CN112080463B - Method for promoting osteogenic differentiation of mesenchymal stem cells - Google Patents
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Abstract
The invention discloses a method for promoting osteogenic differentiation of mesenchymal stem cells, which comprises the following steps: (1) culturing human bone marrow mesenchymal stem cells in a DMEM/F12 culture medium, and digesting and passaging with 0.25% pancreatin when the cell fusion degree reaches 80-90%; (2) inoculating the mesenchymal stem cells obtained by subculturing in the step (1) to a differentiation medium for induced differentiation; the differentiation culture medium comprises DMEM/F12 culture medium, insulin 18-22 mu g/mL, dexamethasone 5-10ng/mL, ascorbic acid 40-50 mu g/mL, sulfobutyl-beta-cyclodextrin 15-23 mu g/mL, betaine 45-52 mu g/mL, indium acetate 10-15ng/mL and glutathione 50-60 mu g/mL. The mesenchymal stem cells obtained by subculture are inoculated on a differentiation medium, and sulfobutyl-beta-cyclodextrin, betaine and indium acetate are added into the differentiation medium to realize synergistic effect, so that the osteogenic differentiation efficiency of the mesenchymal stem cells is effectively improved, the osteogenic differentiation time is shortened, and the guarantee is provided for the clinical application of the mesenchymal stem cells in tissue engineering bone construction.
Description
Technical Field
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
The stem cells are the first cells of human body during the process from proliferation, migration, differentiation to maturation, and are the traditional Chinese medicine sources of cell therapy and tissue engineering seed cells, and common mesenchymal stem cells are derived from bone marrow, peripheral blood, umbilical cord, fat and the like. In stem cell biology, it is of great significance to explore the relationship between cell proliferation and differentiation.
The mesenchymal stem cells are mesenchymal stem cells which exist in bone marrow tissues and have self-renewal, strong proliferation capacity and multidirectional differentiation capacity. The bone marrow stem cells can be differentiated into cells of various mesenchymal sources, such as osteoblasts, adipocytes, chondrocytes, hepatocytes, myocytes, nerve cells and the like, and are ideal seed cells with huge potential in medical application of the stem cells in the future. The growth and differentiation direction of bone marrow stem cells depends on the microenvironment of the external part, and a culture medium serving as an important regulatory factor of an extracellular chemical microenvironment is known to be one of simple and effective methods for inducing stem cells to differentiate directionally in vitro at present, and the differentiation direction of the stem cells can be determined by the in vitro induction condition. In the application of bone tissue engineering, the differentiation of bone marrow mesenchymal stem cells into osteoblasts is a key step for constructing bone tissue engineering by using the osteoblasts.
Exogenous serum such as fetal calf serum and the like is often required to be added in the process of inducing in the osteogenesis direction, so that the safety is reduced, and the problems of low induced differentiation efficiency, long time and the like exist, thereby causing inconvenience to clinical application. Therefore, establishing a simple, safe and effective osteogenic differentiation induction method is the key point of the construction of stem cells in tissue engineered bones.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a method for promoting osteogenic differentiation of bone marrow mesenchymal stem cells, which effectively improves differentiation efficiency and shortens differentiation time.
The purpose of the invention is realized by adopting the following technical scheme:
a method for promoting osteogenic differentiation of mesenchymal stem cells comprises the following steps:
(1) culturing human bone marrow mesenchymal stem cells in a DMEM/F12 culture medium, and digesting and passaging with 0.25% pancreatin when the cell fusion degree reaches 80-90%;
(2) inoculating the mesenchymal stem cells obtained by subculturing in the step (1) to a differentiation medium for induced differentiation;
the differentiation culture medium comprises DMEM/F12 culture medium, insulin 18-22 mu g/mL, dexamethasone 5-10ng/mL, ascorbic acid 40-50 mu g/mL, sulfobutyl-beta-cyclodextrin 15-23 mu g/mL, betaine 45-52 mu g/mL, indium acetate 10-15ng/mL and glutathione 50-60 mu g/mL.
Further, the differentiation medium comprises DMEM/F12 medium, insulin 20 mu g/mL, dexamethasone 8ng/mL, ascorbic acid 45 mu g/mL, sulfobutyl-beta-cyclodextrin 20 mu g/mL, betaine 50 mu g/mL, indium acetate 12ng/mL, and glutathione 55 mu g/mL.
Further, the inoculation density of the mesenchymal stem cells in the step (1) is 1-2 x 104one/mL.
Further, the seeding density of the mesenchymal stem cells in the step (2) is 2-4 x 105one/mL.
Further, the mesenchymal stem cells subcultured to 3-5 th generation in the step (2) are inoculated into a differentiation medium.
Further, the ratio of subculture is 1: 3-4.
Compared with the prior art, the invention has the beneficial effects that: the invention provides a method for promoting osteogenic differentiation of mesenchymal stem cells, which is characterized in that mesenchymal stem cells obtained by subculture are inoculated on a differentiation culture medium, the culture medium takes DMEM/F12 as a basic culture medium, and sulfobutyl-beta-cyclodextrin, betaine and indium acetate are added into the basic culture medium while insulin, dexamethasone, ascorbic acid and glutathione are added to realize synergistic action, so that the osteogenic differentiation efficiency of the mesenchymal stem cells is effectively improved, the osteogenic differentiation time is shortened, the guarantee is provided for the clinical application of the mesenchymal stem cells in tissue engineering bone construction, and a new way is provided for the treatment of orthopedic diseases.
Drawings
FIG. 1 is ALP activity in induced differentiation culture of mesenchymal stem cells according to example 1 of the present invention and comparative examples 1 to 5.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and the detailed description, and it should be noted that any combination of the embodiments or technical features described below can be used to form a new embodiment without conflict.
Example 1
A method for promoting osteogenic differentiation of mesenchymal stem cells comprises the following steps:
(1) inoculating human bone marrow mesenchymal stem cells into a basal medium DMEM/F12 in a 12-well culture plate at an inoculation density of 1 × 104one/mL, 5% CO at 37 ℃2When the cell fusion degree reaches 80%, digesting and passing by 0.25% pancreatin, wherein the ratio of passage culture is 1: 3;
(2) inoculating the 3 rd generation mesenchymal stem cells obtained by subculture in the step (1) to a differentiation medium of a 6-well culture plate, and culturing at 37 ℃ and 5% CO2The culture box of (2) is used for inducing differentiation, and the inoculation density is 2 multiplied by 105Changing the solution every other day for one/mL;
the differentiation culture medium comprises DMEM/F12 culture medium, insulin 20 mu g/mL, dexamethasone 8ng/mL, ascorbic acid 45 mu g/mL, sulfobutyl-beta-cyclodextrin 20 mu g/mL, betaine 50 mu g/mL, indium acetate 12ng/mL and glutathione 55 mu g/mL.
Example 2
A method for promoting osteogenic differentiation of mesenchymal stem cells comprises the following steps:
(1) inoculating human bone marrow mesenchymal stem cells into a basal medium DMEM/F12 in a 12-well culture plate at an inoculation density of 1.5 × 104one/mL, 5% CO at 37 ℃2When the cell fusion degree reaches 85%, digesting and passing by 0.25% pancreatin, wherein the ratio of passage culture is 1: 4;
(2) inoculating the 4 th generation of mesenchymal stem cells obtained by subculture in the step (1) to a differentiation medium of a 6-well culture plate, and culturing at 37 ℃ and 5% CO2The culture box of (2) is used for inducing differentiation, and the inoculation density is 3 multiplied by 105Changing the solution every other day for one/mL;
the differentiation culture medium comprises DMEM/F12 culture medium, insulin 18 mu g/mL, dexamethasone 5ng/mL, ascorbic acid 40 mu g/mL, sulfobutyl-beta-cyclodextrin 15 mu g/mL, betaine 45 mu g/mL, indium acetate 10ng/mL and glutathione 50 mu g/mL.
Example 3
A method for promoting osteogenic differentiation of mesenchymal stem cells comprises the following steps:
(1) inoculating human bone marrow mesenchymal stem cells into a basal medium DMEM/F12 in a 12-hole culture plate at an inoculation density of 2 × 104seed/mL at 37 deg.C，5％CO2When the cell fusion degree reaches 90%, digesting and passing by 0.25% pancreatin, wherein the ratio of passage culture is 1: 4;
(2) inoculating the 5 th generation mesenchymal stem cells obtained by subculture in the step (1) to a differentiation medium of a 6-well culture plate, and culturing at 37 ℃ and 5% CO2The culture box of (2) is used for inducing differentiation, and the inoculation density is 4 multiplied by 105Changing the solution every other day for one/mL;
the differentiation culture medium comprises DMEM/F12 culture medium, insulin 22 mu g/mL, dexamethasone 10ng/mL, ascorbic acid 50 mu g/mL, sulfobutyl-beta-cyclodextrin 23 mu g/mL, betaine 52 mu g/mL, indium acetate 15ng/mL and glutathione 60 mu g/mL.
Comparative example 1
Comparative example 1 provides a method for promoting osteogenic differentiation of mesenchymal stem cells of bone marrow, which is different from example 1 in that: the sulfobutyl-beta-cyclodextrin was omitted and the procedure was as in example 1.
Comparative example 2
Comparative example 2 provides a method for promoting osteogenic differentiation of mesenchymal stem cells of bone marrow, which is different from example 1 in that: the sulfobutyl-beta-cyclodextrin was replaced with hydroxypropyl-beta-cyclodextrin, and the rest was the same as in example 1.
Comparative example 3
Comparative example 3 provides a method for promoting osteogenic differentiation of mesenchymal stem cells of bone marrow, which is different from example 1 in that: betaine was omitted and the procedure was as in example 1.
Comparative example 4
Comparative example 4 provides a method for promoting osteogenic differentiation of mesenchymal stem cells of bone marrow, which is different from example 1 in that: the indium acetate was omitted and the procedure in example 1 was repeated.
Comparative example 5
Comparative example 5 provides a method for promoting osteogenic differentiation of mesenchymal stem cells of bone marrow, which is different from example 1 in that: indium acetate was replaced with indium sulfate, and the rest was the same as in example 1.
ALP Activity assay: the cells of example 1 and comparative examples 1 to 5 were taken to induce differentiation culture of 1d, 4d, 8d and 12d, respectively, the cells were lysed in Tris-HCl buffer pH7.4, centrifuged at 10000r/min for 5min, the supernatant was collected, and ALP activity of each group of cells was measured using ALP kit, and the results are shown in FIG. 1.
The cells of example 1 and comparative examples 1 to 5 were respectively taken, subjected to induced differentiation culture for 12 days, the culture medium was discarded, the cells were washed with 6 ℃ precooled PBS, fixed with 4% paraformaldehyde at normal temperature for 10min, the paraformaldehyde was discarded, the cells were washed with PBS for three times, stained with 0.5% alizarin red for 10min, the staining solution was discarded, the cells were washed with water for 3 times, the staining condition was observed on a microscope line, and the percentage of calcified area was counted, and the results are shown in Table 1.
TABLE 1
ALP is one of early markers of osteogenic differentiation of the mesenchymal stem cells, and the higher ALP activity indicates that the more mesenchymal stem cells are differentiated in the osteogenic direction. As can be seen from fig. 1: the ALP activity of the cells of example 1 was the highest, indicating that more mesenchymal stem cells differentiated into the osteogenic direction.
Calcification of osteoblasts can occur, the calcified osteoblasts can be stained red by alizarin, and the percentage of calcified area can be calculated according to the area of stained cells. As can be seen from Table 1: the cells of example 1 had the largest calcified area after 12 days of culture, and further, it was demonstrated that many osteoblasts were obtained by inducing differentiation in example 1.
The cells of comparative examples 1 to 5 also had a certain ALP activity and percent calcified area, but all were inferior to example 1. The composition of the differentiation medium was adjusted in comparative examples 1 to 5, in which sulfobutyl- β -cyclodextrin, betaine, and indium acetate were omitted in comparative example 1, comparative example 3, and comparative example 4, respectively, sulfobutyl- β -cyclodextrin was replaced with hydroxypropyl- β -cyclodextrin in comparative example 2, indium acetate was replaced with indium sulfate in comparative example 5, ALP activity of the mesenchymal stem cells during induced differentiation was lower than that of example 1, and calcified area after alizarin staining after 12d culture was lower than that of example 1, indicating that the number of mesenchymal stem cells differentiated in the osteogenic direction in comparative examples 1 to 5 was less than that of example 1, and the number of osteoblasts finally obtained was less than that of example 1, indicating that the present invention synergistically acts by adding three components of sulfobutyl- β -cyclodextrin, betaine, and indium acetate to the differentiation medium, after any one component is omitted or one component is replaced, the effect is not as good as that of the simultaneous addition of the three components, and further, the addition of the sulfobutyl-beta-cyclodextrin, the betaine and the indium acetate into a differentiation medium can effectively promote the osteogenic differentiation of the mesenchymal stem cells and improve the differentiation efficiency.
The above embodiments are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited thereby, and any insubstantial changes and substitutions made by those skilled in the art based on the present invention are within the protection scope of the present invention.
Claims (6)
1. A method for promoting osteogenic differentiation of mesenchymal stem cells, comprising the steps of:
(1) culturing human bone marrow mesenchymal stem cells in a DMEM/F12 culture medium, and digesting and passaging with 0.25% pancreatin when the cell fusion degree reaches 80-90%;
(2) inoculating the mesenchymal stem cells obtained by subculturing in the step (1) to a differentiation medium for induced differentiation;
the differentiation culture medium comprises DMEM/F12 culture medium, insulin 18-22 mu g/mL, dexamethasone 5-10ng/mL, ascorbic acid 40-50 mu g/mL, sulfobutyl-beta-cyclodextrin 15-23 mu g/mL, betaine 45-52 mu g/mL, indium acetate 10-15ng/mL and glutathione 50-60 mu g/mL.
2. The method for promoting osteogenic differentiation of mesenchymal stem cells according to claim 1, wherein the differentiation medium comprises DMEM/F12 medium, insulin 20 μ g/mL, dexamethasone 8ng/mL, ascorbic acid 45 μ g/mL, sulfobutyl- β -cyclodextrin 20 μ g/mL, betaine 50 μ g/mL, indium acetate 12ng/mL, glutathione 55 μ g/mL.
3. The method for promoting osteogenic differentiation of mesenchymal stem cells according to claim 1, wherein the mesenchymal stem cells are seeded at a density of 1-2 x 10 in the step (1)4one/mL.
4. The method for promoting osteogenic differentiation of mesenchymal stem cells according to claim 1, wherein the mesenchymal stem cells are seeded at a density of 2-4 x 10 in the step (2)5one/mL.
5. The method for promoting osteogenic differentiation of mesenchymal stem cells according to claim 1, wherein the mesenchymal stem cells subcultured to passage 3-5 in the step (2) are inoculated into the differentiation medium.
6. The method for promoting osteogenic differentiation of mesenchymal stem cells according to claim 1, wherein the ratio of subculture is 1: 3-4.
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| CN113215093A (en) * | 2021-04-08 | 2021-08-06 | 华中科技大学同济医学院附属协和医院 | Method for stimulating and activating aged mesenchymal stem cells by pulse current |
| CN113005079B (en) * | 2021-05-08 | 2022-12-06 | 威海见生生物技术有限公司 | Additive for human bone marrow mesenchymal stem cell in vitro amplification and amplification method |
| CN113652396B (en) * | 2021-08-24 | 2023-08-29 | 赛尔医学科技(山东)有限公司 | Adipose-derived mesenchymal stem cell osteogenesis inducing composition and osteogenesis inducing method |
| CN115634221A (en) * | 2022-10-21 | 2023-01-24 | 拜澳泰克(沈阳)生物医学集团有限公司 | Application of betaine in regulating mesenchymal stem cell adipogenic and osteogenic differentiation |
| CN116376815B (en) * | 2023-02-15 | 2024-05-31 | 山东科金生物发展有限公司 | Culture medium for promoting osteoblast differentiation of mesenchymal stem cells |
| CN116606806A (en) * | 2023-07-20 | 2023-08-18 | 北京益华生物科技有限公司 | Method for improving activity rate of bone marrow mesenchymal stem cells in low-oxygen environment |
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Granted publication date: 20211026 |