[Isolation, expansion, and identification of the perivascular adipose-derived mesenchymal stem cells from rat aortas]

Objective To establish a method for isolating and expanding perivascular adipose-derived mesenchymal stem cells (PV-ADSC) from rat aortas and to provide a reliable cellular model for investigating vascular remodeling and the related cardiovascular diseases. Methods Perivascular adipose tissue (PVAT) was aseptically collected from the aortas of 1-month-old Sprague Dawley (SD) rats, then rinsed, minced, digested, filtered, and seeded for culture. Upon reaching 80%-90% confluence, the cells were passaged for expansion, and their morphology and proliferation were continuously monitored. Passage 5 (P5) cells were harvested for flow cytometric analysis of surface CD marker expression, and directed differentiation assays with lineage-specific staining were performed to assess the trilineage (adipogenic, osteogenic, chondrogenic) differentiation potential. Results On day 4 of the primary culture, spindle-shaped, stellate, and polygonal cells were observed migrating from the explant edges. Between day 5 and 6, island-like cell clones emerged, and by day 7 to 8, the colonies had coalesced into a confluent monolayer with swirling or parallel alignment. After being passaged, the cells exhibited a homogeneous fibroblast-like spindle morphology and maintained robust proliferation, with no evident senescence or aberrant differentiation through P8. P5 cells showed high expression of the mesenchymal stem cells (MSC) surface markers CD90 (93.60±0.62)%, CD73 (86.80±2.40)%, CD44 (97.77±0.58)%, and CD29 (98.60±0.30)%, yielding a mean positivity rate of (94.19±5.00)%. In contrast, expressions of the hematopoietic stem cells (HSC) surface markers CD45 (5.42±0.78)%, CD34 (7.15±0.49)%, and CD11b/c (3.74±0.08)% were significantly lower, with a mean rate of only (5.44±1.55)%. Directed differentiation assays confirmed the cells' ability to differentiate into adipocytes, osteoblasts, and chondrocytes, as demonstrated by positive staining with Oil Red O, Alizarin Red, and Alcian Blue, respectively. Conclusion Using the method established in this study, rat aortic PV-ADSC were successfully isolated and expanded. These cells exhibited the morphological and surface marker profiles typical of MSC, possessed robust self-renewal capacity, and retained the potential for trilineage differentiation into adipogenic, osteogenic, and chondrogenic lineages. Collectively, these characteristics establish a reliable cellular model for studying vascular remodeling and the related cardiovascular diseases.