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Pre-processing culture method for enhancing immune regulation capacity of mesenchymal stem cells

A quality stem cell and immune regulation technology, applied in the field of stem cells, can solve the problems of MSCs quality differences, large differences in immune regulation effects, and effect uncertainty, and achieve the effect of enhancing ability and enhancing immune suppression ability

Inactive Publication Date: 2016-10-05
睿尔(天津)生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, there are still some problems to be solved urgently in the application of MSCs in the field of immune regulation
First of all, due to the differences in sources, methods of cell separation and expansion, and the quality of MSCs, there are also great differences in the quality of MSCs, which leads to great differences in the immune regulation effect of MSCs in different laboratories
In addition, due to the characteristics of cell therapy itself, there are still uncertainties about the effect of MSCs in the treatment of some autoimmune diseases

Method used

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  • Pre-processing culture method for enhancing immune regulation capacity of mesenchymal stem cells
  • Pre-processing culture method for enhancing immune regulation capacity of mesenchymal stem cells
  • Pre-processing culture method for enhancing immune regulation capacity of mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Morphology, differentiation ability and immunophenotype of mesenchymal stem cells

[0032] The second-generation bone marrow-derived mesenchymal stem cells were normally subcultured in vitro to the P2 generation. The basic cell culture medium was DMEM / F12, and 10% fetal bovine serum (volume ratio) was added to the medium. The culture conditions were 37°C, saturated humidity, and static culture in a 5% CO2 incubator. When the cell clone grows to 80% confluence, it is digested with 0.05% trypsin and passaged into a new culture flask. Analyze the cell morphology, differentiation ability, immunophenotype, etc. to determine whether the cells meet the quality requirements.

[0033] It was found that mesenchymal stem cells adhered to the wall in the form of long spindle cells and could form cell clones, such as figure 1 As shown in A. Immunophenotype analysis found that mesenchymal stem cells highly expressed adhesion molecules, such as integrin (CD29); receptor m...

Embodiment 2B

[0034] Example 2 BrdU absorption method detects that mesenchymal stem cells inhibit lymphocyte response in vitro

[0035] Three different batches of mesenchymal stem cells were divided into 0, 1×10 4 , 2×10 4 , 4×10 4 and 8×10 4 Seed in a 24-well plate at a density of / well, make 3 parallel wells for each of the same cells, and add 8×10 4 Peripheral blood lymphocytes (PBLC) were stimulated with PHA at the same time and co-cultured for 48 hours. The BrdU method was used to detect the proliferation of PBMCs after co-cultivation with MSCs in the above groups, and the proliferation amount was represented by OD value (A450nm-A690nm).

[0036] Such as figure 2 As shown, peripheral blood lymphocytes can proliferate significantly after being stimulated by PHA-P (group without MSC), however, after co-cultured with MSCs, lymphocyte proliferation is obviously inhibited. And the inhibition rate of lymphocyte proliferation is directly proportional to the concentration of MSCs, that ...

Embodiment 3

[0037] Example 3 Detection of Protein Factor Pretreatment MSCs Immunomodulatory Ability

[0038]Three different batches of MSCs were cultured in vitro. After the basal culture medium was cultivated to the logarithmic phase, the culture medium was discarded and replaced with a pretreatment medium (DMEM / F12 medium containing 2% fetal bovine serum), and 1000IU / ml IL- 2. Three cytokines, 20ng / ml TNF-α and 15ng / ml IFN-γ, were added singly or in combination of three factors or three factors were added to the pretreatment medium at the same time, and the MSCs were pretreated for 48 hours Afterwards, the above groups of pretreated or untreated BM-MSCs were divided into 1×10 4 Seed in a 24-well plate at a density of / well, make 3 parallel wells for each of the same cells, and add 8×10 4 Peripheral blood lymphocytes (MSC:PBLC ratio 1:8) were stimulated with PHA at the same time and co-cultured for 48 hours. The BrdU method was used to detect the proliferation of PBMCs after co-cultiva...

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Abstract

The invention provides a pre-processing method for enhancing immune regulation capacity of mesenchymal stem cells. The mesenchymal stem cells are pre-processed in vitro by virtue of various protein factor combinations, so that an optimum factor proportion is found, and subsequently, the immune regulation capacity of the mesenchymal stem cells is rapidly enhanced. With the application of the short-time pre-processing method disclosed by the invention, the immune regulation capacity of the mesenchymal stem cells to peripheral blood lymphocytes is significantly enhanced; therefore, the culture method is conducive to the improvement of a clinical treatment effect on autoimmune diseases and immune disorder related diseases.

Description

technical field [0001] The invention relates to the technical field of stem cells, in particular to a pretreatment and culture method for enhancing the immune regulation ability of mesenchymal stem cells. Background technique [0002] Mesenchymal stem cells (MSCs) are an important class of adult stem cells derived from the mesoderm in early development. They have a variety of tissue repair capabilities and have been widely used in clinical treatment research. MSCs were originally non-hematopoietic pluripotent stem cells isolated from bone marrow stroma. It has been confirmed that MSCs also exist in adipose tissue, umbilical cord and other tissues. Studies have shown that an important application of mesenchymal stem cells (Mesenchymal Stem Cells, MSCs) is in the field of immune regulation. A large number of studies have confirmed that MSCs can induce immunosuppression in vivo, reduce GVHD and rejection during hematopoietic stem cell transplantation treatment, and have anti-i...

Claims

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Application Information

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IPC IPC(8): C12N5/0775
Inventor 刘广洋
Owner 睿尔(天津)生物科技有限公司
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