Optimization of the isolation and culture system for mesenchymal stem cells derived from rat thoracic aorta media based on the dual-phase harvesting method
10.19405/j.cnki.issn1000–1492.2026.09.015
- VernacularTitle:基于双相收获法优化大鼠胸主动脉中膜来源间充质干细胞的分离培养体系
- Author:
Suyu LAI
1
;
Jing WU
2
;
Ming CHI
2
;
Zhuo WANG
1
;
Yixian ZENG
1
;
Xiaofeng WANG
1
Author Information
1. Department of Anorectal Surgery， Guang'anmen Hospital， China Academy of Chinese Medical Sciences， Beijing 100053
2. Institute of Integrated Traditional Chinese and Western Medicine， College of Integrated Traditional Chinese and Western Medicine， Fujian University of Traditional Chinese Medicine， Fuzhou 350122
- Publication Type:Journal Article
- Keywords:
thoracic aorta;
mesenchymal stem cells;
dual-phase harvesting method;
flow cytometry;
trilineage differentiation;
cell purity
- From:
Acta Universitatis Medicinalis Anhui
2026;61(9):1626-1633
- CountryChina
- Language:Chinese
-
Abstract:
ObjectiveTo address the challenge of balancing efficiency and purity in isolating MSCs from rat thoracic aorta using traditional methods and to establish and validate an optimized isolation and culture system based on the dual-phase harvesting method. MethodsThoracic aortas were aseptically isolated from 3 to 4-week-old SD rats and minced. Three experimental groups were included： simple enzymatic digestion， pre-explant-enzymatic digestion sequential method， and the dual-phase harvesting method. Growth kinetic parameters， including time to initial cell migration， time to primary confluence， and cell yield， were systematically compared among the three groups. Passage‑4 cells were examined for surface marker expression （CD73， CD44， CD29， CD45， CD34， and CD11b/c） by flow cytometry. Their stemness was evaluated through osteogenic， adipogenic， and chondrogenic trilineage differentiation assays. ResultsThe time to primary confluence in the dual-phase harvesting method group （8.5 ± 1.0 ） d was significantly shorter than that of the simple enzymatic digestion group （11.2±1.2） d and the pre‑explant enzymatic digestion sequential group （12.8±1.5） d. Flow cytometric analysis revealed that the harvested cells consistently showed high expression of CD73 （87.2±1.5）%， CD44 （87.1±0.8）%， and CD29 （96.8±1.3）%， with low expression of CD45 （3.22±0.2）%， CD34 （2.46±0.5）%， and CD11b/c （2.53±2.1）%. Upon directed induction， the cells formed Alizarin Red‑positive calcium nodules， Oil Red O‑positive lipid droplets， and Alcian Blue-positive glycosaminoglycans， confirming complete trilineage differentiation potential. The cells could be stably expanded to passage 8 while maintaining a uniform elongated spindle-shaped morphology. ConclusionThe dual‑phase harvesting method established in this study is an efficient and stable optimized protocol for isolating MSCs from the rat thoracic aorta media. This approach synergistically enhances cell yield， purity， and stemness through its temporally designed two‑step process， thereby providing a high-quality in situ cell source for vascular biology research and regenerative medicine.
