Culture method for increasing amplification efficiency and activity of Vgamma9Vdelta2T cells
A technology of expansion efficiency and culture method, applied in the field of in vitro induction culture, can solve the problems of Vγ9Vδ2T cell induction expansion efficiency and limited activity, and achieve the effect of simple and easy induction culture process, improved curative effect and high induction efficiency.
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Embodiment 1
[0032] Embodiment 1: the preparation of human mononuclear cells:
[0033] 1. Take 10ml of peripheral blood sample and anticoagulate with heparin;
[0034]2. Add 5ml of lymphocyte separation medium to a 15ml centrifuge tube;
[0035] 3. Fully mix 10 ml of peripheral blood with the same amount of Hank's solution, use a dropper to slowly superimpose on the layered liquid surface along the tube wall with twice the volume of the lymphocyte separation solution, pay attention to keep a clear interface, and centrifuge horizontally at 600g× 20 minutes;
[0036] 4. After centrifugation, the tube is divided into three layers, the upper layer is plasma and Hank's solution, the lower layer is mainly red blood cells and granulocytes, the middle layer is lymphocyte separation fluid, and there is a white cloud layer mainly composed of mononuclear cells at the interface of the upper and middle layers narrow band;
[0037] 5. Use a dropper to insert into the cloud layer, absorb mononuclear c...
Embodiment 2
[0039] Example 2: Grouping of mononuclear cells and induction of Vγ9Vδ2 T cells
[0040] The cells obtained in Example 1 were inoculated into 12-well plates, and divided into conventional method induction group and improved method induction group. The conventional method induction group was induced with interleukin-2 (IL-2) and zoledronic acid alone, and Methods The induction group was synergistically induced by IL-2, zoledronic acid combined with 10nmol / L dasatinib. Three replicate wells were set up in each group, each well was 2ml, and IL-2 and zoldronic acid were added to each well on the 0th day (the day of mononuclear cell inoculation was calculated as the 0th day), the amount and final concentration are shown in Table 1 As shown; each well of the improved method induction group was added dasatinib on the first day, the amount was shown in Table 1, and the final concentration was 10nmol / L; Half of the medium was changed, 1ml of culture medium was removed from each well, ...
Embodiment 3
[0043] Example 3: Detection of Expansion Efficiency of Vγ9Vδ2T Cells
[0044] Detecting the Purity of Vγ9Vδ2 T Cells by Flow Cytometry
[0045] 1. Harvest the cells cultured for 9, 12, 15, and 18 days in the conventional method induction group and the improved method induction group respectively, 10 cells per tube 6 , one tube for each group, wash twice with 2ml of 1× phosphate buffer solution, centrifuge at 300g×5 minutes before pouring each time, and blot the liquid at the mouth of the tube with filter paper after pouring;
[0046] 2. Resuspend the cells in 95 μl of 1× phosphate buffer, add 5 μl of anti-TCRVδ2-PE monoclonal antibody, mix well, and incubate at 4°C in the dark for 30 minutes;
[0047] 3. Wash twice with 2 ml of 1× phosphate buffer solution, and pour out the liquid at the mouth of the tube with filter paper after each wash;
[0048] 4. Add 500 μl 1× phosphate buffer;
[0049] 5. On-board detection by flow cytometry; use CELL-QUEST software to obtain 10,000 c...
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