Mesenchymal stem cell with immunosuppression and anti-inflammatory functions, and preparation method and application thereof

A stem cell, immunosuppressive technology, applied in biochemical equipment and methods, anti-inflammatory agents, animal cells, etc., can solve the problems of inability to inhibit T cell proliferation, not exerting anti-inflammatory effect as expected, and achieve excellent therapeutic effects, Simple preparation method and good therapeutic effect

Pending Publication Date: 2022-05-06
重庆睿仕康生物医药有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, recent studies have found that IFN-γ is involved in the induction of MSCs immunosuppression is very problematic
Clinically, when IFN-γ pretreated MSCs were used to treat Crohn’s disease, the infused MSCs did not play an anti-inflammatory effect as expected, and when IFN-γ combined with other cytokines (such as TNF-α, IL-1α and IL -1β, etc.) to induce MSCs, sometimes it can not inhibit T cell proliferation but promotes its activation

Method used

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  • Mesenchymal stem cell with immunosuppression and anti-inflammatory functions, and preparation method and application thereof
  • Mesenchymal stem cell with immunosuppression and anti-inflammatory functions, and preparation method and application thereof
  • Mesenchymal stem cell with immunosuppression and anti-inflammatory functions, and preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Example 1 Isolation, cultivation and identification of human umbilical cord mesenchymal stem cells

[0034] Using existing technology to isolate, culture and identify human umbilical cord mesenchymal stem cells from neonatal umbilical cord, the specific steps are:

[0035] 1. Isolation and culture of MSCs

[0036] 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 the ultra-clean workbench with normal saline to remove surface debris, then soak it in alcohol for 1 minute, then transfer it to clean normal saline to wash off the alcohol on the umbilical cord; then cut off both ends of the umbilical cord and remove the umbilical cord as much as possible coagulated blood, and cut off about 2cm of umbilical cord; p...

Embodiment 2

[0045] Example 2 Induction, culture and identification of human umbilical cord mesenchymal stem cells with enhanced immunosuppressive or anti-inflammatory functions

[0046] On the basis of Example 1, hUC-MSCs were jointly induced by ATP and IFN-γ to obtain hUC-MSCs with enhanced immunosuppressive or anti-inflammatory functions, and the specific steps were:

[0047] 1. Preparation of hUC-MSCs with enhanced immunosuppressive or anti-inflammatory function induced in vitro

[0048] 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 75cm 2 culture flasks, and divide the cells into ATP and IFN-γ single and joint treatment gr...

Embodiment 3

[0097] Example 3 Application of Human Umbilical Cord Mesenchymal Stem Cells with Enhanced Immunosuppressive or Anti-inflammatory Function in the Treatment of Collagen-Induced Arthritis Mice

[0098] 1. Establishment and evaluation of collagen induced a4th4itis (CIA) model

[0099] ⑴Preparation of collagen-induced emulsion: Use a homogenizer (5mm or less in diameter) or a stirrer to emulsify 4mg / ml Complete Freund's Adjuvant (CFA) and collagen solution in a three-way tube sealed syringe (use The same is true for booster injections of incomplete Ford's adjuvant IFA). Since heating denatures collagen, which does not induce arthritis, the syringe is clamped to a ring holder and kept cold by immersing it in an ice-water bath.

[0100] (2) Add a maximum of 2.5ml of CFA to the syringe, and then slowly add an equal volume of collagen solution (2mg / ml collagen dissolved in 0.05M acetic acid), and stir slowly (1000-3000rpm) while adding. Note: To ensure a high quality emulsion, the ma...

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Abstract

The invention relates to mesenchymal stem cells with immunosuppression and anti-inflammatory functions as well as a preparation method and application thereof, and the preparation process mainly comprises the following steps: step 1, separating, purifying and culturing mesenchymal stem cells to obtain mesenchymal stem cells of third to eighth generations for later use; step 2, inoculating the third-generation to eighth-generation mesenchymal stem cells obtained in the step 1 into a culture flask, putting the culture flask into an incubator for culturing, and adding ATP and IFN-gamma induction factors in proportion for induction when the cell fusion degree reaches 70%-80%, so as to obtain the mesenchymal stem cells of the third-generation to the eighth-generation mesenchymal stem cells of the third-generation to the eighth-generation mesenchymal stem cells of the third-generation to the eighth-generation mesenchymal stem cells of the third-generation to the eighth-generation mesenchymal stem cells; and step 3, withdrawing the inducer, and continuously culturing to obtain the mesenchymal stem cells with immunosuppression and anti-inflammatory functions. The mesenchymal stem cells are subjected to in-vitro induction by adopting ATP (adenosine triphosphate) and IFN-gamma (interferon-gamma) to obtain the mesenchymal stem cells with enhanced immunosuppression or anti-inflammatory function, and the mesenchymal stem cells are proved to be capable of remarkably improving the treatment effect on autoimmune diseases and inflammatory related diseases.

Description

technical field [0001] The invention relates to a mesenchymal stem cell with immunosuppressive and anti-inflammatory functions, its preparation method and application, and belongs to the field of biotechnology. Background technique [0002] Mesenchymal stem cells (MSCs) are pluripotent stem cells that are widely distributed in tissues such as periosteum, bone marrow, dental pulp, fat, umbilical cord, and placenta, and have powerful tissue regeneration and anti-inflammatory functions. Tissue damage is often accompanied by an inflammatory response. At this time, MSCs can be mobilized to the damaged site, which can not only replace the damaged cells through differentiation, but also promote tissue repair by promoting the differentiation of related cells to strengthen angiogenesis and extracellular matrix remodeling. It can also regulate the inflammatory process by producing immune regulatory factors, correct the unbalanced inflammatory response, form a microenvironment conduciv...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775A61K35/28A61P37/06A61P29/00
CPCC12N5/0662A61K35/28A61P37/06A61P29/00C12N2501/24C12N2500/40
Inventor 邵文陶李伟强周兴龙皎月申沛鑫
Owner 重庆睿仕康生物医药有限公司
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