Improved high-efficiency human autologous Terg (Regulatory T cell) separation and amplification method

Abstract

The invention discloses an improved high-efficiency human autologous (Regulatory T cell) separation and amplification method. The improved high-efficiency human autologous separation and amplificationmethod comprises the following steps: S1, treating 5000mL of PB (Peripheral Blood) through a blood cell separator (single sampling machine) in 1 to 2 cycles, obtaining PBMCs (Peripheral Blood Mononuclear Cells) in order magnitude of 10*9 to 10*10, sieving separated PBMCs through a nylon net of which the pore diameter is 30 mum, and removing a cell cluster in suspension cells; S2, labeling non-CD4+ cells; S3, removing the non-CD4+ cells through negative selection of an LD separation column; S4, labeling cd25+ cells on an antibody; S5, positively selecting the CD25+ cells through an MS column.According to the improved high-efficiency human autologous separation and amplification method disclosed by the invention, 5000mL of peripheral blood can be treated through the blood cell separator (single acquisition machine) in 1 to 2 cycles, the PBMCs (Peripheral Blood Mononuclear Cells) in order magnitude of 10*9 to 10*10 can be obtained, and mononuclear cells of which the number is 100 timesor above of the number of the mononuclear cells obtained through a traditional blood sampling method can be easily obtained; other blood components are in direct feedback, so that waste is avoided, and the influence on total blood component and blood cell count of a patient is more slight.

Description

technical field [0001] The invention relates to the technical field of Treg cell separation, in particular to an improved and efficient method for separating and expanding human autologous Treg cells. Background technique [0002] CD4+CD25+Foxp3+ regulatory T cell (Regulatory T cell, Treg) is a cell population with immune regulation function, which can inhibit the immune response of autoreactive T cells, inhibit the activation of traditional T cells and promote some inhibitory functions. The secretion of cytokines, etc., play an important role in maintaining the stability of the internal environment of the body and inducing the tolerance of the graft. It is an important way for the body to actively acquire and maintain self-tolerance. [0003] Treg can secrete IL-10, TGF-B, express Foxp3 and CTLA-4 molecules, etc., inhibit the immune response of self-reactive T cells, inhibit the activation of traditional T cells, and promote some inhibition through direct cell-to-cell conta...

AI Technical Summary

Problems solved by technology

However, the proportion of autologous Treg cells in peripheral blood is small (this is more prominent in patients), the isolation efficiency is low, and the expansion is difficult, which ultimately leads to the low yield of cells and becomes the main bottleneck limiting Treg cell immunotherapy.

This protocol aims to solve the problem of low Treg cell isolation rate