NK cell culture solution and cell culture method
A cell culture and NK cell technology, applied in the field of cells, can solve the problems of increasing cost, increasing separation steps, and easy introduction of exogenous cells, etc., to reduce risks, simplify separation steps, optimize cytokine combination and culture The effect of the process
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Embodiment 1
[0049] A cell culture solution for culturing NK cells, comprising: culture solution A and culture solution B.
[0050] The preparation of culture medium A is as follows: add OKT3, CD16, IL-2, IL-15 and 4-1BBL to the basal medium and mix well; among them, OKT3, CD16, IL-2, IL-15 and 4- The final concentrations of 1BBL in culture solution A were: 50ng / mL, 50ng / mL, 100U / mL, 50ng / mL and 50U / mL.
[0051] The preparation of culture medium B is as follows: add IL-2, IL-15 and 4-1BBL to the basal medium and mix well; wherein, the final concentration of IL-2, IL-15 and 4-1BBL in culture medium B Concentrations are: 100U / mL, 50ng / mL and 50U / mL.
[0052] The above-mentioned cytokines and basal medium are commercially available. The basal medium is the NK cell serum-free medium produced by Beijing Youkang Biotechnology Co., Ltd., and OKT3, CD16, IL-2, IL-15 and 4-1BBL were purchased from Beijing Tong Lihaiyuan Biotechnology Co., Ltd.
Embodiment 2
[0054] Under sterile conditions, collect 100 mL of peripheral blood from normal volunteers with a blood collection bag, dilute it with a mixed solution of 0.9% normal saline and peripheral blood at a ratio of 1:1 under an ultra-clean workbench, and blow it evenly with a straw to obtain diluted blood. ;
[0055] Take another new 50mL centrifuge tube, add lymphocyte separation medium, and slowly add the diluted blood to the surface of lymphocyte separation medium according to the ratio of diluted blood: lymphocyte separation medium of 2:1, so that the gap between the two Form a clear interface, and then centrifuge at 2000r / min for 20min at room temperature;
[0056] After taking it out, it can be seen that the liquid in the tube is divided into four layers. From top to bottom, they are plasma (including platelets), middle cloud layer cells (ie mononuclear cells), lymphatic separation fluid, red blood cells and granulocytes. Use a straw to carefully suck out the middle cloud laye...
Embodiment 3
[0058] Take lymphocytes, add NK cell serum-free medium to suspend the cells, and perform cell counting, then use NK cell serum-free medium to adjust the cell density to 2×10 6 / mL, transferred to two cell culture flasks, then added culture solution A, diluted to a cell density of 0.5×10 6 / mL-1.5×10 6 / mL or so, placed at 37°C, 5% CO 2 cultured in a cell culture incubator;
[0059]On the 3rd, 5th, and 7th day of cell culture, supplement culture solution A to maintain the cell density at 0.5×10 6 / mL-1.5×10 6 / mL at 37°C, 5% CO 2 cultured in a cell culture incubator;
[0060] After the medium was supplemented on the seventh day, the cells were transferred to cell culture bags for culture, and then culture solution B was supplemented on the 9th, 11th, 13th, 15th, 17th, and 19th days of culture, so that the cell density was 0.5×10 6 / mL-1.5×10 6 / mL, continue at 37°C, 5% CO 2 Cells were cultured in a cell culture incubator until the 21st day, and the cells were collected....
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