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Differentiation culture method for directionally inducing mesenchymal stem cells

A bone marrow mesenchymal and osteoblast differentiation technology, which is applied in the direction of cell culture active agents, bone/connective tissue cells, biochemical equipment and methods, etc., can solve the problems of poor transformation effect and achieve the improvement of ALP level and increase The effect of expression level

Active Publication Date: 2020-12-18
四川省恩乐生物工程有限公司
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Problems solved by technology

[0005] At present, although many reports provide methods for inducing transformation of bone marrow mesenchymal stem cells into osteoblasts, the transformation effect is often poor.

Method used

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  • Differentiation culture method for directionally inducing mesenchymal stem cells

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Embodiment 1

[0023] 1. Materials:

[0024] Cells: MSCs were extracted and cultured from tibia and femur bone marrow cavity of newborn SD rats by whole bone marrow adherent method.

[0025] Detection method:

[0026] ALP activity: histochemical method

[0027] Gene expression: detected by electrophoresis.

[0028] 2. Experimental steps

[0029] (1) Bone marrow mesenchymal stem cells were taken and placed in serum-free medium for serum starvation treatment for 12-24 hours; then placed in medium A for pre-culture for 5-6 hours;

[0030] (2), after the pre-cultivation, transfer to culture medium B for 2-4 weeks;

[0031] The medium A is based on DMEM / F12 medium, and 8 μmol of very low-density lipoprotein, 2nmol of fibroblast growth factor-2 and 11nmol of L-glutamine are added;

[0032] The medium B is based on DMEM / F12 medium, and added 6ng / ml nicotinamide, 14ng / ml activin A, 1.5ng / ml dexamethasone, 120ng / ml bone morphoprotein-2 and 6ng / ml ml sodium beta-glycerophosphate.

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Abstract

The invention provides a differentiation culture method for directionally inducing mesenchymal stem cells. The method comprises the following steps of: (1) taking mesenchymal stem cells, and putting the mesenchymal stem cells in a serum-free culture medium to perform serum starvation treatment for 12-24 hours; and putting the mesenchymal stem cells in a culture medium A to perform preculture for 5-6 hours; and (2) after the preculture finishes, transferring the mesenchymal stem cells into a culture medium B to perform culture for 2-4 weeks, wherein the culture medium A uses a DMEM / F12 culturemedium as a basal culture medium, and 5-10 micromoles of very low density lipoprotein, 1-3nmol of fibroblast growth factor-2 and 8-12nmol of L-glutamine are added. The method can effectively enhance the ALP level of the induced cells, and can obviously enhance the expression level of Runx2, thereby effectively and directionally inducing the mesenchymal stem cells to differentiate into osteoblasts.

Description

technical field [0001] The invention belongs to the field of biotechnology, relates to cell culture technology, in particular to a differentiation culture method for directional induction of bone marrow mesenchymal stem cells. Background technique [0002] Mesenchymal stem cells are a type of stem cells with self-replication ability and multi-directional differentiation ability, which can repair the damage of seed tissues and organs, and are the preferred seed cells for stem cell therapy. Mesenchymal stem cells exist in various tissues and organs, including bone marrow, hair follicles, umbilical cord, dental pulp, heart and other tissues, especially bone marrow mesenchymal stem cells and umbilical cord mesenchymal stem cells are the most common. [0003] Bone marrow mesenchymal stem cells (dental pulp stem cells, DPSCs) are a type of mesenchymal stem cells that exist in bone marrow tissue with self-renewal, strong proliferation ability, and multilineage differentiation abili...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077C12N5/0775
CPCC12N5/0654C12N5/0663C12N2501/998C12N2501/115C12N2501/39C12N2501/16C12N2501/155C12N2500/42C12N2500/38C12N2500/32
Inventor 宋筱荭邹弼丞钟立武汪敬雄王峰
Owner 四川省恩乐生物工程有限公司
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