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Optimized method for inducing amplification of regulatory T cells from naive T cells

A cell induction and regulation technology, applied in the field of bioengineering, can solve problems such as poor stability, long amplification induction cycle, complicated operation, etc., to avoid biosafety problems and immune rejection, amplification and therapeutic effects Stability, operation time saving effect

Inactive Publication Date: 2017-02-15
SHENZHEN ZHONGSHAN INST OF GENETICS & REPRODN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, nTreg only accounts for normal peripheral blood CD4 + 5‐15% of T cells, greatly limiting its clinical application
Among the reported Treg amplification methods, iTreg mostly has the following problems: the use of anti-CD3 / CD28-magnetic beads as an inducer leads to high amplification costs; the use of heterologous or allogeneic serum has potential biosafety issues; multiple analyses are required. selection, complex operation and increased cost; in addition, there are other problems such as long amplification induction period and poor stability

Method used

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  • Optimized method for inducing amplification of regulatory T cells from naive T cells
  • Optimized method for inducing amplification of regulatory T cells from naive T cells
  • Optimized method for inducing amplification of regulatory T cells from naive T cells

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Embodiment 1

[0023] According to the optimized method of inducing and expanding regulatory T cells from naive T cells of the present invention, the specific steps are as follows:

[0024] Step 1. Coating with anti‐CD3 antibody

[0025] 24h to 1h before cell culture, add PBS (phosphate-buffered saline) containing 0.1-2 μg / ml (preferably 1 μg / ml) anti-CD3 (OKT3, eBioscience) antibody to each well of a U-shaped or flat-bottomed culture plate, Add 1ml for 24-well plates, and convert according to volume or bottom area for other well plates. Store at 4°C until use.

[0026] Step 2. Isolation of Peripheral Blood Mononuclear Cells (PBMCs)

[0027] Draw 20ml of heparin anticoagulated venous blood, take three 15ml centrifuge tubes, add 4ml of lymphocyte separation solution to each tube, slowly place 6-7mL peripheral blood on the layer of lymphocyte separation solution along the tube wall, and centrifuge at 800g for 17min, respectively. Plasma and PBMC were collected, plasma was inactivated in a w...

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Abstract

The invention provides an optimized method for inducing amplification of regulatory T cells from naive T cells. The method comprises the following steps: (1) coating an anti-CD3 antibody; (2) isolating peripheral blood mononuclear cells; (3) sorting the naive T cells by virtue of magnetic beads; and (4) inducing amplification culture. The method provided by the invention is simple and convenient to operate, safe and effective, relatively short in amplification inducing cycle, relatively good in stability and low in cost.

Description

technical field [0001] The invention belongs to the field of bioengineering, and relates to a cell expansion method in vitro, in particular to an optimized method for inducing and expanding regulatory T cells from immature T cells. Background technique [0002] Immune tolerance refers to an active non-response state displayed by immune active cells when exposed to antigenic substances, which is a biological phenomenon different from immunodeficiency and immunosuppression. Immune intolerance is associated with many diseases, such as autoimmune diseases, graft versus host disease (GVHD) and maternal-fetal immune rejection. In organ or tissue transplantation, although traditional immunosuppressive drugs can significantly prolong the survival time of grafts, their side effects are decreased overall immune function of patients, increased chances of infection, and may induce tumors, etc., which greatly limit the clinical organ transplantation. Further development of transplantati...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
CPCC12N5/0637C12N2501/15C12N2501/2302C12N2501/51C12N2501/515C12N2501/999C12N2509/00
Inventor 刁梁辉黄春宇李玉叶连若纯林嘉音何来宾张谞陈聪
Owner SHENZHEN ZHONGSHAN INST OF GENETICS & REPRODN
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