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A method and application of umbilical cord blood Treg cell expansion in vitro based on trophoblast cells

A technology of trophoblast cells and cell bodies, which is applied in the field of biomedicine, can solve the problems of reduced cell activity, unfavorable clinical application, unfavorable expansion of Treg cells in vitro, etc., and achieves low immunogenicity, reduced quality fluctuations, and enhanced activity.

Active Publication Date: 2021-11-02
成都云测医学生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the process of amplification, the expression of FoxP3 is also prone to instability, resulting in decreased cell viability, which is not conducive to clinical application
At the same time, due to the abnormal number and function of Treg cells in patients with autoimmune diseases, it is very unfavorable for the in vitro expansion of autologous Treg cells.

Method used

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  • A method and application of umbilical cord blood Treg cell expansion in vitro based on trophoblast cells
  • A method and application of umbilical cord blood Treg cell expansion in vitro based on trophoblast cells
  • A method and application of umbilical cord blood Treg cell expansion in vitro based on trophoblast cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Prepare umbilical cord blood Treg cells according to the following steps, including the following steps:

[0058] Step 1: Collect 20 mL of fresh umbilical cord blood, dilute it with 2 times the volume of PBS, and then use density gradient centrifugation to separate umbilical cord blood mononuclear cells to obtain 6.2×10 7 cells.

[0059] Step 2: Adjust the mononuclear cells to 1 x 10 with culture medium 6 cells / mL, inoculate it into the culture flask; specifically, take 5×10 7 Cord blood mononuclear cells were resuspended in 50 mL X-VIVO 15 medium and inoculated into T175 culture flasks, and 1×10 8 CD3 / CD28 immunomagnetic beads (the ratio of magnetic beads to cells is 2:1); add human AB serum and IL-2 at the same time; after adding human AB serum is 5 vol%, the final concentration of IL-2 is 500 IU / mL.

[0060] The 3rd generation umbilical cord Wharton's jelly mesenchymal stem cells were resuscitated as trophoblast cells and inoculated with 1×10 7 Cells were co-...

Embodiment 2

[0074] Cord blood Treg cells were prepared according to the same steps as in Example 1, except that the final concentration of each component after adding the optimized amplification factor in step 5 and step 6 was: human AB serum was 10 vol%, and the concentration of IL-2 was 1000 IU / mL, the concentration of rapamycin was 100 nmol / L, the concentration of RARA agonist was 10 nmol / L, and the concentration of DNA methyltransferase inhibitor was 10 μmol / L. The results show that the phenotype of Treg cells in this example is similar to that of Example 1, in which CD4 + CD25 + The positive rate was greater than 90%, and the positive rate of FoxP3 was greater than 80%. On the 14th day, the expansion factor of Treg cells was 3059.

Embodiment 3

[0076] Cord blood Treg cells were prepared according to the same steps as in Example 1, except that the final concentration of each component after adding the optimized amplification factor in step 5 and step 6 was: human AB serum was 5 vol%, and the concentration of IL-2 was 300 IU / mL, the concentration of rapamycin was 100 nmol / L, the concentration of RARA agonist was 1 nmol / L, and the concentration of DNA methyltransferase inhibitor was 1 μmol / L. The results show that the phenotype of Treg cells in this example is similar to that of Example 1, in which CD4 + CD25 + The positive rate was greater than 90%, and the positive rate of FoxP3 was greater than 80%. On the 14th day, the expansion factor of Treg cells was 1446.

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Abstract

The invention discloses an in vitro amplification method and application of umbilical cord blood Treg cells based on trophoblast cells. The specific technical method is as follows: firstly, using umbilical cord Wharton's jelly mesenchymal stem cells as trophoblast cells to induce Treg in umbilical cord blood mononuclear cells Initial proliferation of cells; then purer Treg cells were obtained by magnetic bead sorting; finally, optimized expansion factors were used to stimulate the rapid expansion of Treg cells. The present invention adopts human AB plasma, IL-2, rapamycin, RARA agonist and DNA methyltransferase inhibitor as optimized amplification factors, and can prepare a large amount, high purity and high activity within 2 weeks. Strong cord blood Treg cells. Using umbilical cord blood as the raw material for Treg cell expansion can be prepared in batches, and can reduce the quality fluctuation of Treg cells caused by individual differences in samples. Umbilical cord blood Treg cells have low immunogenicity and can be used as general-purpose cells for clinical research, such as autoimmune diseases and graft-versus-host disease.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a trophoblast cell-based umbilical cord blood Treg cell expansion method in vitro and its application. Background technique [0002] Regulatory T cells (Treg) are CD4 + An important cell subset in T cells, Treg cells play an important role in inducing the body's immune tolerance, maintaining the homeostasis of the immune environment, and preventing the occurrence of autoimmune diseases. Shimon Sakaguchi's team first identified CD25 (IL-2 receptor α-chain, IL-2RA) as a surface marker of Treg cells in 1995, and researchers found that CD4 would be removed + CD25 + Transplantation of subsets of lymphocytes into nude mice can cause a variety of autoimmune diseases, while infusion of CD4 + CD25 + Treg cells can inhibit the occurrence of disease. At the same time, Treg cells also specifically express the transcription factor FoxP3, which plays an important regulatory role in th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0783A61K35/17A61P37/02A61P37/06A61P29/00A61P19/02A61P17/06A61P1/00
CPCA61K35/17A61P1/00A61P17/06A61P19/02A61P29/00A61P37/02A61P37/06C12N5/0637C12N2500/84C12N2501/04C12N2501/2302C12N2501/385C12N2501/51C12N2501/515C12N2501/72C12N2502/1388
Inventor 陈勇军刘少先
Owner 成都云测医学生物技术有限公司
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