An enabling mesenchymal stem cell additive and its application

A technology of MSCs and additives, applied in the field of energized mesenchymal stem cells additives, can solve the problems of hindering the commercialization of MSCs, loss of MSCs differentiation potential, slow cell growth, etc., and achieve strong proliferation ability and multi-directional differentiation potential. , Improve autophagy and proliferation ability, overcome the effect of cell senescence

Active Publication Date: 2022-08-05
珠海贝索细胞科学技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, limited by existing technologies, there are still some problems hindering the commercialization of MSCs
For example, under normal physiological conditions in vivo, most MSCs are in the quiescent phase, but when cultured in vitro, although MSCs can rapidly divide and proliferate due to being separated from the in vivo environment, as the number of passages increases, the MSCs cultured in vitro gradually appear as senescent cells. The characteristics of MSCs, that is, slow cell growth, nuclear pyknosis, nuclear fragmentation, and falling off from the bottom of the culture bottle. Cell senescence will lead to the loss of differentiation potential and self-differentiation of MSCs, which will limit its application and reduce its economic benefits.

Method used

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  • An enabling mesenchymal stem cell additive and its application
  • An enabling mesenchymal stem cell additive and its application
  • An enabling mesenchymal stem cell additive and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] (1) Prepare additives

[0034]The Energizing Mesenchymal Stem Cell Supplement consists of the following concentrations of components: Wnt3a 100ng / mL, Wnt3b 50pg / mL, Wnt8c 50pg / mL, BMP Protein 10pg / mL, Rapamycin 10nmol / L, Pluripotin 5nmol / L, TGF -β1 50IU / mL, FGF-2 100IU / mL, β-catenin 20IU / mL, PDGF 10IU / mL, PI3K 100IU / mL, heat shock protein 2ng / mL, chitosan 0.5μg / mL, glycosaminoglycan 2μg / mL, cold agglutinin 1μg / mL, integrin β1 2μg / mL and thrombin 2μg / mL, after the configuration is completed, added to the MSC-T4 medium at a ratio of 1:50;

[0035] In the control group, an equal proportion of FBS with a concentration of 10% by mass was added.

[0036] (2) Stem cell recovery

[0037] 1. Preheat the constant temperature automatic heater to 37°C;

[0038] 2. Prepare a 50mL centrifuge tube, take the stem cell complete medium according to the ratio of frozen cell solution: stem cell complete medium = 1:9, and preheat it to 37°C;

[0039] 3. Take out the frozen stem cells f...

Embodiment 2

[0049] (1) Prepare additives

[0050] 1. The enabling mesenchymal stem cell supplement is composed of the following components: Wnt3a 70ng / mL, Wnt3b 80pg / mL, Wnt8c 60pg / mL, BMP protein 17pg / mL, Rapamycin 10nmol / L, Pluripotin 3nmol / L , TGF-β1 80IU / mL, FGF-2 150IU / mL, β-catenin 30IU / mL, PDGF 18IU / mL, PI3K 120IU / mL, heat shock protein 8ng / mL, chitosan 0.6μg / mL, glycosamine Polysaccharide 2 μg / mL, cold agglutinin 1.8 μg / mL, integrin β1 5 μg / mL and thrombin 3 μg / mL, were added to the MSC-T4 medium at a ratio of 1:50 after the preparation was completed;

[0051] 2. Comparative Example 1: The difference from Example 2 is that the composition of the additive is different, and the Wnt3a, Wnt3b, Wnt8c and BMP proteins in the additive are removed, and the rest are the same as in Example 2;

[0052] 3. Comparative Example 2: The difference from Example 2 is that the components of the additives are different, except that the rapamycin and Pluripotin in the additives are removed, and the r...

Embodiment 3

[0068] (1) Prepare additives

[0069] The Energizing Mesenchymal Stem Cell Supplement consists of the following concentrations of components: Wnt3a 80ng / mL, Wnt3b 60pg / mL, Wnt8c 55pg / mL, BMP Protein 15pg / mL, Rapamycin 15nmol / L, Pluripotin 4nmol / L, TGF -β1 65IU / mL, FGF-2 120IU / mL, β-catenin 35IU / mL, PDGF 16IU / mL, PI3K 150IU / mL, heat shock protein 5ng / mL, chitosan 1μg / mL, glycosaminoglycan 3μg / mL, cold agglutinin 1.5 μg / mL, integrin β1 6 μg / mL, and thrombin 4 μg / mL, were added to the MSC-T4 medium at a ratio of 1:50 after the preparation was completed. (2) Isolation and culture of primary cells (climbing method)

[0070] 1. Take the umbilical cord from healthy newborns who have normal cesarean section on the operating table, soak in MSC-T4 basal medium, store and transport at room temperature or 4°C;

[0071] 2. Take out the umbilical cord from the ultra-clean bench, wash off the residual blood in the umbilical cord with PBS, and cut it into small pieces of 3-4 cm;

[0072] ...

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Abstract

The invention relates to the field of biotechnology, in particular to an energizing mesenchymal stem cell additive and its application. The invention provides an enabling mesenchymal stem cell additive, which comprises Wnt3a protein, Wnt3b protein, Wnt8c protein, morphogenetic protein BMP, rapamycin, Pluripotin protein, TGF-β1 , FGF-2, β-catenin, PDGF, PI3K, heat shock protein, chitosan, glycosaminoglycan, cold agglutinin, integrin β1 and thrombin. A variety of components act synergistically, so that this enabling mesenchymal stem cell additive not only effectively promotes the in vitro proliferation and culture of mesenchymal stem cells, but also overcomes the shortcomings of traditional mesenchymal stem cells that appear cell senescence after multiple passages in vitro. Mesenchymal stem cells have always maintained a strong proliferation ability and multi-directional differentiation potential.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an energizing mesenchymal stem cell additive and its application. Background technique [0002] Mesenchymal stem cells (MSCs) are the most widely studied and used type of stem cells in recent years. The therapeutic effect and clinical application prospect of MSCs are mainly determined by their basic characteristics. [0003] MSCs have the ability of self-replication, multi-directional differentiation, the ability to secrete cytokines and exosomes, as well as low immunogenicity, inhibition of inflammation, immune regulation and homing effects. Fibrosis, anti-apoptosis, hematopoietic support, and participation in or promotion of angiogenesis. These advantages endow it with broad prospects for clinical application. In recent years, clinical research on MSCs has developed rapidly and achieved remarkable results. For Alzheimer's disease, ALS, diabetes, stroke, osteoarthritis, lupus er...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0775
Inventor 孔伟圣蓝欣黄海娟冉红
Owner 珠海贝索细胞科学技术有限公司
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