Mesenchymal stem cell population with high expression of CD106 and/or reduced expression of CD142 as well as preparation method and application of mesenchymal stem cell population

A high-quality stem cell and high-expression technology, which is applied in the field of stem cell culture and biomedicine, can solve the problems of patients with potential risks, uneven ratio of MSCsCD142, high requirements for culture environment and detection environment, etc., and achieve increased cell migration/chemotactic ability , Improving the ability of immune regulation and avoiding the effect of blood coagulation cascade reaction

Active Publication Date: 2022-02-25
铂生卓越生物科技(北京)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is an existing method for improving the expression of purified CD106 positive cells by flow sorting technology, but this method has many defects: first, the cost of sorting antibodies is high, second, it is easy to contaminate and the yield is low and often cannot meet the needs of treatment, Third, the CD106 expression of the sorted CD106 positive cells will also be lost during the in vitro culture and expansion process.
Hypoxia / low oxygen treatment has high requirements on the culture environment and detection environment, and the labor cost and pollution problems caused by flow sorting
However, methods for reducing tissue factor in mesenchymal stem cells have rarely been reported.
[0008] The mesenchymal stem cells isolated by the prior art have strong immunosuppressive ability in vitro, but the curative effect in the clinical treatment of diseases is not uniform. The proportion of the surface antigen CD274 of the isolated MSCs is ≤ 2%, and the proportion of CD106 positive subpopulation is 15 to 40 %, and the proportion of CD142 in MSCs from different tissue sources is not uniform, which may have potential risks to patients when administered intravenously

Method used

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  • Mesenchymal stem cell population with high expression of CD106 and/or reduced expression of CD142 as well as preparation method and application of mesenchymal stem cell population
  • Mesenchymal stem cell population with high expression of CD106 and/or reduced expression of CD142 as well as preparation method and application of mesenchymal stem cell population
  • Mesenchymal stem cell population with high expression of CD106 and/or reduced expression of CD142 as well as preparation method and application of mesenchymal stem cell population

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0162] Example 1. Establish a three-level cell bank process to produce sufficient amounts of cell banks at all levels

[0163] According to the existing technology (such as CN103266081B), the primary cells of the human umbilical cord mesenchymal stem cell group are separated under GMP conditions, and the P2 generation cells pass the inspection and become the original cell bank; the P2 generation cells grow to the P4 generation and pass the inspection. Afterwards, the main cell bank; the P4 generation cells were thawed and inoculated at a certain concentration, and the P5 generation cells were harvested after being treated with inflammatory factors for 22 ± 2 hours. After passing the inspection, they were used as the working cell bank.

[0164] The P2 original cell bank inspection includes but is not limited to: cell morphology, cell number and viability, cell phenotype, differentiation ability, genetic markers, sterility, mycoplasma, endotoxin, special human virus, immunologica...

Embodiment 2

[0167] Example 2. Phenotype changes of CD106, CD274, CD80 and CD142 after umbilical cord mesenchymal stem cells were treated with inflammatory factors

[0168] Training method:

[0169] 1) Thawing and resuscitating: thaw the P4 main bank cell bank in a water bath at 37 ~ 40°C, and resuspend the cell suspension in buffer (the buffer here can be PBS, normal saline, or normal saline containing 1% human serum albumin) , the resuspension ratio is at least 2 times buffer, 300 ~ 500 × g, centrifuge for 8 - 10 min, discard the supernatant and add Sanli culture medium;

[0170] 2) Culture: according to 5000 ~ 6000 cells / cm 2 Inoculate in T75 cell culture flasks, place the culture flasks at 37°C, 5% CO 2 cultivated in an incubator;

[0171] 3) Add inflammatory factors to continue culturing: After 48 ± 2 h, replace the Sanli MSC serum-free medium containing 10 ng / mL IFN-γ or / and TNF-α (see Table 1 for grouping information), 22 ± 2 h After harvesting the cells separately, take 2 × 10 ...

Embodiment 3

[0181] Example 3: Changes in cell phenotype before and after cryopreservation of hUC-MSCs treated with inflammatory factors

[0182] Culture method: inoculate, process and harvest cells according to the method of Example 2. Cell pellets after harvesting were taken in 2 × 10 6 One cell was analyzed by flow cytometry, and the rest were cryopreserved. Phenotypes were detected after being frozen in liquid nitrogen for two months.

[0183] result:

[0184] Table 3: Detection results of cell phenotype after cryopreservation and thawing

[0185]

[0186] Such as image 3 , 4 As shown in Table 3, there was no significant change in the phenotypes of CD106, CD274, CD80, and CD142 before and after cryopreservation, indicating that various phenotypes were stably expressed after treatment with inflammatory factors.

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Abstract

The invention provides a mesenchymal stem cell population with high expression of CD106 and/or reduced expression of CD142, a preparation method and application of the mesenchymal stem cell population, and particularly, the expression quantity of CD106 of the mesenchymal stem cell population cultured by inflammatory factors (IFN-gamma and/or TNF-alpha) is increased, the expression quantity of tissue factors (CD142/TF) is reduced, and the expression quantity of CD274 is increased while the expression quantity of CD80 is not changed. By utilizing the mesenchymal stem cell population, the blood coagulation cascade reaction excited by tissue factors can be reduced, and the immunosuppression capability and the cell migration/chemotaxis capability can be enhanced.

Description

technical field [0001] The invention relates to the fields of stem cell culture and biomedicine. Specifically, it relates to a group of mesenchymal stem cells with high expression of CD106 and / or reduced expression of CD142, its preparation method, culture and application thereof. After the mesenchymal stem cell population is cultured with inflammatory factors (IFN-γ and / or TNF-α), the expression of the immunoregulatory factor CD106 increases, and / or the expression of CD142 tissue factor (CD142 / TF) decreases, optional , At the same time, the expression of immunoregulatory factor CD274 and migration / chemotactic ability of mesenchymal stem cells increased, but the expression of CD80 did not change. Background technique [0002] Mesenchymal Stem Cells (Mesenchymal Stem Cells, MSCs) are adult stem cells that originate from the mesoderm and have strong self-renewal ability and multilineage differentiation potential. Mesenchymal stem cells have the ability to promote angiogenesi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775A61K35/28A61P37/02A61P29/00
CPCC12N5/0662C12N5/0668A61K35/28A61P37/02A61P29/00C12N2501/24C12N2501/25
Inventor 盛宏霞时兆田乐宝玉陈晓颖侯小强吉建飞王凤李梁
Owner 铂生卓越生物科技(北京)有限公司
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