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Method used 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 problems such as high cost and insufficient amplification multiples

Inactive Publication Date: 2014-04-30
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 used for in vitro amplification of gamma-delta-T cells
  • Method used for in vitro amplification of gamma-delta-T cells
  • Method used for in vitro amplification of gamma-delta-T cells

Examples

Experimental program
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Effect test

Embodiment 1

[0026] 1. Isolation and culture of peripheral blood mononuclear cells (PBMC)

[0027] 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 to 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 test group was supplemented with zoledron...

Embodiment 5

[0044] Example 5: Determination of immunological response results of all γδT subjects cultivated by conventional methods and experimental protocols

[0045] All the test subjects were in stage IV and had no other treatment, and their legal representatives were informed and agreed to receive this immunotherapy.

[0046] The cells obtained from the two schemes were reinfused intravenously to 11 test subjects, and the peripheral blood CD3 was detected before and after treatment (2 months after the end of treatment). + , CD8 + , CD56 + Variety.

[0047] All data are expressed as mean and standard deviation, t test, and SPSS18.0 software package is used for analysis.

[0048] After 2 months of treatment by conventional methods, all patients with CD3 + , CD8 + and CD56 + All were significantly higher than before treatment (P<0.001) (Table 3).

[0049] Table 3 Statistical analysis results of immune indexes of T lymphocyte subsets before treatment and after 2 months of treatmen...

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Abstract

The invention relates to a method for culturing gamma-delta-T cells, and more specifically relates to a method for in-vitro amplification of gamma-delta-T cells. The method comprises the following operating steps: pre-coating a T75 culture bottle with a TCR-gamma-delta resisting antibody and CD28McAb for later use; isolating peripheral blood mononuclear cells (PBMC) of patients; regulating PBMC concentration to 1*10<6> / ml with a serum-free culture medium which contains 5% of autologous plasma, and transferring the PBMC cell suspension into the T75 culture bottle; adding an initial culture medium which contains proper concentrations of Zoledronat, HSP70, Toll-like Receptors7 (TLR7) ligand, Levamisole (LMS), IL-2, IL-7 and IL-15; culturing in a saturated humid environment containing 5% of CO2 at 37 DEG C; according to growth situation of the cell, changing the culture medium every 2 to 3 days so as to control the cell concentration at about 2.5*10<6> / ml; meanwhile, compensating full doses of Zoledronat, HSP70, Toll-like Receptors7 (TLR7) ligand, Levamisole (LMS), IL-2, IL-7 and IL-15;; and continuously culturing for 12to 16 days so as 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 group 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-killing eff...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
Inventor 解尚云叶永清谭令兵
Owner SHANGHAI CLAISON BIOTECH
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