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Cord blood regulative T-cell in-vitro amplification method

A regulatory and cell body technology, applied in the direction of blood/immune system cells, animal cells, vertebrate cells, etc., can solve the problems of high cost and biological safety.

Inactive Publication Date: 2018-05-08
SHANDONG QILU STEM CELL ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although Tregs have a high expansion factor in vitro, the cost of traditional Tregs in vitro expansion methods is high, and the biological safety needs to be studied

Method used

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  • Cord blood regulative T-cell in-vitro amplification method
  • Cord blood regulative T-cell in-vitro amplification method
  • Cord blood regulative T-cell in-vitro amplification method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 In vitro expansion and culture of Tregs cells derived from fresh umbilical cord blood

[0027] Coat the culture dish with 5 μg / ml purified anti-CD3 antibody solution: dilute the purified anti-CD3 antibody to 5 μg / ml with PBS buffer, add 6 ml of 5 μg / ml anti-anti-CD3 antibody solution to the 100mm culture dish, Place in a 37°C incubator for 4 hours, and discard the antibody. Rinse the Petri dish once with PBS buffer, and air dry the bottom of the Petri dish in the operating table.

[0028] Umbilical cord blood was taken from the umbilical cord of full-term healthy newborns, anticoagulated with sodium citrate, and separated within 24 hours after collection.

[0029] The umbilical cord blood was diluted with PBS at a ratio of 1:1, slowly added to an equal volume of Ficoll lymphocyte separation medium, centrifuged at 2000 rpm for 20 min, at a speed up of 1, and down in a speed of 1. Collect the mononuclear cell buffy coat into a new 50ml centrifuge tube, centrif...

Embodiment example 2

[0034] Example 2 Flow cytometric detection of cell surface antigens obtained from in vitro amplification of fresh cord blood

[0035] The amplified cells were harvested, resuspended in PBS buffer, and counted. Take some cells through a 100-mesh cell sieve, centrifuge, collect the cell pellet, resuspend the pellet with PBS buffer, and adjust it to 1×10 6 cells / ml. Add 200 μl of cell suspension to each tube, and add 5 μL of flow cytometry antibody. Use FITC-labeled anti-CD4 antibody and PE-labeled anti-CD25 antibody to label cell surface antigens at room temperature for 30 minutes. Cells are treated with permeabilization solution and fixative solution for 1 hour. Afterwards, the intracellular antigen was labeled with Alexa488-labeled anti-Foxp3 antibody for 30 min. The result is as figure 2 showed that CD4 + CD25 + The proportion of T cells was only 0.19% compared to ( figure 2 , 0day), CD4 in cells obtained after expansion + CD25 + The proportion of T cells increased ...

Embodiment 3

[0036] Example 3 Amplified and Obtained Cells Inhibition Effector Cell Proliferation Function Detection

[0037] Mononuclear cells in adult peripheral blood were separated by Ficoll lymphocyte separation medium density gradient centrifugation, and CD4 obtained by immunomagnetic bead separation and purification + CD25 - Cells were used as effector T cells, after staining with CFSE (5 μM), 1×10 5 Cells / well were seeded in anti-CD3 antibody-coated 96-well plates, according to the ratio of Tregs: effector T cells = 0:1, 1:1, 1:2, 1:5, the expanded cells were added to the 96-well plates Fresh umbilical cord blood Tregs cells were added to each well with Gibco RPMI 1640 + 10% FBS + 2000IU / ml IL-2 + 100ng / ml anti-CD28 culture solution, and cultured at 37°C, 5% and saturated humidity for 4 days, 4 The cells in the 96-well plate were collected one day later, and the results were analyzed by flow cytometry. Such as image 3 As shown, Tregs cells obtained by in vitro expansion can ef...

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Abstract

The invention discloses a cord blood regulative T-cell in-vitro amplification method. The method comprises the following steps: separating fresh cord blood by virtue of a ficoll density gradient centrifugal method to obtain a single karyocyte, inoculating a culture dish, which is previously coated with anti-CD3 antibody, with the single karyocyte, adding induction culture liquid, and inducing thein-vitro amplification of Tregs cells. By adopting the method provided by the invention, the Tregs cells with high purity can be obtained from the fresh cord blood by virtue of high-efficient in-vitroamplification.

Description

technical field [0001] The invention relates to an in vitro expansion method of regulatory T cells, in particular to a method for in vitro expansion of umbilical cord blood regulatory T cells. Background technique [0002] CD4 + CD25 + Foxp3 + Regulatory T cells (regulatory T cells, Tregs) are a subset of T cells that can suppress immune responses in various ways, including secreting inhibitory cytokines IL-10 and TGF-β; The cytokine IL-2; contact inhibition on the cell surface. [0003] When the body lacks Tregs or lacks cell function, the body will produce autoimmune diseases, such as rheumatoid arthritis (RA), myasthenia gravis (MG), multiple sclerosis (MS), diabetes, systemic lupus erythematosus ( SLE), etc. Tregs, especially antigen-specific Tregs, have good development prospects in the treatment of autoimmune diseases. Anderton and Bykovskaya et al. Amplified CD4 from peripheral blood of SLE patients in vitro + CD25 + Foxp3 + CD127 low After Tregs were infus...

Claims

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Application Information

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IPC IPC(8): C12N5/0783
CPCC12N5/0637C12N2501/15C12N2501/2302C12N2501/51C12N2501/515
Inventor 付亚茹孙阳阳王洪星曲廷瑜
Owner SHANDONG QILU STEM CELL ENG
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