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Adult regulatory T cell in-vitro amplification culture medium and application method thereof

A medium and regulation technology, applied in the field of cell culture, can solve the problems of low cell proliferation ability, small number, low purity, etc., and achieve the effect of high expansion rate

Active Publication Date: 2015-01-14
HUNAN XENO LIFE SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the number of regulatory T cells in the body is very small, accounting for only 5% to 10% of the CD4+ T cells in the peripheral blood of normal people. After in vitro acquisition, the number is small, the purity is not high, and the cell proliferation ability is low. The instability of regulatory T cells in certain stages of the disease makes it difficult to expand in vitro

Method used

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  • Adult regulatory T cell in-vitro amplification culture medium and application method thereof
  • Adult regulatory T cell in-vitro amplification culture medium and application method thereof
  • Adult regulatory T cell in-vitro amplification culture medium and application method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Embodiment 1 Culture medium of the present invention and culture method test

[0032] In the present invention, Treg cells are isolated from adult peripheral blood in vitro and cultured, and the initial Foxp3CD4+CD25+CD127-regulatory T cells and CD3CD28 magnetic beads are mixed in a ratio of 1:3 (CD3CD28 magnetic beads are purchased from Invitrogen Corporation ), add transforming growth factor-β (TGF-β) 1-4ng / ml (preferably 2ng / ml), bone morphogenetic protein 4 (BMP-4) 8-12ng / ml (preferably 10ng / ml) in the medium without phenol red 1640 / ml), all-trans retinoic acid (ATRA) 1-4uM / mL (preferably 2uM / mL), recombinant human interleukin-2300-500U / ml, rapamycin 80-100nM (preferably 100nM), 4-hydroxyethyl Piperazine ethanesulfonic acid 20-30mM (preferably 25mM), L-glutamine 2mM, 2-mercaptoethanol 40-50uM (preferably 50uM), human AB serum 5%, penicillin 50U / ml, streptomycin 50ug / ml.

[0033] Resuspend the isolated starting cells in the above medium and place them in a U-bott...

Embodiment 2

[0035] Example 2 Comparison of the culture effects of the culture medium of the present invention and the common regulatory T cell culture medium of the present invention without adding transforming growth factor-β, bone morphogenetic protein-4 and all-trans retinoic acid.

[0036] Peripheral blood was collected from adult lupus erythematosus patients who voluntarily donated blood. Systemic lupus erythematosus is a typical autoimmune disease. Although the etiology is complex, immune regulatory dysfunction plays an important role in the pathogenesis of lupus. Due to the impairment of the body's immune regulation function, normal autoimmune tolerance cannot be maintained, so many self-antigens become target antigens, autoreactive T cells and B cells increase, and excessive autoantibodies are produced, resulting in damage to organs and tissues. Compared with normal people, the ratio of regulatory T cells is lower, and the level of TGF-β is lower than normal people. Therefore, the...

Embodiment 3

[0040] Example 3 Comparison of the culture effects between the medium of the present invention and the common regulatory T cell medium of the present invention without adding bone morphogenetic protein-4 and all-trans retinoic acid.

[0041] Similarly, Foxp3CD4+CD25+CD127-regulatory T cells were isolated from the peripheral blood of adult lupus erythematosus patients, and the culture method was as in Example 1. After three weeks of culture, the present invention had 20 more cells than the culture medium without BMP-4 and ATRA. times,

[0042] The purity ( Figure 4 ) compared with the purity of normal culture medium ( Figure 5 ) high, CD4+CD25+T cells 94.3% after culture in the present invention, among them the cells of Foxp3+ expression in the double-positive cells are 98.7% ( Figure 4 ), 90.8% of CD4+CD25+ T cells cultured in the medium without BMP-4 and ATRA, among which the cells expressing Foxp3+ in the double positive cells were 93.0% ( Figure 5 ).

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Abstract

The invention provides an adult regulatory T cell in-vitro amplification culture medium and an application method thereof. The adult regulatory T cell in-vitro amplification culture medium comprises transform growth factor-beta (1-4 ng / ml), bone morphogenesis protein 4 (8-12 ng / ml), recombinant human interleukin-2 (300-500U / ml), rapamycin (80-100nM / ml), al-trans vitamin A acid (1-4 uM / mL), 4-hydroxyethylpiperazinoethylsulfonic acid (20-30mM / ml), L-glutamine (2mM / ml), 2-mercaptoethanol (40-50uM / ml), 5% human AB type serum, CD3CD28 magnetic bead, penicillin (50U / ml) and streptomycin (50ug / ml). When being used for performing amplification culture and induced differentiation on regulatory T cells, the culture medium can shorten the amplification and differentiation time of the regulatory T cells, and can obtain the high-purity Foxp3CD4+CD25+CD127-regulatory T cells. Therefore, the regulatory T cells can be used in the aspect of clinical treatment of anti-transplantation immunity rejection, autoimmune disease, allergic disease and the like to perform a novel clinical cell therapy.

Description

technical field [0001] The invention belongs to the technical field of cell culture, and in particular relates to a culture medium for in vitro expansion of adult Treg cells and a use method thereof Background technique [0002] Regulatory T cells are a class of cell populations that control autoimmune reactivity in the body and are an important part of the body's maintenance of self-tolerance. Treg cells are currently known to be the most powerful CD4+ T cell subsets in the human body that can exert immunosuppressive functions. % to 10%, but it is closely related to tumors, autoimmune diseases, and graft acceptance. In addition to expressing CD4 and CD25 molecules, Treg cells also highly express Foxp3, and CD127 can also be used as a specific marker of this group of cells. There is a good correlation between the high expression of Foxp3 and the low expression of CD127. Foxp3 is a forked head transcription factor A member of the family is considered to be a marker molecul...

Claims

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Application Information

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IPC IPC(8): C12N5/0783
Inventor 王维易受南暨明董琼
Owner HUNAN XENO LIFE SCI
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