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Method for detecting immunomodulatory ability of mesenchymal stem cells

A technology of mesenchymal stem cells and detection method, applied in the field of detection of mesenchymal stem cells immune regulation ability, can solve the problem of lack of mesenchymal stem cells immune regulation ability, etc., and achieve the effect of strong immune regulation ability, simple operation and good quality.

Inactive Publication Date: 2019-04-12
TIANJIN CHANGHE BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] There are various methods for preparing mesenchymal stem cells, such as tissue block adherent culture method, umbilical cord homogenate collagenase digestion method and improved collagenase digestion method, etc., but most of the current research focuses on the clinical application of mesenchymal stem cells, and There is no good evaluation mechanism to identify the immunomodulatory ability of mesenchymal stem cells prepared by many preparation methods

Method used

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  • Method for detecting immunomodulatory ability of mesenchymal stem cells
  • Method for detecting immunomodulatory ability of mesenchymal stem cells
  • Method for detecting immunomodulatory ability of mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1 Effect of Mesenchymal Stem Cells on Specific Lymphocyte Subgroups

[0067] The mesenchymal stem cells used in this example are umbilical cord mesenchymal stem cells cultured in vitro without serum and with serum respectively, and bone marrow mesenchymal stem cells cultured in vitro without serum and with serum respectively.

[0068] Mesenchymal stem cells were co-cultured with PBMC, and CD4 was quantified by flow cytometry + CD25 + CD127 - The ratio of Treg to detect CD3 + CD8 - INF-γ + Th1, CD3 + CD8 - IL-17 + Th17 cell proliferation.

[0069] Experimental procedure: Dilute 2mL concentration to 1×10 8 L -1 MSCs were inoculated in 24-well plates (pre-treated with 25 μg / L mitomycin at 37°C for 30 min to inhibit their excessive differentiation), and after they were completely attached to the wall on the third day, 1 mL of 2×10 9 L -1 T lymphocytes were stimulated with PMA (25μg / L) and ionomycin (1g / L) as the experimental group; T cells without stimul...

Embodiment 2

[0073] Example 2 Mesenchymal stem cells inhibit the secretion of TNF-α from lymphocytes

[0074] The mesenchymal stem cells used in this example are the same as those in Example 1.

[0075] Mesenchymal stem cells were divided into 5.0 × 10 4 / hole, 1.0×10 5 / hole, 2.0×10 5 3 concentrations per well, the cell suspension was inoculated in 96-well culture plates according to different concentrations, at 37°C, the volume fraction was 5% CO 2 , cultured in a saturated humidity incubator, after 24 hours, treated with mitomycin at a final concentration of 30 μg / mL at 30°C for 30 minutes, discarded mitomycin, and washed the culture plate with RPMI-1640 culture medium containing 10% FBS once. Add 1.0×10 to each cell well 6 PBMC suspension per well and PHA at a final concentration of 2.5 μg / mL to stimulate lymphocyte proliferation. Take the lymphocytes added with PHA as the control, at 37°C, the volume fraction is 5% CO 2 , cultured in a saturated humidity incubator, and after 72 ...

Embodiment 3

[0079] Example 3 Inhibition test of mesenchymal stem cells on lymphocyte proliferation

[0080] The mesenchymal stem cells used in this example are the same as those in Example 1.

[0081] Mesenchymal stem cells (2.5×10 4 , 5.0×10 4 , 2.5×10 5 , 5.0×10 5 ), seeded in a 96-well plate, and irradiated with cobalt-60 at 3000rad. Then with PBMC (5.0×10 4 ) co-culture, stimulate its proliferation with PHA with a final concentration of 2.5 μg / mL, and take the lymphocytes added with PHA as a control, at 37°C, the volume fraction is 5% CO 2 1. After culturing in a saturated humidity incubator for 68 hours, add 10 μL / well CCK-8 solution and continue to incubate for 4 hours. Measure the absorbance (A) value at 450 nm with a microplate reader to detect the effect of umbilical cord mesenchymal stem cells on the proliferation ability of allogeneic T lymphocytes.

[0082] Umbilical cord and bone marrow mesenchymal stem cells cultured with or without serum were tested for inhibition of ...

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Abstract

The present invention relates to the field of biotechnology, and in particular to a method for detecting the immunomodulatory ability of mesenchymal stem cells. The method for detecting the immunomodulatory ability of the mesenchymal stem cells conducts co-culture on an original sample and the mesenchymal stem cells to be evaluated to obtain a sample to be tested; and in comparison with the original sample, an amount of proportion change of a lymphocyte subpopulation, an amount of change in lymphocyte secretion of TNF-alpha, and an amount of change in lymphocyte proliferation ability in the sample to be tested are detected to evaluate the immunomodulatory ability of the mesenchymal stem cells. The method can evaluate the immunomodulatory ability of a certain mesenchymal stem cell. If the proportion of lymphocyte subpopulation, the amount of change in lymphocyte secretion of TNF-alpha and the amount of change lymphocyte proliferation are greater, the immunomodulatory ability and qualityof the mesenchymal stem cells are better. The detection method provided by the present invention is also suitable for differential administration.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for detecting the immune regulation ability of mesenchymal stem cells. Background technique [0002] Mesenchymal stem cells (MSCs) are a type of pluripotent stem cells, derived from the mesoderm and ectoderm in the early stages of development, and can be isolated from various tissues such as bone marrow, fat, synovium, bone, muscle, and umbilical cord. It has the potential to differentiate into osteoblasts, chondrocytes, adipocytes, bone marrow stroma, nerve cells, liver cells, islet cells and cardiomyocytes. The source is convenient, easy to separate, cultivate, expand and purify, and still has the characteristics of stem cells after multiple passages and expansions, without the characteristics of immune rejection. [0003] Mesenchymal stem cells can not only promote the regeneration and repair of damaged tissues, but also have good immune regulation ability, and can also ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/02
CPCC12N2503/00G01N33/5005
Inventor 徐永胜王秀娟倪琳杨莹
Owner TIANJIN CHANGHE BIOLOGICAL TECH
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