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Method for promoting osteogenic differentiation of bone marrow mesenchymal stem cells

A bone marrow mesenchymal and osteogenic differentiation technology, applied in the field of stem cells, can solve the problems of low induction differentiation efficiency, long time, and inconvenient clinical application, and achieve the effect of shortening the time of osteogenic differentiation and improving efficiency

Active Publication Date: 2020-12-15
深圳市旷逸生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In the process of induction towards osteogenesis, it is often necessary to add exogenous serum such as fetal bovine serum, which reduces the safety, and there are problems such as low induction differentiation efficiency and long time, which cause inconvenience to clinical application.

Method used

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  • Method for promoting osteogenic differentiation of bone marrow mesenchymal stem cells
  • Method for promoting osteogenic differentiation of bone marrow mesenchymal stem cells
  • Method for promoting osteogenic differentiation of bone marrow mesenchymal stem cells

Examples

Experimental program
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Effect test

Embodiment 1

[0020] A method for promoting osteogenic differentiation of bone marrow mesenchymal stem cells, comprising the following steps:

[0021] (1) Human bone marrow mesenchymal stem cells were inoculated in the basal medium DMEM / F12 in a 12-well culture plate at a seeding density of 1×10 4 cells / mL, at 37°C, 5% CO 2 cultured in an incubator, and when the cell confluency reached 80%, it was digested and subcultured with 0.25% trypsin, and the ratio of subculture was 1:3;

[0022] (2) Inoculate the third-generation bone marrow mesenchymal stem cells obtained from the subculture in the above step (1) on the differentiation medium of a 6-well culture plate, at 37°C, 5% CO 2 Induced differentiation in an incubator with a seeding density of 2×10 5 cells / mL, change the solution every other day;

[0023] The above differentiation medium includes DMEM / F12 medium, insulin 20 μg / mL, dexamethasone 8 ng / mL, ascorbic acid 45 μg / mL, sulfobutyl-β-cyclodextrin 20 μg / mL, betaine 50 μg / mL, indium a...

Embodiment 2

[0025] A method for promoting osteogenic differentiation of bone marrow mesenchymal stem cells, comprising the following steps:

[0026] (1) Human bone marrow mesenchymal stem cells were inoculated in the basal medium DMEM / F12 in a 12-well culture plate at a seeding density of 1.5×10 4 cells / mL, at 37°C, 5% CO 2 cultured in an incubator, and when the cell confluency reached 85%, it was digested and subcultured with 0.25% trypsin, and the subculture ratio was 1:4;

[0027] (2) Inoculate the fourth-generation bone marrow mesenchymal stem cells obtained from the subculture in the above step (1) on the differentiation medium of a 6-well culture plate, and inoculate at 37°C, 5% CO 2 Induced differentiation in an incubator with a seeding density of 3×10 5 cells / mL, change the solution every other day;

[0028] The above differentiation medium includes DMEM / F12 medium, insulin 18 μg / mL, dexamethasone 5 ng / mL, ascorbic acid 40 μg / mL, sulfobutyl-β-cyclodextrin 15 μg / mL, betaine 45 μ...

Embodiment 3

[0030] A method for promoting osteogenic differentiation of bone marrow mesenchymal stem cells, comprising the following steps:

[0031] (1) Human bone marrow mesenchymal stem cells were inoculated in the basal medium DMEM / F12 in a 12-well culture plate at a seeding density of 2×10 4 cells / mL, at 37°C, 5% CO 2 cultured in an incubator, and when the cell confluency reached 90%, it was digested and subcultured with 0.25% trypsin, and the subculture ratio was 1:4;

[0032] (2) Inoculate the fifth-generation bone marrow mesenchymal stem cells obtained from the subculture in the above step (1) on the differentiation medium of a 6-well culture plate, and inoculate at 37°C, 5% CO 2 Differentiation was induced in an incubator with a seeding density of 4×10 5 cells / mL, change the solution every other day;

[0033] The above differentiation medium includes DMEM / F12 medium, insulin 22 μg / mL, dexamethasone 10 ng / mL, ascorbic acid 50 μg / mL, sulfobutyl-β-cyclodextrin 23 μg / mL, betaine 52...

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Abstract

The invention discloses a method for promoting osteogenic differentiation of bone marrow mesenchymal stem cells. The method comprises the following steps of (1) culturing human bone marrow mesenchymalstem cells in a DMEM / F12 medium, and performing digestion with 0.25% pancreatin for passage when the cell fusion degree reaches 80-90%; and (2) inoculating a differential medium with the bone marrowmesenchymal stem cells obtained by subculture in the step (1), and carrying out induced differentiation, wherein the differential medium comprises the DMEM / F12 medium, 18-22 [mu]g / mL of insulin, 5-10ng / mL of dexamethasone, 40-50 [mu]g / mL of ascorbic acid, 15-23 [mu]g / mL of sulfobutyl-beta-cyclodextrin, 45-52 [mu]g / mL of betaine, 10-15 ng / mL of indium acetate and 50-60 [mu]g / mL of glutathione. According to the invention, the differential medium is inoculated with the bone marrow mesenchymal stem cells obtained through subculture, and the sulfobutyl-beta-cyclodextrin, the betaine and the indiumacetate are added into the differential medium to achieve a synergistic effect, so that the osteogenic differentiation efficiency of the bone marrow mesenchymal stem cells is effectively improved, the osteogenic differentiation time is shortened, and a guarantee is provided for clinical application of the bone marrow mesenchymal stem cells to tissue-engineered bone construction.

Description

technical field [0001] The invention relates to the field of stem cells, in particular to a method for promoting osteogenic differentiation of bone marrow mesenchymal stem cells. Background technique [0002] Stem cells are a type of cells with self-renewal ability and multi-directional differentiation potential. They are the first cells in the process of human newborn cells from proliferation, migration, differentiation to maturity. They are the source of traditional Chinese medicine for cell therapy and tissue engineering seed cells. The common Mesenchymal stem cells are derived from bone marrow, peripheral blood, umbilical cord, fat, etc. In stem cell biology, it is of great significance to explore the relationship between cell proliferation and differentiation. [0003] Bone marrow mesenchymal stem cells are a type of mesenchymal stem cells that exist in bone marrow tissue and have self-renewal, strong proliferation ability, and multilineage differentiation ability. Bo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077C12N5/0775
CPCC12N5/0654C12N5/0663C12N2500/38C12N2501/33C12N2501/39C12N2501/90C12N2501/999C12N2509/00
Inventor 于梦梦马超群
Owner 深圳市旷逸生物科技有限公司
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