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Method for in-vitro amplification of gamma-delta-T cells

An in vitro amplification and cell technology, applied in animal cells, vertebrate cells, blood/immune system cells, etc., can solve the problems of insufficient amplification and high cost.

Inactive Publication Date: 2013-03-27
SHANGHAI CLAISON BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In this field, people have made many beneficial attempts. In China, IPP and IL-2 co-stimulation methods are usually used to culture and expand γδT cells in vitro, but they are not widely used due to relatively expensive costs and insufficient expansion times.

Method used

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  • Method for in-vitro amplification of gamma-delta-T cells
  • Method for in-vitro amplification of gamma-delta-T cells
  • Method for in-vitro amplification of gamma-delta-T cells

Examples

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Embodiment 1

[0028] 1. Isolation and culture of peripheral blood mononuclear cells (PBMC)

[0029] The machine collects PBMC, transfers the collected blood sample to a centrifuge tube; centrifuges at 700g for 10 minutes, absorbs the upper layer of plasma for later use; restores the blood sample to its original volume and mixes; slowly adds diluted blood to Ficoll, 900g, and centrifuges for 20 minutes; absorbs Milky white mononuclear cell layer at the separation liquid interface; centrifuged and washed twice and counted; cells were resuspended to 1×10 with γδT initial medium 6 / ml; According to the growth of the cells, replace the medium every 2-3 days, and the medium used is a serum-free medium containing 5% autologous plasma. At 37°C, 5% CO 2 According to the growth of the cells, the medium was replaced every 2-3 days, and the cell concentration was controlled at 2.5×10 6 At the same time, various factors were supplemented in full amount: the experimental group was supplemented with z...

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Abstract

The invention relates to a method for culturing gamma-delta-T cells, and in particular relates to a method for in-vitro amplification of gamma-delta-T cells, wherein the method comprises the following operating steps of: pre-coating a T75 culture bottle by a TCR-gamma-delta resisting antibody and CD28McAb for later use use; isolating the peripheral blood mononuclear cell (PBMC) of a patient; regulating the PBMC concentration to 1*10<6> 6 / ml by a serum-free culture medium which contains 5% of autologous plasma, and transferring PBMC cell suspension into the T75 culture bottle; adding an initial culture medium which contains proper concentrations of Zoledronat, HSP70, 1L-2, 1L-7 and 1L-15; culturing in a saturated humid environment containing 5% of CO2 at 37 DEG C; depending on growth situation of the cell, changing the culture medium every 2-3days, to control the cell concentration at about 2.5*10<6> / ml; meanwhile, compensating full doses of Zoledronat, HSP70, 1L-2, 1L-7 and 1L-15; and continuously culturing for 12-16days, to obtain a great amount of gamma-delta-T cells which are comparatively high in purity.

Description

[technical field] [0001] The invention relates to a method for culturing γδT cells, in particular to a method for expanding γδT cells in vitro. [Background technique] [0002] According to the T cell surface receptor (TCR), T lymphocytes can be divided into two types: αβT lymphocytes and γδT lymphocytes. In the human body, αβT lymphocytes are the main cell population involved in the immune response, accounting for 90-95% of mature T cells, while γδT cells only account for 0.5%-5% of peripheral blood T lymphocytes, but γδT cells are prior to αβT Cells appear, mainly distributed in epithelial tissues such as skin, small intestine, esophagus, lung and reproductive organs. γδT cells are the first-line defense cells of the body, and play an important role in anti-microbial infection immunity, especially in the immune defense function on the skin and mucous membrane surface, and in anti-mycobacterial infection. In addition, the study also found that γδT cells have a tumor-killin...

Claims

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Application Information

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IPC IPC(8): C12N5/0783
Inventor 解尚云叶永清谭令兵
Owner SHANGHAI CLAISON BIOTECH
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