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Inducing culture method for regulatory T cell

A culture method and regulatory technology, applied in the field of immunology and epigenetics research, can solve the problem of low induction efficiency, and achieve the effect of high induction efficiency, simple and easy enrichment process, and good promotion value.

Active Publication Date: 2011-08-31
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The purpose of the present invention is to overcome the shortcoming of low induction efficiency in the existing research scheme, to provide a method for inducing and culturing regulatory T cells, which involves providing a regulation The induction and culture method of regulatory γδ T cells can provide a research platform for further clarifying the biological characteristics of regulatory γδ T cells

Method used

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  • Inducing culture method for regulatory T cell
  • Inducing culture method for regulatory T cell
  • Inducing culture method for regulatory T cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: Induction of Regulatory γδ T Cells

[0022] (1) Add 7ml of lymphocyte separation medium to a 15ml centrifuge tube;

[0023] (2) Take 10 ml of heparin anticoagulated venous blood and mix it well with the same amount of Hank's solution, slowly superimpose on the layered liquid surface along the tube wall with a dropper, pay attention to keep a clear interface, and centrifuge horizontally at 600g×20 minutes;

[0024] (3) 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 mainly composed of mononuclear cells at the interface of the upper and middle layers Layer narrow band;

[0025] (4) Use a dropper to insert into the cloud layer, absorb mononuclear cells, put them into another 15ml centrifuge tube, add 5 times the volume of Hank's solution, centrifuge at 300g×5 minutes, ...

Embodiment 2

[0030] Example 2: Detection of regulatory γδ T cells by flow cytometry

[0031] (1) Harvest the cells cultured for 10 days in the conventional method induction group and the improved method induction group respectively, 10 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;

[0032] (2) Resuspend the cells in 40 μl of 1× phosphate buffer, add 20 μl of anti-TCR γδ FITC monoclonal antibody, mix well, and incubate at 4°C in the dark for 30 minutes;

[0033] (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;

[0034] (4) Add 1ml of Fix / Perm buffer, mix well, and incubate at 4°C for 30 minutes;

[0035] (5) Add 2ml of 1×Perm Wash buffer solution to wash twice, pay attention to the action to be gentle, after each washing, ...

Embodiment 3

[0039] Example 3: Enrichment of Regulatory γδ T Cells by Immunomagnetic Beads Cell Sorting Technology

[0040] (1) Prepare the buffer, which is composed of pH 7.2 1× phosphate buffer, 0.5% bovine albumin and 3M ethylenediaminetetraacetic acid (EDTA), hereinafter referred to as buffer; count cells, take 10 7 ;

[0041] (2) Centrifuge at 300×g for 10 minutes, remove the supernatant;

[0042] (3) Cells were resuspended in 40 μl buffer;

[0043] (4) Add 10 μl anti-TCR γδ hapten antibody;

[0044] (5) Incubate at 4°C for 10 minutes after mixing;

[0045] (6) Add 90 μl buffer and 60 μl anti-hapten microbeads-FITC;

[0046] (7) Mix well and incubate at 4°C in the dark for 15 minutes

[0047] (8) Add 2 ml buffer, centrifuge at 300×g for 10 minutes, discard the supernatant;

[0048] (9) Resuspend cells in 500 μl buffer;

[0049] (10) Put the MS column in the corresponding MACS sorter, and rinse the sorting column with 0.5ml buffer;

[0050] (11) Add the cell suspension to the s...

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Abstract

The invention provides an inducing culture method for regulatory T cells and relates to an inducing culture method for regulatory gamma delta T cells. In the method, cell factors are applied and deoxyribonucleic acid (DNA) demethylated medicament decitabine is combined so as to cooperatively induce the generation of the regulatory gamma delta T cells, and an enrichment process of magnetic cell sorter (MACS) positive sorting is adopted; after enrichment, detections of cell growth state and activity are carried out so as to ensure the next experience. The method has the advantages of simple and practicable inducing and enrichment processes, short period, low cost, high inducing efficiency and good repeatability; and after enrichment, cell activity is good, the quantity and activity of cells can ensure the next in vivo and in vitro studies, thereby providing a good study platform for defining the biological characteristics of the gamma delta T cells. Thus, the method has a good popularization value.

Description

technical field [0001] The invention belongs to the field of immunology and epigenetics research, and relates to a culture method for synergistically inducing the generation of regulatory γδ T cells by using cytokines combined with DNA demethylation drugs. Background technique [0002] γδ T cells have unique structures and biological functions, and account for a small proportion (1-5%) in peripheral blood and lymphoid organs, which has been ignored by researchers before. Since Kunzmann et al. first discovered that γδ T 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. At present, the in vitro expansion and purification of γδ T cells has become a routine technique; therefore, the biological functions of this type of cells are becoming increasingly clear. Researchers have found that γδ T cells play a pivotal role in anti-infection and anti-tumor, and its adoptive immunotherapy has...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783G01N15/10
Inventor 黄河胡永仙吴康妮
Owner ZHEJIANG UNIV
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