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Immunosuppression or anti-inflammatory function enhanced PD-L1 positive mesenchymal stem cells (MSCs), induction kit and application

A PD-L1, stem cell technology, applied in anti-inflammatory agents, cell culture active agents, animal cells, etc., can solve problems such as differences in clinical efficacy, immune regulation ability or weak anti-inflammatory function

Inactive Publication Date: 2020-08-11
重庆福美干细胞生物科技发展有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the immunoregulatory ability of MSCs is not constitutive, that is, the immunoregulatory ability or anti-inflammatory function of MSCs in the natural state is weak, and the acquisition of its immunomodulatory or anti-inflammatory function needs to be stimulated by inflammatory cytokines (such as inflammatory cytokines in the inflammatory microenvironment cytokines) induced to "enable"
However, during the occurrence and development of inflammatory diseases, there are huge differences in the number and types of inflammatory cytokines at different development stages of the disease, and these changes seriously affect the activation efficiency of MSCs in the direction of immunomodulatory or anti-inflammatory functions. Therefore, the state of the immune microenvironment of the patient's body becomes an important determinant of the immune regulation or anti-inflammatory function of MSCs and its clinical efficacy, which is also an important reason why there are still huge differences in clinical efficacy among patients with the same disease treated with MSCs

Method used

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  • Immunosuppression or anti-inflammatory function enhanced PD-L1 positive mesenchymal stem cells (MSCs), induction kit and application
  • Immunosuppression or anti-inflammatory function enhanced PD-L1 positive mesenchymal stem cells (MSCs), induction kit and application
  • Immunosuppression or anti-inflammatory function enhanced PD-L1 positive mesenchymal stem cells (MSCs), induction kit and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Isolation, cultivation and identification of human umbilical cord mesenchymal stem cells

[0041] The preparation process of human umbilical cord mesenchymal stem cells was established.

[0042] Isolate, culture and identify human umbilical cord mesenchymal stem cells from neonatal umbilical cord, the specific steps are:

[0043] (1) Isolation and culture of MSCs

[0044] Isolation and culture of human umbilical cord mesenchymal stem cells (hUC-MSCs): Fresh umbilical cord samples collected under sterile conditions (the samples were from healthy puerperas who were negative for infectious diseases such as hepatitis B, hepatitis C, HIV and syphilis and had no genetic diseases) in Wash in normal saline on the ultra-clean workbench, wash the blood and other substances on the surface, transfer it to an alcohol dish to soak for about 1min, and then transfer it to a clean physiological saline dish to wash off the alcohol on the umbilical cord; and put the umbilical c...

Embodiment 2

[0053] Example 2 Induction, culture and identification of human umbilical cord mesenchymal stem cells with enhanced immunosuppression or anti-inflammatory function

[0054] The hUC-MSCs prepared in Example 1 were jointly induced by IFN-γ and TNF-α to obtain hUC-MSCs with positive expression of PD-L1, immunosuppression or enhanced anti-inflammatory function. The specific steps are:

[0055] 1. Preparation of PD-L1-positive immunosuppressive or enhanced anti-inflammatory function hUC-MSCs induced in vitro

[0056] The hUC-MSCs cryopreserved cells obtained in Example 1 were resuscitated, and 4×10 4 cells / cm 2 The cell density was seeded at 75cm 2 Place the culture bottle in an incubator (37°C, 5% CO 2 incubator), when the cultured cells grew to a cell density >80%, they were digested and dissociated with TrypLE and subcultured, and the digested cells were divided into 4×10 4 cells / cm 2 The cell density was seeded at 25cm 2 culture flasks, and divide the cells into IFN-γ (0,...

Embodiment 3

[0067] Example 3 Application of human umbilical cord mesenchymal stem cells with enhanced immunosuppression or anti-inflammatory function in the treatment of mice with collagen-induced arthritis

[0068] 1. Establishment and evaluation of mouse collagen induced arthritis (CIA) model

[0069] A CIA mouse model was constructed, and the CIA mice were evaluated using the paw swelling for arthritis clinical score. Clinical arthritis was assessed using the following system: 0 points, no swelling; 1 point, mild swelling and erythema; 2 points, marked edema; 3 points, joint stiffness. Each limb is scored and aggregated to a maximum possible score of 12 per animal.

[0070] 2. Application of TNF-α combined with IFN-γ-induced immunosuppressive or anti-inflammatory enhanced hUC-MSCs in collagen-induced arthritis mouse model

[0071] 1) Protocol for treating collagen model mice with MSCs: Treat hUC-MSCs with 20ng / ml TNF-α combined with 20ng / ml IFN-γ as used in Example 2 for 24 hours, co...

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Abstract

The invention discloses immunosuppression or anti-inflammatory function enhanced PD-L1 positive mesenchymal stem cells (MSCs), an induction kit and application. The immunosuppression or anti-inflammatory function enhanced PD-L1 positive MSCs are prepared through the following steps: separating and culturing MSCs; and inducing the cultured MSCs with an immune factor induction complexing agent to obtain the immunosuppression or anti-inflammatory function enhanced PD-L1 positive MSCs, wherein the induction complexing agent comprises 5-20 ng / ml IFN-gamma and 0-20 ng / ml TNF-alpha. The MSCs can efficiently express a PD-L1 receptor, and compared with wild type MSCs, the immunosuppression or anti-inflammatory function enhanced PD-L1 positive MSCs have better treatment effects on collagen-induced arthritis mice and CLP sepsis model mice, and have no side effects. The safe and effective immunosuppression or anti-inflammatory function enhanced MSCs have great application potentials in clinical treatment of autoimmune diseases and inflammation related diseases.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a PD-L1-positive mesenchymal stem cell with enhanced immunosuppression or anti-inflammatory function, and its in vitro induction kit, quality assessment method and application. Background technique [0002] With the deepening of stem cell research, mesenchymal stem cells (Mesenchymal Stem Cells, MSCs) have been considered as an ideal tool for the repair and treatment of tissue damage-related diseases. MSCs are a kind of precursor cells with self-renewal and multi-lineage differentiation potential, widely distributed in adult bone marrow, fat, dental pulp, placenta and umbilical cord and other tissues. De Miguel et al. found in their research (Immunosuppressive properties of mesenchymal stem cells: advances and applications.) that MSCs not only have strong self-renewal ability and multidirectional differentiation potential to mesoderm, ectoderm and endoderm, but also have good...

Claims

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Application Information

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IPC IPC(8): C12N5/0775C12Q1/02A61K35/28A61P29/00A61P37/02
CPCA61K35/28A61P29/00A61P37/02C12N5/0665C12N2501/24C12N2501/998G01N33/5005
Inventor 冉启平邵文陶李伟强王光福
Owner 重庆福美干细胞生物科技发展有限公司
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