Preparation method of CIK (Cytokine Induced Killer) cells with high proliferation capacity, high cytotoxic activity and high survival rate, associated CIK cells and application
A proliferative and cytotoxic technology, applied in the field of surgery, L and C, can solve the problems of low cell viability and tumor killing ability, no significant increase in the number of cells, and expansion of CIK cells, etc., and is suitable for large-scale promotion and application. , Best tumor killing efficiency, cleverly designed effect
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[0036] The preparation method of the present invention uses a sorting method to remove CD4 before culturing + CD25 + For Treg cells, add IFN-γ (1000U / ml) to the culture, and coat with anti-CD3 monoclonal antibody (1ug / ml) to improve cytotoxic activity, and add IL-6 (100ng / ml) and PGE2 (10ng / ml) to inhibit CD4 + CD25 + Treg cell differentiation, adding insulin to the final concentration of 1ug / ml, IL-2 (1000U / ml) to promote cell proliferation.
[0037] Therefore, one of the technical solutions adopted by the present invention to solve the above-mentioned technical problems is: a method for inhibiting monocytes to CD4 + CD25 + The method of Treg cell differentiation is to add IL-6 (100ng / ml) and PGE2 (10ng / ml) to the culture medium.
[0038] The second technical solution adopted by the present invention to solve the above-mentioned technical problems is: a method for promoting the proliferation of CIK cells, that is, adding a cell culture solution containing insulin to a fi...
Embodiment 1
[0041] The preparation of embodiment 1CIK cell
[0042] 1. Preparation of peripheral blood mononuclear cells (PBMC) (Ficoll density gradient method)
[0043] Use a sterile syringe to collect 30-50ml of anticoagulant blood from the patient under aseptic conditions, centrifuge at 1500rpm / min for 15 minutes, draw the upper layer of plasma, inactivate at 56°C for 30 minutes, put it at 4°C, and centrifuge at 3000rpm / 8min after 30 minutes to remove the precipitate , put in a 4°C refrigerator for later use. Double dilute the blood cell pellet with normal saline, put the human lymphocyte separation medium with a specific gravity of 1.077 and the diluted blood at a ratio of 1:2 into the centrifuge tube, centrifuge at 2000rpm / min for 20 minutes, carefully extract the white blood cell layer, and wash it twice with normal saline , PBMC were obtained after low-speed centrifugation.
[0044] 2. Mini MACS to remove CD4 + CD25 + Treg cells (Mini MACS High Magnetic Beads Separation Column ...
Embodiment 2
[0047] Example 2: Detection of CIK cells
[0048] 1. CIK cell live cell detection
[0049] After adding PHA and INF-γ, most of the cells were still in a suspended state; after 3 days, the cell volume increased, the cells gradually aggregated into clusters, the cells were translucent, and the cytoplasm was abundant; from the 5th day, the cells began to proliferate in large quantities, and the cell shapes were diverse. The number of cell colonies increases; take 100ul of CIK cells cultured on days 1, 5, 7, 9, 11, 13, 15, 17, and 19, add 100ul of 0.4% placenta blue staining solution, the living cells will not be stained, and the dead cells will be stained into blue. See figure 1 As shown (wherein the experimental group is the CIK cell prepared by the present invention, that is, the CIK cell prepared in Example 1, and the control group is conventional CIK culture (according to the Chinese invention titled "lymphocyte culture medium and method and application for culturing lympho...
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