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A technology of NKT cells and culture methods, which is applied in the field of NKT cell culture, and can solve the problems that cells are easy to adhere to the wall, NKT cells cannot fully contact the culture medium, and are easy to sink to the bottom.
Active Publication Date: 2016-12-07
北京景达生物科技有限公司
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[0005] However, when NKT cells are cultured in traditional culture flasks, some cells are easy to adhere to the wall
When the cells are proliferating and cultured, a large number of clumps of cells are easy to sink to the bottom, which can easily cause NKT cells to not fully contact the culture medium, slow down cell proliferation, and easily cause clumps of cells to die
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Embodiment 1
[0034] Example 1: Isolation of mononuclear cells
[0035] In the case of voluntary blood donors, the inventor collected 50 mL of human venous peripheral blood, diluted it with 10 mL of normal saline, and slowly added it to a centrifuge tube containing 15 mL of lymphocyte separation liquid, so that the layers of the diluted blood and lymphocyte separation liquid were clear , 500g-800g centrifugal force, centrifuge for 20-30min, extract the middle buffy coat cells, and obtain mononuclear cells, add normal saline to wash twice, count, the cell volume is 2×10 7 ~4×10 7 indivual.
Embodiment 2
[0036] Example 2 Cell Culture
[0037] 1. Preparation of NKT primary cells
[0038] 1. Divide 50-100mL of peripheral blood from the patient evenly into 2 tubes, use a pipette to draw a small amount of original blood (about 300μL) to streak or drop into a plate for bacterial inspection. Then, at room temperature, centrifuge at 865g for 15 minutes (9,7, meaning up 9 down 7, the same below).
[0039] 2. Transfer the upper plasma into a centrifuge tube, inactivate at 56°C for 30 minutes, centrifuge at 865g for 10 minutes, and take the supernatant for later use.
[0040] Use an equal volume of normal saline to mix with the blood cell pellet, and carefully add to the 15mL Ficoll layer that was divided into 50mL centrifuge tubes to keep the layering clear. Centrifuge at 865 g at room temperature for 20 minutes (9, 3).
[0041] 3. Aspirate the peripheral blood mononuclear cell (PBMC) layer, try to absorb the cell layer at the junction of the two liquid surfaces, add 5mL of normal s...
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Abstract
The invention discloses a novel NKT cell culture method. The method comprises NKT primary cell culture and NKT cell subculture. The NKT primary cell culture comprises inoculating a serum-free medium with peripheral blood mononuclear cells. The NKT cell subculture comprises adding a promoter humanized CD3 monoclonal antibody, a humanized CD28 monoclonal antibody and IL-2 into the serum-free medium in the first day of the NKT cell subculture and adding amplification factors IL-2 and IL-7 into the later amplified solution. The NKT cells cultured by the novel NKT cell culture method have a good cell state, a fast propagation speed, high proliferation multiple, high purity and killing effects obviously better than those of NKT cells obtained by the existing culture method.
Description
technical field [0001] The invention relates to the technical field of cell biology, in particular to a novel NKT cell culture method. Background technique [0002] Cellular immunotherapy is an emerging treatment technology. Cellular immunotherapy is to collect the body's own immune cells, culture them in vitro, increase their number by hundreds of times, and enhance their targeted killing function, and then reinfuse them back into the human body to kill pathogens, cancer cells, and mutants in blood and tissues. Cells, breaking immune tolerance, activating and enhancing the immune ability of the body, taking into account the dual effects of treatment and health care. Cellular immunotherapy can restore and enhance the immune monitoring and tumor killing functions of tumor patients, effectively kill the remaining tumor cells in patients after surgery, radiotherapy and chemotherapy, and achieve the purpose of treating tumors, preventing recurrence and metastasis, and finally e...
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