Method and medium for amplifying bone-marrow mesenchymal stem cells and application

A bone marrow mesenchymal and stem cell technology, applied in the field of bioengineering, can solve the problems of limiting the research and clinical application of BMSCs, deterioration of biological activity and function, and decreased proliferation ability, achieving good application prospects and promoting the formation and proliferation of blood vessels. Ability Increase Effect

Pending Publication Date: 2019-08-27
SOUTH CHINA INSTITUDE OF BIOMEDICINE +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the content of BMSCs in the bone marrow is very small, and as the age of the donor increases, the number of BMSCs in the body gradually decreases, and the proliferative ability decreases, and the biological activity and function deteriorate during the conventional culture in vitro. To a certain extent, it limits the further research and clinical application of BMSCs

Method used

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  • Method and medium for amplifying bone-marrow mesenchymal stem cells and application
  • Method and medium for amplifying bone-marrow mesenchymal stem cells and application
  • Method and medium for amplifying bone-marrow mesenchymal stem cells and application

Examples

Experimental program
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Effect test

Embodiment 1

[0060] 1. Isolation of rat BMSCs

[0061] 1) Male SD rats (purchased from the Experimental Animal Center of Guangzhou University of Traditional Chinese Medicine) were killed with ether, soaked in 75% alcohol for 5 minutes; a surgical towel was placed in the ultra-clean table, and the skin of the rat was cut in a sterile tray.

[0062] 2) Use sterile scissors and other surgical instruments to separate the rat femur, and use a syringe to absorb α-MEM culture solution (purchased from GIBCO) and repeatedly rinse the bone marrow cavity until the color turns white (about 4-5 times).

[0063] 3) Filter the washing solution with a 400-mesh sieve, centrifuge, discard the supernatant, and collect the cells.

[0064] 4) Add 5mL of rat lymphocyte separation medium with a density of 1.077g / mL (purchased from Tianjin Haoyang Company) into a centrifuge tube, resuspend the bone marrow cell pellet with 5mLα-MEM culture medium, and then lightly Lightly added to the rat lymphocyte separation me...

Embodiment 2

[0080] 1. Functional identification of BMSCs after combined treatment with bFGF+VEGF factors

[0081] 1) Take the BMSCs obtained from the bFGF treatment group, the bFGF+VEGF treatment group and the control group respectively in Example 1, digest and collect the cells by blowing and centrifuging.

[0082] 2) After resuspending with PBS, divide into 4 equal portions and add to 4 1.5mLEP tubes respectively.

[0083] 3) Centrifuge and remove the supernatant.

[0084] 4) Add antibodies to each tube, and incubate at 4 degrees for 30 minutes. Add 2 μL of PE-CD90 (purchased from Biolegend), PE-CD45 (purchased from Biolegend), and FITC-CD105 (purchased from Millipore) respectively, the system is 100 μL), and the other is the control group.

[0085] 5) After washing with PBS, the surface molecules of BMSCs were detected by flow cytometry (Becton Dickinson, USA). The expression results of the cell surface molecules are shown in Table 1.

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Abstract

The invention relates to the technical field of bioengineering, and particularly relates to a method and a medium for amplifying bone-marrow mesenchymal stem cells and application. The method for amplifying bone-marrow mesenchymal stem cells comprises the steps of treating the bone-marrow mesenchymal stem cells with bFGF and VEGF to amplify the bone-marrow mesenchymal stem cells. The bFGF and VEGFare added to the medium. The bone-marrow mesenchymal stem cells cultured by the method provided by the invention can increase the proliferation ability of the cells, and maintain the biological characteristics and functions of the cells, and also can increase the expression rate of the cell surface molecule CD105, thereby being useful for transplant treatment of acute myocardial infarction, chronic myocardial infarction, and ischemic heart disease caused by cardiac failure.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a method, culture medium and application for expanding bone marrow mesenchymal stem cells. Background technique [0002] Bone marrow mesenchymal stem cells (BMSCs) are a group of heterogeneous cells existing in the bone marrow, which have the ability of self-renewal and multi-directional differentiation potential, and can differentiate into osteoblasts, chondrocytes, myocardial Cells, adipocytes, endothelial cells, etc., and can form a variety of connective tissues, such as skeletal muscle, tendon, ligament, dermis, fat, and bone marrow matrix, etc., which have a huge role in tissue regeneration, wound repair, and the treatment of various diseases. potential. [0003] BMSCs are a type of adult stem cells that have been studied more deeply. The morphology of BMSCs cultured in vitro is fusiform or spindle-shaped, and the growth pattern is generally swirl-like and fish-like...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
CPCC12N5/0663C12N2501/115C12N2501/165
Inventor 周军年裴雪涛黄媛媛岳文周月环
Owner SOUTH CHINA INSTITUDE OF BIOMEDICINE
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