72 results about "Lymphocyte culture" patented technology

The invention discloses a culturing method of an NK (natural killer) cell. The culturing method comprises the following steps of (1) separating a mononuclear cell from peripheral blood or umbilical cord blood of a human body; (2) inoculating the mononuclear cell into a culture medium suitable for culturing lymphocyte, adding a CD3 monoclonal antibody, recombinant human interleukin 2 and autologous plasma, and culturing for 3 to 5 days; (3) adding the recombinant human interleukin 2 and the autologous plasma, and culturing; (4) harvesting the NK cell. According to the culturing method of the NK cell, the culturing cost is reduced, the amplification multiple and cell toxicity of the NK cell are guaranteed, the operation time is saved, meanwhile the probability of error operation is reduced, the obtaining efficiency of the NK cell is higher, and the safety of the NK cell is better.

The invention discloses an amplification method for an NK cell without trophocyte. The method comprises the following steps: adopting a blending culture solution for culturing the NK cell and culturing for 7 days, and then using a self-plasma culture medium for culturing, supplying the fluid per 2-3 days in the culturing process, maintaining the cell density of 2.0*106/mL and culturing for more than 20 days. The blending culture solution is composed of 1-10% of self-plasma, 200-1500 IU/mL IL-2, 10-50ng/mL IL-15 and 10-50ng/mL IL-21 added into a lymphocyte culture medium. The self-plasma culture medium is composed of 1-10% of self-plasma and 200 IU/mL IL-2 added into a serum-free medium. According to the amplification method for the NK cell without trophocyte disclosed by the invention, cord blood or autologous peripheral blood is taken as a cell source, no trophocyte is required in the culturing process, the amplification efficiency is high and the purity of the acquired NK cell is high. The NK cell acquired according to the method can be used as a medicine compound for cell immunization therapy and is mainly used for treating and/or preventing infectious diseases and/or cancer.

The invention provides a kit for separated culture of DC-CIK cells, and an application thereof. The kit comprises a lymphocyte separation liquid, a peripheral blood sample treatment liquid, a CIK cell induced propagation system, a DC induced propagation system, a cell culture bottle coating system, a lymphocyte culture medium GT-T551 and a cell culture bag. The application of the kit in separating and culturing the DC-CIK cells comprises the following steps of separation of peripheral blood sample mononuclear cells, separation of the DC cells and CIK cells, induced propagation of the DC cells, the induced propagation of the CIK cells and co-culture of the DC cells and CIK cells. The lymphocytes obtained from the separation by the kit have very high purity and activity; the propagation rate of the CIK cells is fast; the proportion of CD3+CD56+ double positive cells is high; operation method is simple; and conditions are easy to control. The kit can be widely applied in the separated culture for the DC-CIK cells of human and mammals.

The invention provides a lymphocyte serum-free medium which comprises the following materials per liter: a basic culture medium, 10 mg of transferrin, 0.01 mg of sodium selenite, 110 mg of pyroracemic acid, 1.5 mg of ethanolamine, 10 mg of insulin, 10 to 30 mg of glutamine substitute, 300 mg of lipid-type bovine serum albumin, 0.001 mg of cupper sulfate, 0.0005 mg of ammonium meta-vanadate, 0.00005 mg of manganese chloride, 0.8 mg of zinc sulfate, 2 mg of Vitamin E, 100 mg of PHA, and 10 to 25 mg of an MHEPES/NaHCO3 buffering system. The invention further provides a preparation method and application to lymphocyte culture of the lymphocyte serum-free medium. The lymphocyte serum-free medium has the advantages that the repeatability of lymphocyte culture is high; poisoning and polluting of blood serum to lymphocyte are avoided; convenience is brought for differentiation of the lymphocyte, so as to obtain various mitosis phases.

The invention provides a method of producing a stable lymphocyte culture and methods of producing monoclonal antibodies.

The invention discloses a preparation method for a monoclonal antibody. According to the method, magnetic granular microballoon spheres of an immobilized antigen are added into B lymphocyte culture micropores, magnetic separation is carried out on the magnetic granular microballoon spheres so as to allow the spheres to enter into corresponding microplates, a single B lymphocyte which secretes a specific antibody is detected by using the method of immunochemiluminescence or immunofluorescence, a light chain variable region gene and a heavy chain variable region gene of the antibody are amplified by using the method of single-cell RT-PCR and nested RT-PCR and are recombined to expression plasmid containing a constant domain of the antibody, and a host cell is transfected to express the monoclonal antibody.

The invention relates to the technical field of stem cell culture, in particular to a medium and application thereof in central memory T lymphocyte culture. The medium provided by the invention is composed of a basic medium, an mTOR inhibitor, interleukin-2, interleukin-7, interleukin-12, interleukin-15, interleukin-1a, an Anti-CD3 antibody and an Anti-CD28 antibody. By means of the medium and a culture method provided by the invention, great numbers of high-proportional central memory T lymphocytes can be provided, experiments show that by using the medium provided by the invention, TCMs (central memory T cell) can amplify around 100 times, and in 20 days of culture, the number of the cells is the highest on the twentieth day, while the purity of the TCMs (sample B) is the highest on thetwelfth day.

The invention relates to a method of extracting phytohemagglutinin and belongs to the technical field of phytohemagglutinin extraction. The phytohemagglutinin can be extracted from beans by soaking in a buffer solution, acid conditioning to settle protein and adding 0.1-0.4% of sodium chloride solution to settle the protein; the extraction method is simple and convenient; and a reagent used in an extraction process is cheap, easy to obtain and harmless to a human body. Extracted PHA (phytohemagglutinin) protein is high in purity and good in activity; and when the extracted PHA protein is applied to a lymphocyte culture medium, blood coagulation or hemolysis can be avoided, and a good effect of stimulating lymphocyte growth is achieved. The extraction method is suitable for large-scale production of the PHA protein.

The invention provides a culture medium with a stable pH value. The culture medium is composed of a 1640 base culture medium, a 100 mg/L phytohemagglutinin (PHA), a calf serum with the volume percentage of 20% and a 5-50 mM sodium glycerophosphate/4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) dual-buffering system. The sodium glycerophosphate/HEPES dual-buffering system is composed of a phosphate buffer solution, sodium glycerophosphate and 4-HEPES, wherein the phosphate buffer solution contains Na2HPO4 and KH2PO4. The invention also provides a preparation method of the culture medium and an application of the culture medium in lymphocyte culture. The pH value of the culture medium is not affected by gas composition in the environment, and the pH value during the lymphocyte culture also can be stabilized; and long-term preservation of the culture medium is facilitated.

The invention relates to a teaching agent box of human peripheral haemolymph cell train and colorant layer inspection, which packages the agent and the material in the agent box by small package type and dose the optimum allocation by the experiment teaching standard class of ten groups, each group of small package comprises: aqua-colchin, an injector, menthol and glacial acetic acid for preparing for the fixing liquid, hypo tonic liquid, improved oxybenzene fuchsine dyeing solution, lens-washing liquid, lens-wiping liquid, 1640 culturing liquid, slide glass, eccentric pipe, coloring frame, long drop pipe, ice box and clamp and so on, wherein each agent box is matched with a culturing frame and a usage handbook.

The invention belongs to the technical field of biological agents and discloses a novel biological agent for treating drug resistant tuberculosis and a preparation method thereof. The novel biologicalagent for treating drug resistant tuberculosis is a tuberculosis specific lymphocyte and is prepared by utilizing IL-1 alpha, IL-2, a CD3 monoclonal antibody and tuberculosis specific antigens (CFP10, ESAT6). Compared with the conventional tuberculosis treatment method, the novel biological agent disclosed by the invention is reported for the first time at home and abroad, and obvious curative effects are achieved when patients with pulmonary tuberculosis are treated by utilizing lymphocytes cultured in vitro. For patients subjected to cell therapy, the number of lymphocytes in peripheral blood is obviously increased, the sputum examination result is rapidly turned to be negative, pulmonary shadow is rapidly absorbed, the course of disease is shortened, the mental state is improved, and the appetite and weight are increased. The living quality of the patients in anti-tuberculosis therapy is obviously improved.

The invention relates to the technical field of cell culture, and in particular, relates to intestinal lymphocyte culture equipment. The intestinal lymphocyte culture equipment comprises a barrel body, a covering mechanism, a stirring mechanism and a driving mechanism. A rotating groove is formed in the bottom of the barrel body, a cavity is formed in the bottom of the barrel body, and the cavityis communicated with the rotating groove; the covering mechanism detachably sleeves the top of the barrel body in a sealing manner; the stirring mechanism comprises a rotating part and a stirring rod,the rotating part is rotatably mounted in the rotating groove in a sealed mode, the stirring rod is located in the barrel body, and the bottom of the stirring rod is fixedly mounted at the top of therotating part; and the driving mechanism is mounted in the cavity, one end of the driving mechanism is fixedly arranged on the rotating part in a sleeving mode, and the driving mechanism is used fordriving the rotating part to rotate. The invention aims to solve the problem that intestinal lymphocytes cannot be shaken up by workers in the process of culturing the intestinal lymphocytes by an existing culture device.

The invention discloses an immune cell culture solution which is prepared by adding autologous plasma, human serum albumin, gentamycin sulfate, immune cell growth factor interleukin-2, sodium bicarbonate, phytic acid and vitamin C into a basic complete solution. The immune cell culture solution has the advantages of overcoming the defects that lymphocyte culture solution available in the existing market is unstable, number of immune cells cultured by the lymphocyte culture solution is small and purity of the immune cells is low, and is capable of culturing sufficient amount of high-activity immune cells.

The invention provides a high cytotoxic CIK (cytokine induced killer) cell preparation and a culture method thereof, and relates to the field of tumor treatment. The culture method comprises: centrifuging anticoagulated peripheral blood, removing a plasma layer, mixing with a lymphocyte separation solution, and separating peripheral blood mononuclear cells; adjusting the cell concentration of theperipheral blood mononuclear cells with a lymphocyte culture medium, adding IL-2, IFN-gamma and autologous plasma, and culturing in a culture flask; performing expansion in vitro for culture, and centrifugally collecting the obtained CIK cells. Through the culture method, the cell culture time can be shortened, the cell activity of the obtained CIK cell preparation is more than 95 percent, the ownimmunity of a patient can be enhanced after reinfusion, and the anti-tumor effect is increased.

The invention discloses a kit for rapidly inducing a large number of DC-CIK and NK cells with a lymphocyte culture medium and a use method of the kit. The kit comprises a DC set box: 10mu g of dry powder DC-A and 5mu g of dry powder DC-B, a CIK set box: 6 pieces of dry powder CIK-A (30mu g/piece), 10mu g of dry powder CIK-B and 20mu g of dry powder CIK-C, an NK set box: 5 pieces of dry powder NK-A (50mu g/piece), 10mu g of dry powder NK-B, and 5mu g of dry powder NK-C. By adopting the DC-CIK and NK cell induction kit, with the combination of the lymphocyte culture medium, lymphocyte more than or equal to 5*10<9> can be induced and amplified within the shortest time, the percentage of CIK cells (CD3<+>CD56<+>) is more than or equal to 25%, and the percentage of NK cells (CD3<->CD56<+>) is more than or equal to 50%. A convenient method is provided for in-vitro large-scale amplification of DC-CIK and NK cells, and convenience can be brought to related research and clinical testes.

The invention relates to a lymphocyte culture method, and in particular discloses a culturing method of adenovirus gene-modifying tumor specificity cytotoxic T lymphocyte. The method comprises: taking 150-200ml of bleeding of the umbilicus or autologous peripheral blood for single karyocyte separation, separately culturing DC cells by adhering to a wall for 2h, and performing T lymphocyte preculture on the non-adhered cells in an IL-2-contained culture medium; collecting mature DC after the DC cells are mature through induction of iAPA factor adenovirus infection, stimulating the autologous primary T lymphocyte to differentiate into cytotoxic T lymphocyte according to an effect target ratio of DC to T being 1 to 10, and observing and recording multiplication capacity of CTL and detecting the cytotoxicity of the CTL. The limitation of a blood component single sampling machine is solved, collected blood volume is less, burden of a patient is reduced, and iAPA-DC can be observed in vitro to stimulate and differentiate the primary T lymphocyte into tumor specificity cytotoxic T lymphocyte.