckoo as well as the technique for domestication and replanting, which comprises the first generation culture of inducing the clump sprout by applying the germ-free seedling as explant, subculture for enrichment-culture, root-growing inducing for the clump sprout, water-planting for domestication before replanting the tissue culture sprout, the final replanting to flowerpot and getting the red-welcome cuckoo. The invention is suitable for the red-welcome cuckoo from different places, the k-factor of the clump sprout is high, the root is uniform, and the survival rate after the water-culture domestication is high and is suitable for the red-welcome cuckoo commercial production.

The invention provides a NK (natural killer) cell culture medium and a culture method of a NK cell, wherein the NK (natural killer) cell culture medium comprises the following components in parts by weight: 83 to 93 parts of serum-free basal culture medium; 4 to 6 parts of plasma; 0.5 to 1.5 parts of interleukin-2; 0.5 to 1.5 parts of anti-CD3 monoclonal antibody; 1 to 5 parts of insulin-like growth factor 1; 1 to 3 parts of lycium barbarum polysaccharide. According to the NK (natural killer) cell culture medium provided by the invention, the safety is better, and the NK cell cultured by the culture medium with the components can quickly and stably proliferate, and has better killing activity to tumor cell. Experimental results show that, after using the NK cell culture medium provided by the invention for culturing the NK cell for two weeks, the proliferation times soars almost fiftyfold; the killing activity to K562 reaches 91 percent at effector-target ratio being 40: 1.

The present invention relates to a cell line derived from the cambium of an herbaceous plant having a storage root and a method for isolating the same. More specifically, relates to a cambium-derived homogeneous cell line having the ability to divide, which is obtained from the cambium-containing storage tissue of an herbaceous plant having a storage root without a separate dedifferentiation process, and to a method for isolating the same. The cell line derived from the cambium of an herbaceous plant having a storage root has active division ability and is homogeneous. Also, it is stable during culture, because it has not undergone a dedifferentiation process. Thus, through the optimization of proliferation thereof, the cell line can be allowed to proliferate in a large amount within a short time. Accordingly, the cell line derived from the cambium of an herbaceous plant having a storage root makes it possible to produce large amounts of useful plants which are difficult to cultivate outdoor due to various problems associated with the period of cultivation, the selection of cultivation land, cultivation cost and the like.

The invention discloses a combined algal reef cluster. Artificial algal reefs in the prior art only can provide attachments for growth of algae but can not make algae at part of sea areas be rapidly multiplied within short time. The combined algal reef cluster comprises floaters, slings, cultivating bodies and positioning anchors. Each cultivating body comprises a circular ring upper frame, a bottom plate, a middle column, a conical surface silica gel base body, a balancing weight and algal reef bricks, wherein the edge of the wide end of the conical structure of the conical surface silica gel base body is fixedly connected with the upper frame; the edge of the narrow end is fixedly connected with the edge of the bottom plate; the algal reef bricks are laid on the inner side surface of the silica gel base body; the top end of the middle column is connected with the corresponding floater through the corresponding sling; the bottom end of the middle column is connected with the corresponding positioning anchor through the corresponding sling. The combined algal reef cluster has the advantages that algal seedlings are protected, and the survival rate of algal seedlings is improved; the algal growing space is extended when algae grow to be a certain scale, and algae are further multiplied; the yield of a unit cultivating area is easy to control, and cultivating industrialization of algae is facilitated; algae automatically sink when big wind and waves come, and losses caused when algae are destroyed are reduced.

The present invention provides a cell culture method for promoting the rapid and stable proliferation of umbilical cord blood hematopoietic stem cells by using a three-dimensional cell culture system. The cell culture method comprises: 1) extracting and preparing cells adopted as matrix cells during co-culture, and identifying with flow cytometry; 2) treating a micro-carrier or other carriers and other materials used during a three-dimensional culture process so as to be spare; 3) inoculating the matrix cells onto the micro-carrier, and culturing in a three-dimensional culture system; 4) after the matrix cells proliferate to cover more than 80% of the micro-carrier surface, treating the cells to damage mitosis; 5) preparing a homogenized hematopoietic stem cell culture medium; 6) separating PBMC from umbilical cord blood, sorting CD34+ cells after obtaining the PBMC, and identifying with flow cytometry; and 7) culturing the sorted CD34+ cells and the treated matrix cells in the three-dimensional culture system by adding the homogenized hematopoietic stem cell culture medium. According to the present invention, the culture system for promoting the rapid and stable proliferation of the hematopoietic stem cells within a certain time by using the three-dimensional cell culture system and by combining the matrix cells and a variety of cytokines is established, and the theoretical basis and the technical platform are provided for the clinical obtaining of the high-quality hematopoietic stem cells.

The invention provides a method for obtaining and cultivating sphagnum protonema. The method comprises the following steps: carrying out tissue smashing on sphagnum cormus used as a raw material; cultivating in a liquid culture medium; growing new protonema from fragments of sphagnum tissue cormus, and carrying out tissue smashing on the protonema; carrying out liquid cultivation, so as to obtain the sphagnum protonema. By adopting the method, a lot of protonemata of the sphagnum can be obtained; the cormus with uniform growth can be largely obtained by using the protonema within a short period of time; application of the sphagnum in a tissue culture physiological experiment and sphagnum protoplast preparation, transformation or regeneration is facilitated.

The invention provides a cell culture method for duck flavivirus. The method comprises the following steps: inoculating to attach an anchorage-dependent kidney cell BHK21 of a baby hamster to the surface of a cell culture container to form a monolayer; inoculating the monolayer with virus; culturing the monolayer inoculated with the virus in an incubator with 5% CO2 at 37 DEG C; and continuously passing by taking a cell culture product as an inoculum for the next passage. According to the cell culture method provided by the invention, after the virus is continuously passed for 5-7 generations on the monolayer, stable multiplication can be obtained; and after the cell is inoculated for 3-4 days, an obvious cytopathic effect can be formed. The method provides a convenient and visual virus operating platform for studying the biological characteristics of the virus and the molecular basis of virus pathopoiesia and for the vaccine research developed for effectively preventing and controlling the disease.

Cultivating a pluripotent stem cell in a medium comprising at least one member selected from the group consisting of ethanolamine, an ethanolamine analog, and a pharmaceutically acceptable salt thereof, and which is substantially free of β-mercaptoethanol or contains β-mercaptoethanol at a concentration of not more than 9 μM, and the like, is effective for the proliferation of a pluripotent stem cell while maintaining an undifferentiated state.

The invention refers to a process for using yeasting method to produce lactic acid. Takes out the mixed liquid of embedding agent and fungus suspending liquid to solidify the cells, the embedding agent is made up of fungus suspending liquid: 2% kala glue liquid: 8-12% polyvinyl alcohol and 2-4 sodium alginate with proportion 1:1:3-5, combines with using pH controller to control the acid rate pH value = 5.0-6.2 automatically, when the pH gauge displays the pH value is 5.0 in the yeasting liquid, the pH control system switches on the power automatically, the circular pump begin to work, when the pH value reaches 6.5, the relay cuts off the power, the circular pump stops, the yeasting is going on, it realizes in situ separation yeast lactic acid generating.

The invention provides a vitro propagation technology of miscanthus anderss giganteus. The technology includes a process of obtaining giganteus regenerated plantlet by induction from the primary culture of buds to the secondary reproduction culture, the root induction of the axillary buds and the acclimation transplant with the giganteus sterile with the sterile giganteus axillary bud as the explants. The technology has a high axillary bud growth coefficient, an orderly rootage, and a high survival rate after the vitro propagation of the acclimation transplant, which is applicable to the commercial production of the giganteus plant seeds.

The present invention relates to a cell line derived from the cambium of an herbaceous plant having a storage root and a method for isolating the same. More specifically, relates to a cambium-derived homogeneous cell line having the ability to divide, which is obtained from the cambium-containing storage tissue of an herbaceous plant having a storage root without a separate dedifferentiation process, and to a method for isolating the same. The cell line derived from the cambium of an herbaceous plant having a storage root has active division ability and is homogeneous. Also, it is stable during culture, because it has not undergone a dedifferentiation process. Thus, through the optimization of proliferation thereof, the cell line can be allowed to proliferate in a large amount within a short time. Accordingly, the cell line derived from the cambium of an herbaceous plant having a storage root makes it possible to produce large amounts of useful plants which are difficult to cultivate outdoor due to various problems associated with the period of cultivation, the selection of cultivation land, cultivation cost and the like.

The invention provides an infectious bursal disease virus Vero cell-adapted strain and belongs to the field of bioengineering. The related infectious bursal disease virus Vero cell-adapted strain is named Ck / Jiangsu / NJ-23 / 2008 and has a collection No. of CGMCCNO.8852. The infectious bursal disease virus Vero cell-adapted strain which can efficiently multiply on serum-free cultured Vero cell is finally obtained through wild strain separation, chick embryo passage, Vero cell passage adaption; the infectious bursal disease virus Vero cell-adapted strain is subjected to continuous passage culture on the serum-free cultured Vero cell and TCID50 can be kept to be higher than 108.5 / mL. Virus culture solution is inactivated and prepared into oil emulsion; after the prepared oil emulsion is used to immunize chicken, detection proves that the prepared oil emulsion has good immunogenicity. The infectious bursal disease virus (IBDV) strain and the production process thereof are simple, safe and efficient and suitable for industrial culture.

The invention discloses a method for preparing rice flour by combining pure strain fermentation with semidry method flour grinding. The method comprises the following steps that rice moistening is performed, so that the moisture content of long-shaped rice grains is 28 to 32 percent; the long-shaped rice grains treated in step 1 are crushed to prepare long-shaped rice flour; the long-shaped rice flour and water are mixed and stirred; long-shaped rice milk with the water content of 48 to 57 percent is prepared; a fermentation agent is added into the long-shaped rice milk for fermentation, wherein the fermentation temperature is 35 to 37 DEG C, and the fermentation time is 4 to 8 h; the fermentation agent is prepared from the following components in parts by mass: lactobacillus plantarum powder, lactobacillus fermenti, yeast and aspergillus oryzae; the fermentation agent accounts for 0.1 to 0.2 percent of the dry weight of the long-shaped rice flour; the fermented long-shaped rice flouris subjected to sheet steaming, extrusion forming, water boiling and cooling to prepare the rice flour. The method has the advantages that the fermentation velocity is improved; the stable fermentation flavor is ensured; the dominant bacterial community in the fermentation process fast proliferates; the strain variation caused by living contaminants and continuous multi-fermentation is inhibited;the stability of the quality and the flavor of the fermented rice flour product is improved.

The invention provides an adapting method of a rabies virus (RV) CTN-1 strain to a primary chicken embryo fibroblast. The adapting method comprises the following steps of carrying out 10 continuous passages on RV CTN-1V5 in a vero cell to obtain a CTN-1V15 strain; carrying out one passage on a virus seed of the CTN-1V15 strain in a chicken embryo to obtain a first-generation virus of the RV CTN chicken embryo; carrying out passage on the first-generation virus of the RV CTN chicken embryo in the chicken embryo fibroblast to enable the first-generation virus of the RV CTN chicken embryo to gradually adapt to the chicken embryo fibroblast. The invention provides the adapting method of the RV CTN-1 strain to CEC (chicken embryo cardiomyocytes); by using the method, the CTN strain can be rapidly and efficiently multiplied in the CEC; moreover, the obtained RV CTN chicken embryo cell adapting strain can be stably multiplied on the CEC, and has favorable stability and immune protection. The invention also provides an inactivated vaccine prepared by using the RV CTN chicken embryo cell adapting strain, and the inactivated vaccine has favorable immune protection and can be used for producing refined and purified RV for people.

The invention discloses an influenza virus vaccine strain. The invention discloses a virus. The amino acid sequence of each of PB2 protein, PB1 protein, PA protein, NP protein, NA protein, M1 protein, M2 protein, NS1 protein, NS2 protein and HA protein are respectively shown as follows: SEQ ID No. 26, SEQ ID No. 34, SEQ ID No. 33, SEQ ID No. 21, SEQ ID No. 29, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 31, SEQ ID No. 32 and SEQ ID No. 16. The recombinant virus disclosed by the invention can be rapidly and stably proliferated in high yield. Compared with the method for preparing the vaccine by utilizing the chicken embryo, the recombinant virus has obvious advantage, and the inactivated vaccine of the recombinant virus can be used for protecting animals against the infection of influenza virus.

The invention provides a human placenta mesenchymal stem cell, a preparation method and an application thereof. The preparation method comprises the following steps: collecting, separating, culturing,cryopreserving, detecting, recovering, and the like. A high-purity high-activity mesenchymal stem cell can be acquired by only performing slide adherent culture after a human placenta chorion tissueblock is acquired in the separating process; the technological process is simplified; the cost of enzymic digestion is saved; the adherence and growth rate of primary cells are accelerated; the cell culture period is shortened; the introduction of more external interference factors is avoided, so that the process stability can be easily controlled. The multiplication capacity of the human placentamesenchymal stem cell acquired according to the invention is more stable than that of other mesenchymal stem cells; after the human placenta mesenchymal stem cell passes to P20 generation, the cell still can stably proliferate, and the cellular morphology, molecular surface antigen and adipogenesis osteogenesis differentiative potential thereof all meet the regulations for minimum standard of MSCidentification from international cell therapy association.

The invention relates to a method for purifying stem cells by utilizing adipose tissues obtained during the weight-loss liposuction of a healthy volunteer for breast augmentation and wrinkle removal. The method comprises the following operation steps of: (1) collecting the adipose tissues; (2) digesting the adipose tissues; (3) purifying the adipose tissues; (4) purifying adipose-derived stem cells; (5) carrying out amplification culture on the adipose-derived stem cells; (6) freezing the stem cells for storage. The ADSCs (Adipose-Derived Stem Cells) obtained through extraction and purification under the condition that a GMP (Good Manufacturing Practice) workshop is sterile has multiple advantages for the breast augmentation or face wrinkle removal: the ADSCs can be stably proliferated in vitro, is low in mortality rate, less in damage on a machine body during transplantation, wide in material source and good in breast augmentation effect, can achieve ideal wrinkle removing effect through a small quantity of cells, is safe and reliable without rejection and can provide a good method for augmenting breasts and restoring the skin elasticity of adolescence.

The invention discloses an establishment method for an immortal bone mesenchymal stem cell line of a Chang-Bai piglet. The method comprises the following steps: taking bone marrow cells of a Chang-Bai boar piglet, culturing the bone marrow cells with a nutrient solution for 20 to 28 h, then replacing a fresh nutrient solution, continuing culture and carrying out passage until F3-generation Chang-Bai piglet bMSCs with uniform cellular morphology is obtained; infecting the F3-generation Chang-Bai piglet bMSCs with a lentivirus liquid carrying a Large T gene and then carrying out cell screening and passage until a cell strain stably expressing Egfp is obtained; and subjecting the cell strain stably expressing Egfp to passage for more than 50 generations and carrying out cytobiological analysis and detection so as to obtain the immortal bone marrow mesenchymal stem cell line of the Chang-Bai piglet. According to the invention, a system capable of supporting stable culture of porcine bMSCs (bone mesenchymal stem cells) is obtained; rapid and stable propagation of the cells in vitro is promoted and biological characteristics of the original generation of the cells are maintained; it is proved that immortal bMSCs of the Chang-Bai piglet are obtained; and in-depth research on biological properties of the porcine bMSCs can be carried out and the bMSCs can be applied to research and development of drugs or vaccines.

The invention discloses an ankara virus strain FAdV-HB and a preparation and application of an inactivated vaccine of the ankara virus strain FAdV-HB. The strain is separated from a liver tissue sample of a diseased chicken house, and is proved to be serum-4 type fowl adenovirus after being subjected to sequencing, evolution analysis and regression experiments, that is, the strain is pathogen of the hydropericardium hepatitis syndrome. After plaque purification, the virus proliferates in a chicken hepatocellular carcinoma cell line (LMH), and a stable filial generation virus with high virus titer is kept for the preparation of the inactivated vaccine. The animal experiment proves that the inactivated vaccine reaches 100% of protection rate over the ankara virus, high content of antibody is produced, the holding time of the antibody is long, the ankara disease is effectively prevented, and the huge economic losses of the breeding industry caused by the infection of the ankara virus are reduced.

The invention discloses a culture method capable of promoting cyclocarya paliurus callus growth and secondary metabolite accumulation. The culture method includes: taking materials, and disinfecting;selecting semi-lignified stems with buds and top-end leaflets of cyclocarya paliurus, and disinfecting; performing callus induced culture: culturing stems and leaves in an induced culture medium to generate callus, wherein culture conditions are continuous dark treatment, and culture temperature is set at 20 DEG C; performing callus multiplication culture: transferring the callus into a transgenerational multiplication culture medium for transgenerational multiplication culture, and using a sealing film to sea, and putting into a culture chamber for culture, wherein culture conditions are continuous dark treatment, and culture temperature is set at 20 DEG C. Induction and multiplication culture of the callus are performed at temperature of 20 DEG C, so that generation of phenolic substances is inhibited while normal growth of cyclocarya paliurus is ensured, and callus growth and secondary metabolite accumulation are promoted at low temperature.

The invention provides a construction method of a sea bass fry cell line. The construction method comprises the following steps: (1) primary culture: carrying out pancreatin digestion on sea bass frytissues to obtain a cell suspension, and adding a primary cell culture solution to culture primary cells; (2) subculture: when passage is carried out for 1-10 generations, the concentration of fetal calf serum in a passage cell culture solution is 20%; when passage is carried out for 11-20 generations, the concentration of fetal calf serum in the passage cell culture solution is 15%; after passageis carried out for 20 generations, the concentration of fetal calf serum in the passage cell culture solution is 10%; and (3) collecting passage cells to obtain the sea bass fry cell line. The sea bass fry cell line obtained by the construction method provided by the invention has a good growth state and stable cell proliferation, can be continuously passed, can be frozen and recovered at ultralow temperature and can be used for researches of exogenous gene expression, virus infection separation and the like.

The invention discloses a Vero-E6 suspension cell strain sVero-E6 adapted to a porcine epidemic diarrhea virus and application of the Vero-E6 suspension cell strain sVero-E6, and belongs to the technical field of biology. The sVero-E6 suspension strain provided by the invention can stably subculture and grow, when the sVero-E6 suspension strain is used for culturing PEDV, the cultured PEDV can stably proliferate, and the sVero-E6 suspension strain can be used for continuously proliferating and culturing the PEDV and repeatedly harvesting a virus culture solution, so that a large amount of production cost and culture time can be saved, and the productivity is improved. The inactivated vaccine prepared from the obtained virus culture solution is good in immunogenicity, stable and safe, and can be effectively used for preventing porcine epidemic diarrhea diseases.

The invention discloses umbilical cord mesenchymal stem cell external gel for promoting trauma healing. The umbilical cord mesenchymal stem cell external gel is prepared by the following steps of carrying out subculture on resuscitated umbilical cord mesenchymal stem cells, and carrying out an enrichment and concentration method to obtain high-concentration umbilical cord mesenchymal stem cells with biological activity; and matching with sodium alginate gel joint preparation to obtain the umbilical cord mesenchymal stem cell gel with high stability and permeability. The gel provided by the invention promotes the growth of the cells at a wound by applying the gel to the local wound in vitro, meanwhile, avoids bacterial invasion, reduces the possibility of infection, and provides a microenvironment beneficial to tissue growth for the wound, thereby promoting trauma healing, shortening the treatment time and reducing the pain of the wounded. The umbilical cord mesenchymal stem cell external gel is simple in operation, the finished product is convenient to store, the treatment cost of the wounded is reduced, and the treatment effect is improved.

The present invention provides an immortalized natural killer cell line retaining the function and characteristics intrinsic to natural killer cells, a method for establishing the same, a method for screening for useful substances using the immortalized natural killer cell line, and a cell vaccine. By culturing natural killer cells obtained by isolating natural killer cells from the spleen of a transgenic mouse to which a large T-antigen gene of SV40 temperature-sensitive mutant tsA58 is introduced, a cell line which proliferates and activates in the presence of Interleukin-2, has azurophilic granules within cytoplasm, and retains an ability to kill a target cell without presensitization and / or an ability to kill target cells coated with an antibody, is established.

The invention provides a method for scaled production of radix astragali hairy roots. The method comprises the steps of preparing a radix astragali bacteria-free strain, activating and culturing agrobacterium, pre-culturing and co-culturing hairy roots, and performing extended culturing on the hairy roots. The radix astragali hairy roots prepared by the method have high astragaloside iv content, and besides, the method has the advantages that based on guaranteeing the content of the astragaloside iv, stable high-yield radix astragali hairy roots can be realized, the production cycle is short,the production quality is unified, and the method is suitable for normal-temperature growth conditions.

The invention discloses an epinephelus lanceolatus head kidney cell line as well as a construction method and application thereof. The ELHK cell line is preserved in the Guangdong Microbial Culture Collection Center (GDMCC) on December 04, 2020, the address is the 5th floor of No.59 building, No.100, Middle Xianlie Road, Guangzhou City, Guangdong Province, the postal code is 510070, and the preservation number is GDMCC No: 61340. The Epinephelus lanceolatus head kidney cell line-ELHK cell line is obtained, the growth state is good, cell proliferation is stable, the cell morphology takes a fibroblast sample as the main morphology, continuous passage can be achieved (at present, the cells have already been transferred to more than 120 generations), ultralow-temperature cryopreservation and recovery can be achieved. The establishment of the cell line lays a foundation for related research of grouper germplasm resource preservation.

The invention belongs to the technical field of fish cell culture, and discloses an establishment method and application of a fish targeted gene nutrition regulation and control cell model. The method comprises fish intestinal epithelial cell primary culture, subculture and identification. In the primary culture (1), a mechanical method and a treatment method of 0.05 vol% of trypsin mild digestive juice are adopted, so that the damage to intestinal cells is less, and the proliferation of the intestinal cells is promoted; in the subculture (2), 0.05 vol% of trypsin mild digestive juice is used for digestion in the subculture process, so that the activity of the intestinal cells is kept, and the subculture of the cells is facilitated; and in the identification (3), the intestinal epithelial cells of a fish are identified through a cell immunofluorescence technology. The fish targeted gene nutrition regulation and control cell model provided by the method can be directly used for researching the action mechanism of nutrient substances on targeted genes and researching the functions of exogenous genes.

The invention provides a biological membrane used in vitro. The biological membrane used in vitro comprises a matrix adsorber, a biological additive, a stent additive, a cross-linking agent and a permeation agent. The biological membrane used in vitro can keep the biological system of culture cells and stabilize growing status of the culture cells, and is suitable for detecting and analyzing drugscreening, drug therapeutic effect evaluation, drug adaptation disease evaluation, drug toxicity and side effect evaluation and drug compatibility study; meanwhile, the biological membrane used in vitro can detect and analyze biological activity and genetic characteristics of the tissue and the cells.

The invention discloses a rabbit hemorrhagic disease virus mutant strain, a construction method and application thereof. The rabbit hemorrhagic disease virus mutant strain contains a genome coded capsid protein VP60, a secondary structure protein VP10 and non-structural protein p11, p28, p35, p32, VPg, 3C like protease and RNA dependent RNA polymerase. Specifically, the capsid protein contains RGD short peptide, which is composed of the 305th arginine, 306th glycine and 307th aspartic acid of capsid protein VP60. The rabbit hemorrhagic disease virus mutant strain constructed by the invention can achieve stable proliferation and stable passage in RK13 cells, and can reach a good protective effect on hosts after cytotoxicity inactivation. The construction method can promote batch production of vaccines and reduce the vaccine production cost.