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Improved expansion culture medium for regulatory T cells of human cord blood origin and application method of expansion culture medium

A technology of umbilical cord blood source and culture medium, applied in the direction of blood/immune system cells, animal cells, vertebrate cells, etc., to achieve superior activity and lymphocyte suppression function

Active Publication Date: 2015-02-18
HUNAN XENO LIFE SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In order to meet the requirements of clinical treatment, it is necessary to expand a large number of human umbilical cord blood-derived T regulatory cells with high purity, good activity, strong function and stable yield in a short period of time; and before Treg is really used in clinical treatment on a large scale There are many challenges to be overcome. At present, there is no optimal method for culturing human cord blood-derived Treg cells that can provide a large amount of high-purity, activity, and strong functions. Therefore, how to establish an optimized optimal method for clinical treatment The composition and culture method of Treg medium have become the most urgent problems in the application of T regulatory cells

Method used

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  • Improved expansion culture medium for regulatory T cells of human cord blood origin and application method of expansion culture medium
  • Improved expansion culture medium for regulatory T cells of human cord blood origin and application method of expansion culture medium
  • Improved expansion culture medium for regulatory T cells of human cord blood origin and application method of expansion culture medium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1. Separation and purification of umbilical cord blood regulatory T cells

[0038] The obstetrics department of our hospital collected umbilical vein blood from 30 cases of normal delivery full-term fetuses. Immediately after ligation and cutting of the umbilical cord, blood was collected by umbilical vein puncture and anticoagulated with heparin. The general blood collection volume was 50-100ml.

[0039] Collected into a 50ml centrifuge tube, centrifuged at 2500rpm for 10 minutes, took the upper plasma layer (the umbilical cord blood autologous plasma as the medium component of the present invention) for subsequent use, and diluted the lower blood cell layer with 3 times the volume of PBS. After the blood cells are resuspended, place 10ml of 1.077-1.080g / ml density lymphatic separation solution on the lower layer of a 50ml centrifuge tube, and gently add 40ml of blood cell suspension to the upper layer, with a clear boundary between the two layers, centrifuge at...

Embodiment 2

[0046] Example 2. Conditional expansion of umbilical cord blood regulatory T cells

[0047] 1. Improved group: collect CD4+CD25+ regulatory T cells every 5×10 5 The cells were resuspended in 250μl RPMI1640 conditioned medium containing 10-12% autologous plasma, 800IU / ml IL-2, 50uM 2-ME, 100nM Rapamycin, and CD3CD28 Microbeads with a ratio of 1:3-1:4 to the number of cells were added.

[0048] After culturing for 3-4 days to form cell colonies, break up the cells and use 250μl of RPMI1640 conditioned medium containing 10-12% autologous plasma, 800IU / ml IL-2, 50uM 2-ME, 100nM Rapamycin., and CD3CD28 Microbeads to continue For subwell expansion, the ratio of CD3CD28 Microbeads to cells is 1:2, and the number of cells at the beginning of the subwell is about 2×10 5 pcs / hole.

[0049] After that, divide the wells once every 1-2 days (the conditions for dividing the wells are the same as before), the total expansion time is 3-4 weeks, and the cell expansion times of 7, 14, 21, and...

Embodiment 3

[0054] Example 3. Detection of the phenotype of regulatory T cells in umbilical cord blood by flow cytometry.

[0055] 1.2.1 Collect the CD4+CD25+ regulatory T cells expanded in vitro for 14 days using the medium of the control group and the modified group, and resuspend the cells to 2×10 with PBS 5 cells / 100 μl.

[0056] 1.2.2 Immunofluorescence staining and intracellular factor staining: add CD4-FITC, CD4-PE, CD45-PE-Cy5, CD19-PE-Cy5, CD25-PE-Cy5, HLA-DR-PE, CD62L-PE, Put 5-20 μl each of GITR-PE, CD127-PE, CD45-RO-PE, CD45-RA-PE-Cy5, and CTLA-PE into each well of the present invention, mix well, incubate at 4°C in the dark for 30 minutes, add Wash once with cold PBS, centrifuge at 1600 r / min for 5 min, discard the supernatant, add 0.5 mL of freshly prepared fixation / permeabilization solution if intracellular staining is required, protect from light at 4°C for 60 min, and use 1 mL of fixation / permeabilization buffer After washing once, resuspend the cells in 100 μl, then di...

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Abstract

The invention relates to an improved expansion culture medium for regulatory T cells of human cord blood origin and an application method of the expansion culture medium. According to the expansion culture medium, heparin anticoagulated autologous cord blood plasma accounting for 10%-12% of the volume of a culture medium, CD3-CD28 antibody co-expressed immunomagnetic beads, recombinant human interleukin 2, 2-mercaptoethanol, rapamycin, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) and gentamicin are added into the RPMI (Roswell Park Memorial Institute)1640 culture medium; and then separated cell suspension is inoculated in a 96-well plates with a U-shaped bottom, hole-division expansion can be performed every 1-2 days, and the expansion period is 3-4 weeks. All reagents in the culture system reach the GMP (good manufacturing practice) level or are originated from autologous cord blood, so that risks caused by ingredients of animal origin are avoided, and the regulatory T cells can be used for a third-party unrelated donor and directly applied to clinical disease treatment; and compared with a traditional culture system, Treg cells (the regulatory T cells) expanded by the improved culture medium is excellent in aspects of growth speed, purity, activity, lymphocyte inhibition function and the like, and the Treg cells are expected to be used as the regulatory T cells of the third-party unrelated donor and applied to the clinical disease treatment.

Description

technical field [0001] The invention relates to an improved expansion medium and a culture method in the production process of human umbilical cord blood-derived CD4+CD25+ regulatory T cells. Background technique [0002] Regulatory T cells are a group of T cell subsets that can suppress autoimmune responses, prevent the occurrence of damaging immune pathology, and maintain the immune balance of the body. They can be used to prevent and treat immune rejection, graft-versus-host disease (GVHD) and autoimmunity. disease. CD4+CD25+T cells are the most important group in the regulatory T cell family, which was established by Sakaguchi et al [SakaguchiS, SakaguchiN, AsanoM, et al. .Breakdown of single mechanism of sel-f tolerancecau-sesvarious autoimmune disease[J].J Immunol,1995,155(3):1151-1164.] It was first discovered in 1995 that it is the A chain of IL-2 receptor, which is continuously expressed in Antigen-induced activated T cells account for about 5% to 10% of normal hu...

Claims

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Application Information

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IPC IPC(8): C12N5/0783
Inventor 王维易受南马小倩张娟
Owner HUNAN XENO LIFE SCI
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