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In-vitro amplification optimization method for regulatory T cells derived from cryopreserved umbilical cord blood

An optimization method, the technology of umbilical cord blood, applied in the field of cell culture, can solve the problem of low purity of Tregs, and achieve the effect of high purity, good in vitro inhibitory function, and inhibition of in vitro proliferation

Pending Publication Date: 2021-10-29
SHANDONG QILU STEM CELL ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the purity of Tregs obtained by the latter induction culture method was low (CD4 + CD25 + CD127 - ratio of about 60%)

Method used

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  • In-vitro amplification optimization method for regulatory T cells derived from cryopreserved umbilical cord blood
  • In-vitro amplification optimization method for regulatory T cells derived from cryopreserved umbilical cord blood
  • In-vitro amplification optimization method for regulatory T cells derived from cryopreserved umbilical cord blood

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Pre-coat the culture dish with Anti-Human CD3 antibody: prepare Anti-Human CD3 antibody working solution (5μg / mL) with PBS buffer, take 5.5mL Anti-Human CD3 antibody working solution to coat a φ100mm culture dish, and let stand at 37°C for 4h Or coat overnight at 4°C, discard the liquid, gently rinse the culture dish once with PBS buffer, dry the bottom of the culture dish on the operating table, and set aside.

[0029] A method for optimizing the in vitro expansion of regulatory T cells derived from cryopreserved umbilical cord blood, comprising the following steps:

[0030] (1) Separation of total nucleated cells: After resuscitating the frozen cord blood, centrifuge and wash with PBS buffer containing 1-3% human serum albumin, discard the supernatant, and set the centrifugation temperature at 4°C, 1500rpm Centrifuge for 10 minutes to obtain total nucleated cell TNCs, separate and wash again to obtain total nucleated cell TNCs precipitate, and the recovery operation i...

Embodiment 2

[0034] Harvest the expanded regulatory T cells (Treg cells), resuspend them with PBS buffer, count them, take some cells and filter them through a 40-100 μm cell mesh, centrifuge, discard the supernatant, resuspend them with PBS buffer again, and adjust Cell density 1-2×10 6 / mL. Take a number of new flow tubes and mark them, divide them into blank tubes, isotype control tubes and test tubes, add 100 μL of cell suspension to each tube, and then add flow cytometry antibodies as required: Anti-human CD4-FITC, Anti-human CD25-APC, Anti-human CD127-PE, isotype antibodies, etc., after mixing, incubate at room temperature in the dark for 15-20 minutes or at 4°C in the dark for 30 minutes. After incubation, centrifuge and wash 1-2 times with PBS buffer, resuspend cells with 200 μL PBS buffer, add 200 μL sheath fluid, and perform flow cytometry detection on the machine. The result is as figure 1 As shown, CD4 in initial frozen cord blood TNCs + CD25 + The ratio is only 0.45% ( f...

Embodiment 3

[0036] Detection of the function of the expanded regulatory T cells in inhibiting the proliferation of effector T cells in vitro

[0037] Adult peripheral blood mononuclear cells (PBMCs) were obtained by density gradient centrifugation using Ficoll lymphocyte separation medium, and CD4 was obtained by separation and purification by immunomagnetic beads + CD25 - T cells were used as effector T cells (Teff), stained with CFSE, according to 1×10 5 Each well was inoculated in 96-well culture plates pre-coated with Anti-human CD3 antibody, and the Tregs:Teff ratios were 0:1, 1:1, 1:2 and 1:5. For the harvested regulatory T cells after expansion, the culture medium was RPMI1640+10% FBS+2000IU / mL IL-2+100ng / mL Anti-human CD28, and the final volume of culture medium added to each well was 200 μL. Placed at 37°C, 5% CO 2 and under saturated humidity conditions for 4 days. After co-cultivation, all the cells in the 96-well culture plate were collected, and the results were analyzed ...

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Abstract

The invention discloses an in-vitro amplification optimization method for regulatory T cells derived from cryopreserved umbilical cord blood. The method comprises the following steps of: resuscitating the cryopreserved umbilical cord blood, and then performing centrifugal washing to obtain total karyocytes; then inoculating the total karyocytes into a culture dish pre-coated with an Anti-human CD3 antibody, and adding an induction medium for induction culture; and performing CD4 magnetic bead sorting and purification on the eighth day of culture to obtain CD4+ regulatory T cells, and then continuing second-stage induction culture amplification to finally obtain high-purity regulatory T cells. The method provided by the invention can obtain the high-purity regulatory T cells from the cryopreserved umbilical cord blood through in-vitro amplification optimization. The method is also suitable for in-vitro amplification optimization culture of the regulatory T cells of fresh umbilical cord blood.

Description

technical field [0001] The invention relates to the technical field of cell culture, in particular to an optimization method for in vitro expansion of regulatory T cells derived from cryopreserved umbilical cord blood. Background technique [0002] CD4 + CD25 + CD127 - Regulatory T cells (Tregs) are a subset of T cells that can exert immunosuppressive responses in a variety of ways in vivo, including direct contact with effector T cells, inhibiting the maturation and presentation of antigen-presenting cells (APCs). It is active, secretes anti-inflammatory factors IL-10, IL-35 and TGF-β1, etc., exerts immunosuppressive effect through perforin, granzyme, etc. [0003] CD4 in cord blood + CD25 + Tregs cells are an independent cell subset, mainly CD4 + CD25 high CD127 low cell. CD4 in cord blood + CD25 + The proportion of Tregs (0.35%-9.07%) was higher than that of adult peripheral blood (1.64%-6.45%). Study finds freshly isolated CD4 in cord blood + CD25 + The inh...

Claims

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Application Information

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IPC IPC(8): C12N5/0783
CPCC12N5/0637C12N2501/515C12N2533/50C12N2501/2302C12N2501/51C12N2501/15
Inventor 于丽丽王龙张倩倩庄肃静雒猛卢正海马琛孙旭燕
Owner SHANDONG QILU STEM CELL ENG
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