53 results about "Established cell line" patented technology

The invention discloses a cell line of pterygiophore tissue of cryprinus carpiod and a construction method. The construction method comprises the following steps of: A, preparing pterygiophore tissue blocks of cryprinus carpiod, namely putting cryprinus carpiod into potassium permanganate solution, disinfecting, transferring pterygiophore tissue to a culture dish, cleaning with an antibiotic, and transplanting the pterygiophore tissue blocks in cell culture flasks; B, preparing proliferation culture solution special for cells of pterygiophore tissue of cryprinus carpiod, namely adding fetal bovine serum, adding alkaline fibroblast growth factors and epidermal growth factors into the culture solution; C, performing primary culture of the cells of pterygiophore tissue of cryprinus carpiod, namely adding the special proliferation culture solution into pterygiophore tissue which is subjected to dry sticking in each culture flask, culturing in the culture box, and adding the proliferation culture solution special for the cells of pterygiophore tissue of cryprinus carpiod; and D, performing the subculture of the cells of pterygiophore tissue of cryprinus carpiod, namely after the cells of pterygiophore tissue of cryprinus carpiod are grown to form a single layer, preparing cell suspension, and performing the subculture. The method is easy and convenient to operate and has a scientific and reasonable process, and the established cell line of pterygiophore tissue of cryprinus carpiod is subcultured for over 60 generations at present.

We have developed an ASFV Georgia strain adapted to grow in Vero cell line. The resulting virus, ASF-GVAV, efficiently grows in Vero cells although it still is able to significantly replicate in primary cell cultures of swine macrophages. ASF-GVAV virus was successfully used as parental virus to develop several recombinant ASF viruses. The development of an ASFV adapted to grow in an established cell line is a significant advance for research and development of vaccine candidate strains using genetic manipulation based in the process of homologous recombination. The GVAVS can be utilized as a basis for large scale production of ASF vaccines.

The invention relates to a method for establishing and identifying a cynoglossus semilaevis testis cell line, which comprises the following steps: adopting a MEM (Minimum Essential Medium), adding 20 percent of fetal calf serum, 0.1 percent of B-mercaptoethanol, 2 ng / ml of human alkaline fibroblastic growth factors, 1 ng / ml of leukemia inhibiting factors, 1 nmol of sodium pyrurate and 1 nmol of glutamine and preparing into a complete medium; inoculating a tissue block into a culture bottle, overturning and inversely placing the bottle to culture for 3 h and positively placing the culture bottle after the tissue block is attached onto a wall, so that the tissue block is soaked into the culture medium for culture. A trypsin digestion method is adopted for subculturing cells. One cynoglossus semilaevis testis cell line is established by adopting the above method and is subcultured to fiftieth generations. Meanwhile, the invention also provides a method for identifying a chromosome of the established cell line and the level of a molecular marker and a method for identifying the sensitivity of the cell line to related viruses. The method for establishing the cynoglossus semilaevis testis cell line can be used for culturing other fish gland cells and establishing the cell line, thereby having wider promotion and application prospect.

The invention relates to a method for constructing a human corneal epithelial cell system. The construction method comprises the following steps of: disinfecting cornea, digesting the cornea, taking off corneal epithelium with a front elastic layer, cutting the corneal epithelium into small tissue blocks, flatly attaching the tissue blocks to the bottom of culture holes downwards, adding culture solution into a carbon dioxide culture box, and performing plate-attaching culture at the temperature of 37 DEG C, changing the culture solution during culturing, performing subculture when the cells grow into a single layer, and presently, completing the construction of the human corneal epithelial cell system above 60 generations, wherein the culture solution contains 20 percent of fetal calf serum, 0.002 to 0.004 percent of human epidermic cell growth factor, 0.001 to 0.002 percent of human alkali fibroblast growth factor, 0.04 to 0.1 percent of carboxymethyl chito-oligosaccharide, 0.025 to0.1 percent of DMEM / F12 culture solution of chondroitin sulfate, and 0.008 to 0.01 percent of IV type collagen. In the construction method, the non-transfection human corneal epithelial cell system is successfully constructed by utilizing the human corneal epithelial tissues, and the constructed cell system can realize continuous transfer of culture, and a great number of human corneal epithelialcells are provided.

The invention discloses a Pseudosciaena crocea muscle cell line and its establishing method. The cell line is preserved in China Center for Type Culture Collection on Oct., 31, 2013, and has a preservation registration number of CCTCC NO:C2013158. The Pseudosciaena crocea muscle cell line can be serially passed, can provide a large amount of the Pseudosciaena crocea muscle cell line, and can be used for researching pathogenic characteristics, vaccines, gene functions and breeding; and the Pseudosciaena crocea muscle cell line has excellent properties, is a fibroblast and is soma-fusiform or anomaly triangular, the cytoplasm outwardly stretches to form a plurality of protrusions with different lengths, and the protrusions are arranged in a radial or spiral manner. The Pseudosciaena crocea muscle cell line has the characteristics of strong splitting, short passage time, easy digestion, good adherent rate and starvation resistance. The establishing method has strong repeatability, and the established cell line has a good stability.

The invention aims at providing an establishment method of an ovary cell line of cynoglossus semilaevis, which comprises the following steps: 1) removing the outermost membrane of the ovary tissue of cynoglossus semilaevis under a sterile condition, cutting the tissue into small blocks in a culture solution, flushing the tissue blocks with a PBS solution and digesting with a trypsin solution, wherein the solution after trypsin digestion contains cells, cell clusters and tissue blocks not completely digested; and 2) centrifuging the solution to remove the supernatant trypsin, and suspending the cells, cell clusters and small tissue block precipitates with a culture solution; after a suspension is obtained, inoculating into a culture plate treated by the type-I collagen; adding a culture solution, and culturing in a biochemical incubator at 24 DEG C; and performing subculture to finish establishment of the ovary cell line. In the establishment method provided by the invention, an ovary cell line of cynoglossus semilaevis is successfully established by use of the ovary tissue of cynoglossus semilaevis, the established cell line can be continuously passed, the passage cells obtained by the method can be passed for over 60 generations, and a great quantity of ovary cells can be provided.

The invention discloses a Pseudosciaena crocea brain cell line and its establishing method. The cell line is preserved in China Center for Type Culture Collection on Oct., 31, 2013, and has a preservation registration number of CCTCC NO:C2013156. The Pseudosciaena crocea brain cell line can be serially passed, can provide a large amount of the Pseudosciaena crocea brain cell line, and can be used for researching pathogenic characteristics, vaccines, gene functions and breeding; and the Pseudosciaena crocea brain cell line has excellent properties, is an epithelioid cell and is flatly anomaly triangular and polygonal. The Pseudosciaena crocea brain cell line has the characteristics of strong splitting, short passage time, easy digestion, good adherent rate and starvation resistance. The establishing method has strong repeatability, and the established cell line has a good stability.

The invention relates to human placenta trophoblastic cell series which is conserved in China Committee for Culture Collection of Microorganisms on September 22th in 2005 with CGMCC No.1461 conserving number. It includes the following steps: separating the trophoblastic cell form the placenta tissue which is pregnant for 6-7 weeks; filtering the cells with multiplication capacity; processing subunit gene transfection by telomere enzyme to gain four long life cell strains. The cell series has normal multiplication capacity and biological chemical function, active cell growth, and reaches the 210 generation. Thus it can be used as target cell model for filtering antifertility drug and curing pregnant correlative disease medicine, provide ideal vitro model for further studying trophoblastic cell, lay important foundation for studying embryo implantation and pregnant physiology and pathological mechanisms.

The invention belongs to the field of microorganism animal cell lines, and relates to a cell system for generating hepatitis B virus (HBV) recombinant cccDNA. Through a transposon system, HBV single-copy genomes with two ends provided with loxP sites are integrated into HepG2 cells, a clone HepG2-HBV / loxP cell line integrated with the different-copy-number HBV genomes is obtained, the cell line does not express HBsAg, after cyclization recombination enzyme (Cre) is introduced through adenovirus transduction, the rcccDNA is generated and the HBsAg is expressed, and expression of the HBsAg is closely relevant to the generation and level of the rcccDNA; the rcccDNA is rapidly and stably generated, the structure feature of the rcccDNA is similar to that of real HBV cccDNA, and transcription, copy and protein expression of viruses can be supported; the rcccDNA can be quantitatively detected through quantitative PCR by designing a specific primer, and Southern Blot detection can be carried out by utilizing a digoxin marking system. The cell system can be used for establishing a platform for HBV cccDNA relevant biological research and screening and evaluation of anti-HBV medicines.

The invention relates to mouse stem cell culture technologies, and specifically discloses a construction method of mouse bone marrow mesenchymal stem cell lines. The construction method of the mouse bone marrow mesenchymal stem cell lines comprises the following steps: extracting bone marrow; carrying out primary culture of mesenchymal stem cells; carrying out subculture; carrying out cryopreservation; carrying out resuscitation; carrying out molecular identification of cell surface; and identifying differentiation into adipocytes and osteoblasts. Cell culture fluid adopted for the construction method of the mouse bone marrow mesenchymal stem cell lines is a low-sugar-content DMEM basic cell culture medium added with fetal bovine serum. The constructed mouse bone marrow mesenchymal stem cell lines are fibroblast-like in morphology. The construction method of the mouse bone marrow mesenchymal stem cell lines disclosed by the invention is simple in operation and high in repeatability; and the constructed mouse bone marrow mesenchymal stem cell lines are feasible for continuous passage, thereby enabling supply of a large number of mesenchymal stem cells. In addition, the constructed mouse bone marrow mesenchymal stem cell lines can differentiate into adipocytes and osteoblasts, and can be directly applied in study on functional genes of mice. The construction method of the mouse bone marrow mesenchymal stem cell lines is expected to become a research platform of stem cell functions and regulation mechanisms, and is capable of promoting practical application of tissue engineering on basis of mesenchymal stem cells.

The invention discloses a porcine immortalized alveolar macrophage cell line for expressing a porcine reproductive and respiratory syndrome virus, PRRSV, acceptor CD163. The cell line is named as PAM-Tang-20 which is preserved in CGMCC, the preservation number of culture of which being CGMCC No. 13591. Experiments verify that the cell line established by the invention can express the CD163 acceptor highly, is a susceptible cell of PRRSV, and can be widely applied to proliferation of PRRSV, separation of PRRSV clinical strains, vaccine production, PRRSV related basic researches and the like. The cell line for provides an effective technical means for in vitro culture of the PRRSV.

The present invention relates to a method for picking a colony of cells and to producing a cell line from this colony, as well as to the cell line thus established and to use of this cell line in a method for producing a biological molecule or biological entity of interest.

The present invention relates to Lymantria xylina cell lines established from pupal tissues of L. xylina Swinhoe, including NTU-LY-1, NTU-LY-2, NTU-LY-3, and NTU-LY-4. These four cell lines were confirmed to be newly established cell lines derived from L. xylina by random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) and isozyme analysis. The genotypes and characteristics of the abovementioned cell lines are totally different from other insect cell lines. In addition, these four L. xylina cell lines are susceptible to insect baculovirus of L. xylina multiple nucleopolyhedrovirus (LyxyMNPV), LdMNPV-like virus, PenuMNPV, as well as microsporidia of Nosema sp. (isolated fromEurema blanda) and N. bombycis and the like. Therefore the invention can be applied in multiplication of the abovementioned insect-pathogenic microorganisms to produce biopesticides in pest control. In addition, the cell lines can also be used as hosts for the expression vectors of said baculovirus to produce recombinant proteins.

The present invention belongs to the field of biotechnology and cell engineering, and relates to monoclonal antibody hybridoma cell line and its construction process. Technology, the present invention features that soluble immunogen is first prepared with human ovary carcinoma tissue sample and then used to immunize BALB / c mouse, and through fusion, screening and repeated cloning in hybridoma technology, hybridoma cell line capable of secreting stably human ovary carcinoma resisting monoclonal antibody is obtained. The hybridoma cell line, named as BUPH: OVCAMab, is preserved in the preservation number of CGMCC No. 1225. The present invention also discloses the method of preparing monoclonal antibody OVCAMab with the cell line and purifying the antibody. The antibody is used in early diagnosis, intracorporeal locating, detection, metastatic focus detecting, etc. of ovary carcinoma in radioimmunoassay.

The invention provides a cell line used for susceptibility detection of lung cancer chemotherapy drugs and establishment thereof. The establishment thereof comprises the following processes: culturingNIH3T3 cell by using 10 percent of calf serum added with DMEM, placing the NIH3T3 cell into an incubator with the temperature of 37 DEG C and 5 percent of CO2 for culturing for 3 days, growing 12-hole plate, and changing with a culture medium without antibiotics to continue culturing for 24h; according to the ratio of 1:2.5, carrying out transfection of 2 he of eIF3a eukaryotic expression vectorpCbA-eIF3a to the NIH3T3 cell, wherein the sequence of the vector pCbA-eIF3a is shown in a sequence table; and after transfection for 48h, according to the volume ration of 1:10, diluting transfer ofculture to a cell culture dish of 100mm, with 1ml of inoculation quantity; adding G418 of 600he / ml, screening for two weeks, selecting monoclone and expanding culture, and establishing the cell line.The establishing method of the cell line is simple and convenient in operation, steady in conditions and low in cost; and the established cell line has stable expression and good prediction effect.

A subcultivatable, established microglia having the following properties. (a) Form: Both or either of a macrophage-like or spherical form in the presence of a granulocyte-macrophage colony stimulation factor and a branched form similar to branched microglia present in the brain in the absence of the factor. (b) Functional characteristics: specific affinity for the brain highly poor phagocytic action. (c) Cell proliferation: proliferative depending upon a granulocyte-macrophage colony stimulation factor. Preparation, separation, and screening methods of the microglia, a pharmaceutical composition using the microglia, and a method for treatment of cerebral diseases using the composition.

The present invention relates to a method for screening a compound for preventing, ameliorating or treating glomerular lesions or disease of kidney, characterized by assaying the regulating activity of compound on 4F2hc expression shown by contacting human peripheral blood mononuclear cells or monocytes or an established line of human cultured cells having a nature of human macrophages with macrophage-activating substances. The screening method is useful for screening compounds capable of preventing, ameliorating or treating glomerular lesions or disease of kidney.

The present invention relates to Lymantria xylina cell lines established from pupal tissues of L. xylina Swinhoe, including NTU-LY-1, NTU-LY-2, NTU-LY-3, and NTU-LY-4. These four cell lines were confirmed to be newly established cell lines derived from L. xylina by random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) and isozyme analysis. The genotypes and characteristics of the abovementioned cell lines are totally different from other insect cell lines. In addition, these four L. xylina cell lines are susceptible to insect baculovirus of L. xylina multiple nucleopolyhedrovirus (LyxyMNPV), LdMNPV-like virus, PenuMNPV, as well as microsporidia of Nosema sp. (isolated from Eurema blanda) and N. bombycis and the like. Therefore the invention can be applied in multiplication of the abovementioned insect-pathogenic microorganisms to produce biopesticides in pest control. In addition, the cell lines can also be used as hosts for the expression vectors of said baculovirus to produce recombinant proteins.

The invention discloses an in-vitro establishing method of an acipenser ruthenus theca cell line and reagents applied to acipenser ruthenus theca cell line preparation. The in-vitro establishing method of the acipenser ruthenus theca cell line includes the steps: 1, a digestive juice is used for digesting acipenser ruthenus ovarian tissue to obtain an acipenser ruthenus ovarian tissue cell suspension; 2, a standing-centrifugation method is adopted to obtain acipenser ruthenus theca cells, wherein the acipenser ruthenus ovarian tissue cell suspension is subjected to standing, a supernatant containing cells and cell masses is collected and then centrifuged, a sediment is collected, a culture solution is added into the sediment to prepare a cell suspension, and inoculation and culture are conducted to obtain the acipenser ruthenus theca cells; and 3, digestion is conducted two times, and subculture is conducted, wherein after the acipenser ruthenus theca cells grow into a single layer, the digestive juice is adopted to carry out digestion two times, and then subculture is carried out to obtain the acipenser ruthenus theca cell line. According to the in-vitro establishing method of theacipenser ruthenus theca cell line, the standing-centrifugation method and a two-time digestion method are adopted to establish the acipenser ruthenus theca cell line, the established acipenser ruthenus theca cell line can be continuously subcultured, the cells obtained through the in-vitro establishing method can be subcultured for 60 passages or above, and a large number of theca cells can be provided in vitro.

Belonging to the technical field of genetic engineering, the invention relates to a SgRNA targeting sequence for knockout of mouse Tet3 gene, a cell line for conditional induced knockout of mouse spermatogonia Tet3 gene, a construction method and application thereof. The gene editing off-target effect is an important aspect that puzzles the application of gene editing technology. The Tet3 gene target screened by the patent is the only sequence-targeted target screened out from the whole mouse genome, no similar sequence exists in the genome, and therefore off-target effect does not exist theoretically. At the same time, an induced knockout method is employed to close Cas9 protein expression and also can effectively prevent off-target, 24h later after adding of Dox, 97% or more of the mousespermatogonia genome can be knocked out, and the cell lines before and after Dox induction can be employed as good experimental group and control group. The cell line and method established by the invention can be widely applied to Tet3 and other genetic functional study.

The present invention relates to cDNA library, and especially human oral cavity epithelial cancerous protuberance cell cDNA library and its preparation process. The library is prepared based on the cell line via separating human oral cavity epithelial cancerous protuberance, and the preparation process includes: extracting cell mRNA, synthesizing cDNA polynucleotide product, and inserting cDNA polynucleotide into carrier. The present invention provides basis for the deep research of the oral cavity cancer mechanism and the search of treating molecule target. In the same time, the present invention provides one kind of effective method of preparing human oral cavity epithelial cancerous protuberance cell cDNA library.

A method for training an animal to detect a condition, such as cancer, in a human or animal individual is disclosed. A training sample is presented to the animal to be trained where, the training sample is a gaseous sample or vapor generated by a cell population associated with the predetermined condition. The population of cells associated with the predetermined condition may be, for example, a culture of an established cell line associated with the predetermined condition. Simultaneously with, or subsequent to, presentation of the training sample, the animal is subjected to an adverse stimulus, such as an electric shock. The animal is allowed animal to perform a first predetermined response in order to avoid, escape or terminate the adverse stimulus. The invention also provides a method for detecting a condition, such as cancer, in an individual in which a trained animal performs the predetermined response upon exposure to vapors from a body fluid of an individual affected with the condition.

It is an object of the present invention to provide host cells, which are able to proliferate in a serum free medium, which are able to synthesize and secrete a large amount of protein of interest, and which are able to produce a glycoprotein having a sugar chain structure. The present invention provides an established cell line, which is established from distal tubular epithelial cells derived from a mammal.

The invention discloses a method for establishing a transgenic removable human skin fibroblast feeder cell line. By utilizing a retrovirus-mediated gene transfer way, the production tool introduces areinforced green fluorescent protein gene, a telomerase reverse transcriptase gene and a herpes simplex virus thymidine kinase gene into human skin fibroblasts to establish a fluorescence-labeled immortalization-removable TERT + TK-D human feeder cell line. After being treated by mitomycin C, the established cell line serving as feeder cells is co-cultured with human limbal stem cells, and the culture result is compared with a culture result of 3T3 feeder cells. Transgenic fluorescence-labeled immortalization-removable human skin fibroblast feeder cells are expected to replace the 3T3 cells for corneal regeneration therapy.

The invention belongs to the field of micro-organic zooblast system. The invention adopts pericardial effusion of human lung adenocarcinoma patients as a material for building the system and establishes a parental human lung adenocarcinoma cell line with high potential of bone metastasis through cell cultivation, which is called CPA-Y1; and the preservation number of the parental human lung adenocarcinoma cell line is CGMCC No.2521. The established cell line can provide reference data for carrying out early diagnose on human lung adenocarcinoma bone metastases; relevant gene chip technology can be further established; and treatment effect of various medicaments comprising chemical medicaments, biological medicaments, immune medicaments, Chinese medicaments, radioactive medicaments and the like can be evaluated and researched. The parental human lung adenocarcinoma cell line provides a technological platform for searching and diagnosing human lung adenocarcinoma bone metastases and systematically studying multiple lung cancer metastasis. Meanwhile, the parental human lung adenocarcinoma cell line can be used for further analyzing functional genome related to lung cancer bone metastasis and genes related to bone metastasis to provide an effective molecular detection method for early diagnosis and evaluation of treatment effect of bone metastasis.

An object of the present invention is to provide a cytotoxic assay using a new established fish cell line. The invention attained according to the object is a cytotoxic assay, wherein an evaluation of toxicity of a specimen is performed on the basis of its toxicity to an established cell line originated from sturgeon, preferably a STIP-1 cell line (FERM BP-8421) and a STIP-3 cell line (FERM BP-8422). Furthermore, another object of the present invention is to provide a new established cell line expected to be used in the diagnosis of viral infection disease of sturgeon and so on. The invention attained according to the object is an established cell line originated from a sturgeon eyeball, particularly a STIP-1 cell line (FERM BP-8421) and a STIP-3 cell line (FERM BP-8422).