73 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 establishes an operation method in T-Lymphocytes culture in vitro by immunomagnetic bead separation technique to maximize the separation of human T-Lymphocytes from the same volume of human peripheral blood to obtain a lot of purified T-Lymphocytes and cover the shortage that lymphocytes can be only primary cultured but not be subcultured to the greatest extent. The method is simple and practicable.

The invention discloses a lymphocyte culture fluid and culture method thereof and application therefor, the culture fluid is adding ATP and coenzyme A in completely culture fluid containing basic culture fluid RPMI-1640, albumin, L-glutamine, glucose, baking soda and HEPES. The culture method includes steps: (1) preparing culture fluid; (2) suspending cell in culture fluid prepared by step (1); (3) activating cell; (4) washing activated cell by the culture fluid prepared by step (1); (5) collecting cell. The culture fluid of the invention cultures cell with higher amplification times and longer generation time.

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.

A method for producing a monoclonal antibody and antigen-specific antibody-producing hybridomas using a single antigen-specific B lymphocyte is provided. The method for producing antigen-specific antibody-producing hybridomas comprises selecting a single B lymphocyte reacting specifically with a certain antigen, an “antigen-specific B lymphocyte”, culturing the antigen-specific B lymphocyte selected, and fusing the cultured antigen-specific B lymphocyte with myeloma cells to obtain the hybridomas. The monoclonal antibody is produced with thus obtained hybridomas.

The invention discloses a culture method for functionally enhanced tumor infiltrating lymphocytes (TILs). The method includes the following steps: separating lymphocytes from tumor tissue, adding a start culture medium, performing inoculation in a 12-well culture plate (4 ml / well), performing start lymphocyte culture for 10 days or 14 days to obtain start TILs, and performing cryopreservation on the obtained start TILs for standby application; suspending the lymphocytes in a 25 cm<2> culture flask (20 ml / flask) by using an induction culture medium, placing the culture flask in an incubator having 5% CO2 at 37 DEG C, and performing TIL induction culture for 1 day; performing half quantity change by using an expansion culture medium, and performing expansion flask culture and expansion bag culture for 13 days or 14 days; on the 14th or 15th day of culture, collecting TILs, performing washing by using normal saline, and resuspending the TILs by using a function enhancement culture medium,and performing incubation for 30 min; and collecting TILs, and performing functional test to obtain the functionally enhanced TILs. The culture method described in the invention can obtain the functionally enhanced TILs with stronger tumor cell killing activity and higher-level anti-tumor cytokine secretion ability.

The invention discloses a method for preparing CIK cells with a killing effect on tumor cells. The CIK cells are obtained through induced culture of 17-20 days by using a ficoll mononuclear cell separation medium with a density of 1.084 in the PBS system to separate mononuclear cells in peripheral blood or umbilical cord blood. According to the present invention, mononuclear cells are separated and obtained efficiently by using the ficoll density gradient centrifugation method, and a sufficient amount of CIK is obtained by using cell culture bags and a CIK cell culture system to meet the needs of clinical treatment. The Takara lymphocyte culture medium and the autologous serum and cytokine co-culture technique are used in the method, thereby preventing application of fetal bovine serum, reducing contamination risks of exogenous pyrogen and sensitinogen, and while maintaining the advantage of CIK cell efficient proliferation. The cell culture bag technology is used to reduce risk of cell contamination, and is suitable for clinical therapeutic application.

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

The invention relates to a biological agent and particularly relates to a lymphocyte culture medium. The lymphocyte culture medium comprises lectin, a basal culture medium and a serum substitution, wherein the lectin is L-type phytohemagglutinin; the basal culture medium is an RPMI1640 basal culture medium; and the serum substitution comprises bovine serum albumin, recombinant human insulin, ferric citrate and an amino acid mother solution. By adopting the serum-free human peripheral blood lymphocyte culture medium, the animal or human serum is not used so that the cost of the culture medium is reduced and the disease dissemination risk is also reduced. The components of the serum-free human peripheral blood lymphocyte culture medium are determined so that the problems of complex traditional serum components, quality differences in different batches and fluctuated culture effect can be avoided. The culture medium provided by the invention is high in lymphocyte conversion rate and lymphocyte metaphase index, low in reagent cost and stable in quality, and the cultured cell can be used for clinical diagnosis of the karyotype analysis.

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.

A preparing method of a human DCCIK immunocompetent cell is disclosed. The method includes steps of: (1) separating a mononuclear cell from human peripheral blood; (2) inoculating the mononuclear cell to a culture medium suitable for lymphocyte culturing, adding IFN[gamma], IL-2, an anti-CD3 monoclonal antibody, GM-CSF and IL-4, and culturing for 3-5 days; (3) adding TNF[alpha], and continuously culturing for 1-2 days; and (4) adding IL-2, and continuously culturing for 7-10 days to obtain the DCCIK cell. A composition of various cytokines and stimulating factors is added directly into the mononuclear cell so that the DC cell and lymphocyte induce at the same time and mutually stimulate maturity, thus simplifying operation steps, reducing experiment consumable items and shortening cell culturing time. In addition, normalized experiment process technology can be easily mastered, thus facilitating clinic popularization and application.

The invention discloses a combined factor for inducing tumor specific T cells and a method for obtaining the tumor specific T cells. The combined factor comprises a tumor specific antigen and cell factors IL-2, IL-7 and IL-15. The method comprises the following steps: suspending peripheral blood PBMC with a fresh serum-free lymphocyte culture solution, wherein the cell density is 2*10<6>-5*10<6> / ml; adding a stimulus of the combined factor for inducing the tumor specific T cells, conducting culturing in a 5% CO2 culturing box at 37 DEG C for 2-10 days, and separating the IFN gamma positive antigen specific T cells through an IFN gamma magnetic bead enrichment method. The method can be used for obtaining the high-purity tumor antigen specific T cells, and is simple, and low in cost. The prepared tumor antigen specific T cells can be applied to preparation of medicines or vaccines for clinical cell treatment and tumor immunization treatment.

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

The invention discloses a composition for autologous lymphocyte culture, a culture solution and application of the composition. The active ingredients of the composition for autologous lymphocyte culture are composed of plasma, an anti-human CD3 monoclonal antibody, IL-2, IL-7, IL-15 and PHA-P. Compared with a composition for lymphocyte culture as a control, the content of a T lymphocyte subset obtained by using a culture medium containing the composition for induced culture of peripheral blood mononuclear cells is significantly increased; the number of lymphocytes is also significantly increased after 14 days of culture, and the number is increased by 1.15 times; and the killing activity of the lymphocytes on K562 cells is also significantly enhanced, and the killing activity is enhancedby 1.6 times. The composition for autologous lymphocyte culture can be used for specific induced amplification of mononuclear cells to produce autologous lymphocytes with high purity and strong biological activity.

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 for amplifying tumor-killing T cells in tumor-infiltrating lymphocytes, and the method comprises the following steps: a, mixing a mononuclear cell suspension with a feeder layer material suspension, and carrying out suspension culture for 96-120 hours to obtain a first culture; wherein the feeder layer material comprises a 3D tumor organ subjected to irradiation inactivation; b, mixing the first culture with a tumor infiltrating lymphocyte culture medium, and carrying out suspension culture for 65-72 hours to obtain a second culture; c, mixing the second culture with the tumor infiltrating lymphocyte culture medium, and carrying out suspension culture for 72-96 hours to obtain a third culture; and d, removing the feeder layer material in the third culture,and separating to obtain the tumor killing T cells. By means of the technical scheme, in-vitro directional amplification of the tumor-killing T cells can be achieved, the amplification efficiency andthe tumor-killing activity of the tumor-killing T cells are greatly improved, and meanwhile the consistency of the amplification efficiency between different amplification batches can be improved.

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 discloses an anti-tumor composition containing immune cells. The composition contains a dendritic cell culture, a T lymphocyte culture, a rosemary herb extract, a daffodil flower extractand a blueberry extract. The anti-tumor combination inhibits tumor growth through the mode that dendritic cells (DC) and T cells are in contact to stimulate an immune response. The dendritic cells (DC cells) express MHC type-I and type-II molecules at a high level, and when the DC cells expressing tumor-related peptide antigens and MHC type-I molecules activate CD4 and CD8 T cells, the T cells can exert effects to kill tumor cells. The composition contains the rosemary herb extract, the daffodil flower extract and the blueberry extract which all have an anti-tumor effect. The anti-tumor composition can protect cells against damage caused by external environmental factors while inhibiting tumors, and the tumors are prevented and resisted.

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 discloses a medium freeze-drying powder production process for lymphocyte culture, which comprises following steps: (1) preparing medium stock solution; (2) filtering medium stock solution, freezing, drying and obtaining the freeze-drying powder; (3) adding mixing gas containing nitrogen and oxygen into the freeze-drying powder, and obtaining the product. According to the process provided by the invention, a mixing gas containing nitrogen and oxygen at a certain ratio is added in the freeze-drying stage after the secondary drying ends, thereby activity and stability of the medium freeze-drying powder are unexpectedly improved. After a period of time,the freeze-drying powder compound solution is used for blood lymphocyte culture and chromosome karyotype analysis, and the two key active indexes such as lymphocyte conversion rate and mitotic phase index remain stable and are substantially higher than those of the traditional liquid medium. The lymphocyte medium freeze-drying powder prepared by the process provided by the invention has the advantages of long preservation time, stable effect, convenient storage, carrying and transport, thereby the commercialization of the lymphocyte medium is realized.

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 discloses a culture medium for lymphocyte culture and application thereof. The culture medium for lymphocyte culture provided by the invention comprises a basic medium, mixed deoxymononucleotides and human blood plasma.

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 belongs to the technical field of in-vitro cell culture, and relates to a lymphocyte culture medium. The lymphocyte culture medium is prepared from the following components: basal culture medium dry powder, insulin, human transferrin, hydrocortisone, casein phosphopeptides, an epidermal growth factor, L-glutamine, ethanol amine, soybean peptides, lecithin, sodium selenite, ferric citrate, zinc sulfate, vitamin E and gentamicin. By adopting the lymphocyte culture medium, the proliferation ability and the sterilizing activity of cultured lymphocytes are enhanced, and the problems that a disease transmission risk exists and that the quality of different batches cannot be unified in the conventional lymphocyte culture medium are solved effectively.

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.