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In-vitro amplification method of cord blood Treg cells

An in vitro expansion and cell technology, applied in the field of in vitro expansion of cord blood Treg cells, can solve the problems of unsatisfactory expansion multiples and expansion cycles, cumbersome operations, increased cell pollution, etc., to achieve immunosuppressive functions, simplifying Amplification method, the effect of a wide range of cord blood sources

Active Publication Date: 2017-09-15
深圳市沃英达生命科学有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods reported so far require purification of cells with immunomagnetic beads before in vitro amplification, which is cumbersome to operate and increases the possibility of cell contamination, which is not conducive to large-scale clinical application.
Moreover, existing studies have shown that CD4+CD25-T cells can be transformed into CD4+CD25+Tregs under the induction of interleukin 7, interleukin 15 and estrogen, etc. Pre-purification, complicated operation
[0006] In addition, there are other limitations in the clinical application of adult peripheral blood Tregs. For example, in patients with autoimmune diseases, the number of cells may be significantly reduced or dysfunctional, so it is impossible to extract their own Tregs for treatment

Method used

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  • In-vitro amplification method of cord blood Treg cells
  • In-vitro amplification method of cord blood Treg cells
  • In-vitro amplification method of cord blood Treg cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] The process of separating cord blood mononuclear cells is as follows: neonatal cord blood is collected from full-term healthy newborns in the Department of Obstetrics and Gynecology. After the fetus is delivered, 100ml of blood is collected from the placenta umbilical vein and placed in a blood collection bag containing citric acid anticoagulant. The blood was sent to the laboratory for processing within 4 hours after collection. Take 100ml of cord blood and centrifuge at 750g / min for 15min. Collect the upper layer of plasma, inactivate the plasma at 56°C for 30 min, centrifuge at 2500 rpm / min and use it for later use; add an equal volume (1:1) of PBS to dilute the remaining blood cells, and separate mononuclear cells by conventional density gradient centrifugation.

Embodiment 2

[0042] Flow cytometric detection of the cell ratio in mononuclear cells: Flow cytometry detects the ratio of CD4+CD25-T cells and CD4+CD25+Tregs in the mononuclear cells isolated in Example 3.

[0043] Collect the separated mononuclear cells, each group of 1x10^6 cells, centrifuge at 750g room temperature for 5min, discard the supernatant; 2ml PBS repeated washing cells 3 times, resuspend the cells with 100ul PBS; and add 10ul APC-CD4 and 10ul PE-CD25 antibody double staining was used as the test group, and 10ul APC-CD4 and 10ul PE-CD25 antibody single staining groups were set for fluorescence compensation, and the unstained cell group was used as the negative background. Incubate in the dark at 4°C for 30 min. After incubation, add 2ml PBS to each tube, centrifuge at 750g for 5min at room temperature, and discard the supernatant. After repeated washing with 2ml PBS for 2 times, add 200ulPBS to resuspend the cells and test on the machine.

[0044] The results showed that the rati...

Embodiment 3

[0046] An in vitro expansion method of cord blood Treg cells, the extraction method comprising the following steps;

[0047] S1: Separate mononuclear cells: Take 100ml of cord blood, centrifuge at 800g / min for 10min, collect the upper plasma, inactivate the plasma at 54°C for 40min, centrifuge at 2500rpm / min, and use it for later use; then add an equal volume of PBS to dilute the remaining blood cells , Use conventional density gradient centrifugation to separate cord blood;

[0048] S2: Adjust the cell density to 0.5x10 with RPMI1640 6 / ml, 5ml per bottle is inoculated into T25 culture flasks; inactivated plasma is added to make the final plasma concentration of 5.5%.

[0049] S3: The first day: add the estradiol, TGF-β, CD3 / CD28 monoclonal antibody and IL-2, and make the final concentration of each component: 100ng / ml of the estradiol, 5ng / ml of TGF-β , CD3 / CD28 monoclonal antibody 2μg / ml, IL-2 2000U / ml;

[0050] S4: Fourth day: Add 3ml RPMI1640, add inactivated plasma, estradiol, ...

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Abstract

The invention relates to an in-vitro amplification method of cord blood Treg cells, comprising the steps of S1, isolating mononuclear cells; S2, adjusting cellular density, inoculating to a culture flask, and adding inactivated plasma; on the first day, adding coumestrol, TGF-beta, CD3 / CD28 monoclonal antibody and IL-2; S3, on the fourth day, supplementing a culture medium, adding inactivated plasma, coumestrol and TGF-beta, and keeping the final concentration constant; S4, on the sixth day, supplementing the culture medium, and keeping the final concentrations of other components unchanged; S5, on the ninth day, supplementing the culture medium, and keeping the final concentrations of other components unchanged; S6, on the twelfth day, collecting cells. The amplification of CD4, CD25 and Tregs is induced under the coaction of coumestrol and TGF-beta, the amplifying efficiency is high, and high-purity Treg cells can be acquired without pre-purification.

Description

Technical field [0001] The invention relates to the field of biotechnology, in particular to an in vitro expansion method of cord blood Treg cells. Background technique [0002] Regulatory cells (Regulatory cells, Tregs for short) are a type of T cell subgroup that can control autoimmune reactivity in the body discovered in recent years. Because of their immunosuppressive effects, they were also called suppressor Tcells in the early stage. Tregs are a subset of CD4+ T cells that highly express CD25 and depend on IL-2 for survival. According to the different sources of Tregs, there are two types of CD4+CD25+Tregs, natural Tregs (nTregs) and inducible Tregs (iTregs). nTregs develop in the thymus and iTregs develop in the periphery. What we usually call regulatory T cells refer to nTregs that develop in the thymus. So far, people have been studying Tregs for more than 20 years. Although the factors affecting the development of Tregs in the body are still not very clear, the immun...

Claims

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Application Information

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IPC IPC(8): C12N5/0783
CPCC12N5/0637C12N2501/15C12N2501/2302C12N2501/392C12N2501/51C12N2501/515
Inventor 林词雄王旭李陶林洁璇朱刚
Owner 深圳市沃英达生命科学有限公司
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