Method for restoring totipotency of mesenchymal stem cell
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A mesenchymal stem cell, totipotent technology, applied in the field of restoring the totipotency of mesenchymal stem cells, can solve the problems of unresearched MSCs pluripotency
Active Publication Date: 2014-03-26
SHENZHEN GRADUATE SCHOOL TSINGHUA UNIV
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[0004] The current 3D culture cannot meet the current demand of cell engineering and genetic engineering for the amount of seed cells to form an industrialization. Moreover, whether 3D culture can fundamentally restore the pluripotency of MSCs has not yet been studied.
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Embodiment 1
[0051] Example 1. Totipotency reduces the acquisition of mesenchymal stem cells
[0052] 1. In vitro passage and adherent culture of mesenchymal stem cells
[0053] The isolated mesenchymal stem cells (MSCs) were adhered and subcultured in low-glucose DMEM medium (Hyclone, SH30021.01B) containing 10% fetal bovine serum (FBS, Gibco), and the time for each subculture was 3 days , to obtain the 1st-10th passage adherent MSCs respectively.
[0054] 2. Detection of cell pluripotency after adherent culture
[0055] 1) Morphological changes of MSCs after adherent culture
[0056] The 1st and 10th passage cultured MSCs cells were digested and seeded in a six-well plate for overnight (12h). The next day, the ordinary optical microscope was observed with a 10× objective lens; the results were as follows: figure 1 As shown in A-B, it can be seen from the results that the 1st and 10th passage cultured MSCs cells were digested and then cultured to obtain the 2nd passage (p2) and 12th pa...
Embodiment 2
[0066] Example 2, Restoring the Culture of Mesenchymal Stem Cells Totipotency
[0067] The mesenchymal stem cells (4th generation adherent MSCs, 6th generation adherent MSCs, and 10th generation adherent MSCs) obtained in Example 1 above with reduced totipotency were subjected to the following two groups of treatments:
[0068] Group A: adherent culture: The culture method was the same as above, and the adherent MSCs of the 5th passage, the adherent MSCs of the 7th passage and the adherent MSCs of the 11th passage were obtained.
[0069] Group B: 3D culture, the culture method is as follows:
[0070] 1. Hanging drop culture
[0071] The 4th passage adherent MSCs, the 6th passage adherent MSCs, and the 10th passage adherent MSCs were digested into single-cell suspensions with digestive juice, and high-sugar DMEM (GIBCO, C11995) + 10% (mass percentage) ) Fetal bovine serum, adjust the cell concentration in the single cell suspension to 1×10 6 cells / mL to obtain a single cell ...
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Abstract
The invention discloses a culture method for restoring totipotency of a mesenchymal stem cell. The method for restoring totipotency of a mesenchymal stem cell, provided by the invention, comprises the following steps: 1) performing hanging-drop culture on an in-vitro mesenchymal stem cell with reduced totipotency, thus obtaining a hanging-drop cultured cell; and 2) further performing suspension culture on the hanging-drop cultured cell, thus realizing totipotency restoration of the mesenchymal stem cell. The experiment of the invention proves that, according to the method for restoring totipotency of a mesenchymal stem cell (specifically 3D culture), after the mesenchymal stem cell (in-vitro mesenchymal stem cell with reduced totipotency) subcultured multiple times is subjected to 3D culture treatment, the increased volume is gradually restored, the cell viability is enhanced, the cloning capability and differentiation capability are enhanced, and the paracrine action is also improved, thereby activating the aged mesenchymal stem cell and restoring the characteristics of the stem cell.
Description
technical field [0001] The invention relates to the field of biotechnology, in particular to a method for restoring the pluripotency of mesenchymal stem cells. Background technique [0002] Mesenchymal stem cells (MSCs) are a kind of adult stem cells, because of their strong proliferation ability and multi-directional differentiation potential, low immunogenicity, convenient source, easy isolation, culture, expansion and purification, etc., It has broad clinical application prospects in autoimmune diseases and various alternative treatments. Although the content of mesenchymal stem cells in bone marrow is relatively large, it only accounts for 0.001% to 0.01%, which is difficult to meet the needs of cell therapy. Therefore, it needs to be separated and purified in vitro, and cultured and expanded to meet the requirements. However, the aging and autonomous differentiation of MSCs into osteoblasts in the process of in vitro expansion seriously affect their therapeutic effect ...
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