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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.

Inactive Publication Date: 2014-02-05
ZHEJIANG UNIV
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Problems solved by technology

[0004] The purpose of the present invention is to overcome the shortcomings of the limited efficiency and activity of Vγ9Vδ2T cell induction expansion in the existing scheme, to provide a culture method for improving the efficiency and activity of cell expansion, and to further improve the curative effect of anti-tumor immunotherapy based on Vγ9Vδ2T cells. support

Method used

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  • Culture method for increasing amplification efficiency and activity of Vgamma9Vdelta2T cells
  • Culture method for increasing amplification efficiency and activity of Vgamma9Vdelta2T cells
  • Culture method for increasing amplification efficiency and activity of Vgamma9Vdelta2T cells

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Experimental program
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Effect test

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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Abstract

The present invention provides a culture method for increasing amplification efficiency and activity of Vgamma9Vdelta2T cells, wherein IL-2 and zoledronic acid are combined with a tyrosine kinase inhibitor dasatinib to carry out in vitro Vgamma9Vdelta2T cell induction production to obtain the Vgamma9Vdelta2T cells with high induction efficiency and high activity. According to the present invention, the method has characteristics of simple and easy performing induction culture process, short period, low cost, high induction efficiency and good repeatability, the cells obtained through induction has enhanced activity, the tumor immunotherapy effect of the current Vgamma9Vdelta2T cells is expected to be increased with the number and the function of the induction-cultured cells, and good application potential is provided.

Description

technical field [0001] The invention belongs to the field of immunology and cell biology research, and relates to an in vitro induction culture method for improving cell expansion efficiency and activity, which is a culture method for synergistically inducing the generation of Vγ9Vδ2T cells by applying cytokines combined with tyrosine kinase inhibitors. Background technique [0002] γδT cells are a subgroup of T lymphocytes with unique structure and biological functions that has attracted much attention in recent years. and anti-tumor aspects play a pivotal role. Vγ9Vδ2T cells are an important subset of γδT cells, accounting for 50-70% of peripheral γδT cells. Since Kunzmann et al. first discovered that Vγ9Vδ2T cells can be expanded in large quantities, more and more scholars have begun to devote themselves to the research of this type of cell population. In vitro experiments and in vivo animal experiments have confirmed that Vγ9Vδ2T cells have a wide range of tumor-killin...

Claims

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Application Information

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IPC IPC(8): C12N5/0783
Inventor 黄河吴康妮胡永仙盛立霞
Owner ZHEJIANG UNIV
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