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Induction culture medium and induction method for directional differentiation of human mesenchymal stem cells towards endothelial cells

A technology for induction medium and bone marrow mesenchyme, which is applied in the direction of vascular endothelial cells, cell culture active agents, artificial cell constructs, etc., can solve the problem of not having a stable, efficient and rapid induction scheme, unclear differentiation efficiency of endothelial cells, Trouble and other problems, to achieve good cell growth status, improve the induction effect, and increase the effect of the acquisition ratio

Active Publication Date: 2019-04-23
PEKING UNIV FIRST HOSPITAL
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  • Description
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Problems solved by technology

However, there are differences in the induction schemes used in different studies, and the specific endothelial cell differentiation efficiency is not clear in many studies, and there is no stable, efficient, and rapid induction scheme so far, which also plagues its application. a problem of

Method used

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  • Induction culture medium and induction method for directional differentiation of human mesenchymal stem cells towards endothelial cells
  • Induction culture medium and induction method for directional differentiation of human mesenchymal stem cells towards endothelial cells
  • Induction culture medium and induction method for directional differentiation of human mesenchymal stem cells towards endothelial cells

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Embodiment 1 to Embodiment 8

[0062] Embodiment 1 to embodiment 8 provide a kind of induction medium respectively, and basal medium is IMDM medium, adds the penicillin-streptomycin that volume fraction is 1% to IMDM medium, and other added components are as shown in Table 1 , added to the IMDM medium according to the content of each component in the table below, prepared as an induction medium on ice, placed in a refrigerator at 4°C, and used within two weeks.

[0063] The addition composition and consumption of the induction medium of table 1 embodiment 1 to embodiment 8

[0064]

Embodiment 9

[0066] Isolation and subculture of BMSCs

[0067] Take 10-20ml of human bone marrow blood, fully mix it with an equal volume of PBS, and slowly superimpose it on the surface of an equal volume of Ficoll separation solution (1.077g / ml), avoiding mixing with human lymphocyte separation solution. Centrifuge for 20 min under the condition. After centrifugation, the liquid is divided into four layers, the uppermost layer is plasma and platelet layer, the second layer is mononuclear cells (MNCs) layer, the third layer is lymphocyte separation liquid layer, and the bottom layer is red blood cell and multinuclear cell layer. Draw the cloudy MNCs layer liquid between the plasma layer and the lymphocyte separation layer into a new centrifuge tube, add an appropriate amount of PBS, mix well, centrifuge at 4°C and 700g / min for 5min, discard the supernatant, and use Remove residual separation solution. Then add 5ml PBS to resuspend the cells, pipette repeatedly 2-3 times, centrifuge at 4...

Embodiment 10

[0070] Detection of surface antigens of BMSCs by flow cytometry

[0071] Select BMSCs in a good growth state, add 0.25% trypsin to digest for about 2 minutes, observe under an inverted phase-contrast microscope, and after the BMSCs shrink and become round and partly fall off, add IMDM medium containing FBS to stop the digestion process, and blow gently, Collect BMSCs into a flow tube, centrifuge at 1200rmp / min for 5min, discard the supernatant, rinse twice with PBS, wash away the residual culture medium, add 100μl PBS to the flow tube, resuspend the cells, press flow Formula antibody instructions Add fluorescently labeled antibodies to the resuspended cells: 5 μl each of APC-CD29, PE-Cy7-CD44, FITC-CD90, PE-CD105, PerCP-Cy5.5-CD31 and APC-Cy7-CD34, placed in Incubate for 20 min at room temperature in the dark. Wash twice with PBS to completely remove unbound fluorescent antibodies, add 200 μl PBS to the flow tube, resuspend the cells, place them on a flow cytometer to detect ...

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Abstract

The invention discloses an induction culture medium and induction method for directional differentiation of human mesenchymal stem cells towards endothelial cells, and belongs to the technical field of cell induction and differentiation. The induction culture medium comprises a basic culture medium and additive components, wherein the basic culture medium is the IMDM culture medium, and the additive components comprise vascular endothelial growth factors, basic fibroblast growth factors, epidermal growth factors, insulin-like growth factors, fetal calf serum, penicillin-streptomycin, heparin,ascorbic acid and / or hydrocortisone. The addition amount ratio of hydrocortisone to ascorbic acid to heparin is 1:(1-3):(8:10). According to the induction culture medium for directional differentiation, the improved induction culture medium is adopted, hydrocortisone, ascorbic acid and heparin can achieve the synergistic effect, the induction effect is improved, the endothelial cells can be stably, efficiently and quickly obtained from the mesenchymal stem cells, and the ratio of the endothelial cells is up to 93.5%.

Description

technical field [0001] The invention belongs to the technical field of cell induction and differentiation, and in particular relates to an induction medium and an induction method for directional differentiation of human bone marrow mesenchymal stem cells to endothelial cells. Background technique [0002] With the aging of the population and changes in people's diet structure, the incidence of ischemic diseases of the lower limbs is increasing year by year, and has become a major disease that threatens human health. The essence of lower extremity ischemic disease is the stenosis and occlusion of lower extremity arteries, resulting in reduced blood supply, often causing intermittent claudication, lower extremity rest pain, and even serious complications such as ulcers and gangrene, which can lead to amputation and death. With the advancement of medical technology, surgical vascular bypass and interventional endovascular angioplasty have become important treatments for ischem...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/069C12N2501/30C12N2501/91C12N2506/1353C12N2500/38
Inventor 李渊杨敏刘绘绘王承恩邹英华任汉云姚子平
Owner PEKING UNIV FIRST HOSPITAL
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