Method for amplification of gamma delta T cells by phosphate and application thereof

A cell and nuclear cell technology, applied in the field of immunology, can solve the problems of poor universality, high production cost, and inability to kill tumors extensively, and achieve the effects of enhanced killing activity, high amplification efficiency, and strong lethality

Inactive Publication Date: 2015-05-27
SHENZHEN INST OF ADVANCED TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

How to efficiently and rapidly expand human peripheral blood γδT cells in large quantities in vitro is very important for the study of γδT cell immune function, improving patient immunity, anti-tumor and anti-infection capabilities, but due to the high content of γδT cells in peripheral blood and tumor tissues Not high, generally no more than 5% of the total number of T cells, it is very difficult to obtain a large number of γδT cells with high cytotoxic activity
[0003] The invention patent with the authorized announcement number CN1306027C provides a method for expanding γδT lymphocytes in vitro. This method uses solid-phase anti-γδTCR antibody to selectively expand γδT cells. However, the anti-γδTCR antibody used is expensive and the production cost is too high.
[0004] The invention patent with the authorized announcement number CN102533649B provides a simple and efficient method for preparing human γδT cells. The method uses Mycobacterium tuberculosis thermostable antigen combined with cytokines human recombinant rhIL-2 and human recombinant rhIL-21 to stimulate and expand γδT cells. However, the specific clones of γδT cells obtained by this method are dominant, but the universality is poor, and it cannot widely kill different types of tumors

Method used

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  • Method for amplification of gamma delta T cells by phosphate and application thereof
  • Method for amplification of gamma delta T cells by phosphate and application thereof
  • Method for amplification of gamma delta T cells by phosphate and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] This embodiment provides a method for isolating human peripheral blood mononuclear cells (PBMCs), comprising the following steps:

[0039] (1) Aseptically collect blood into heparin anticoagulant tubes;

[0040] (2) Take the lymphocyte separation solution and add it to the bottom of a 15ml centrifuge tube, tilt the centrifuge tube at an angle of 45°, draw the blood sample with a straw (separation solution volume: blood sample volume = 1:2), carefully add the separation solution at 1cm above, along the The wall of the centrifuge tube slowly superimposes the blood sample on the separation liquid, and does not damage the interface of the separation liquid layer;

[0041] (3) Centrifuge horizontally at 400g at room temperature for 20min, and slowly increase and decrease the speed;

[0042] (4) Take out the centrifuge tube smoothly. At this time, the bottom layer is red blood cells and granulocytes, the middle layer is lymphocyte separation medium, and the top layer is plas...

Embodiment 2

[0046] This embodiment provides a method for expanding γδ T cells in vitro, comprising the following steps:

[0047] (1) Design the control group and the experimental group, and the experimental group is divided into 3 groups; 10U / ml of rhIL-2 is added to the culture medium of the control group and the experimental group 1-3, and the culture medium of the experimental group 1-3 Add 0.5μM, 1.0μM and 2.0μM IBA respectively;

[0048] (2) Take a 24-well plate containing 500 μL of medium and the peripheral blood mononuclear cell PBMC obtained in Example 1, in which 2×10 5 PBMC; the medium used is RPMI-1640+15%FBS+1% double antibody;

[0049] According to step (1) and step (2), the design of the control group and each experimental group is as follows in Table 1:

[0050]

[0051]

[0052] Table 1 The addition of each component in the control group and the experimental group

[0053] (3) After adding the corresponding substances to the marked culture wells of each group accord...

Embodiment 4

[0066] This embodiment provides a method for expanding γδ T cells in vitro, comprising the following steps:

[0067] (1) Design the control group and the experimental group, and the experimental group is divided into 3 groups; 50 U / ml rhIL-2 is added to the culture medium of the control group and the experimental group 1-3, and the culture medium of the experimental group 1-3 Add 0.5μM, 1.0μM and 2.0μM IBA respectively;

[0068] (2) Take a 24-well plate containing 500 μL of medium and the peripheral blood mononuclear cell PBMC obtained in Example 1, in which 2×10 5 PBMC; the medium used is RPMI-1640+12%FBS+1% double antibody;

[0069] (3) After adding the corresponding substances to the marked culture wells of each group according to the experimental scheme designed in step (1), place the culture plate at 37°C with 5% CO 2 Expanded culture in a cell culture incubator.

[0070] (4) Change the medium in half every two and a half days. Collect the cells on the 10th day and che...

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Abstract

The invention provides a method for in vitro amplification of human gamma delta T cells and application thereof. The method for in vitro amplification of human gamma delta T cells includes the steps of: (1) preparing or providing mononuclear cell; (2) adding the mononuclear cell into a cell culture liquid, and placing the cell culture liquid into a cell incubator to conduct culture; and (3) acquiring the cells. The cell culture liquid contains (1-hydroxy-3-(methyl pentyl amino)propylidene)diphosphonic acid monosodium salt (IBA) and human recombinant interleukin rhIL-2. According to the invention, the method of combining rhIL-2 and IBA to stimulate human peripheral blood mononuclear cells (PBMC) is employed to amplify human gamma delta T cells in vitro. The method has the characteristics of low cost, simple preparation and high amplification efficiency, and can obtain gamma delta T cells with strong tumor lethality and TCR (T cell receptor) cloning diversity.

Description

technical field [0001] The invention relates to the field of immunology, in particular to a method for amplifying γδT cells with phosphate and application thereof. Background technique [0002] T lymphocytes are divided into two types according to the expression of T cell antigen receptor (TCR): TCRαβT cells and TCRγδT cells, referred to as αβT cells and γδT cells, respectively. γδT cells have the ability to specifically recognize antigens without MHC restriction, have anti-infection, anti-tumor and immune regulation functions, and are the first line of defense for the body's immune surveillance. How to efficiently and rapidly expand human peripheral blood γδT cells in large quantities in vitro is very important for the study of γδT cell immune function, improving patient immunity, anti-tumor and anti-infection capabilities, but due to the high content of γδT cells in peripheral blood and tumor tissues It is not high, generally no more than 5% of the total number of T cells...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783A61K35/17A61P35/00
Inventor 金言党利君胡玉婷张茜任广慧万晓春
Owner SHENZHEN INST OF ADVANCED TECH
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