In vitro isolation and characterization of rat adipose-derived stromal vascular fraction and mesenchymal stem cells for mesodermal tissue regeneration

Abstract Background Adipose-derived stromal vascular fraction (AdSVF) and adipose-derived mesenchymal stem cells (AdMSCs) are promising tools in regenerative medicine due to their accessibility and therapeutic potential. This study focuses on the in vitro characterization of rat-derived AdMSCs (r-AdMSCs) isolated from AdSVF (r-AdSVF) for mesodermal tissue regeneration. Results r-AdSVF was isolated through enzymatic digestion of subcutaneous ventral abdominal fat depots, yielding an average of 5.39 ± 0.73 × 10 6 cells per gram of adipose tissue. The r-AdSVF was differentiated into AdMSCs under optimized culture conditions, with P3 cells achieving 90% confluence within five days and exhibiting fibroblast-like morphology. Comprehensive characterization included morphological analysis, immunocytochemistry, gene expression studies, trilineage differentiation (osteogenic, chondrogenic, and adipogenic), and colony-forming unit assays. r-AdMSCs showed positive expression of mesenchymal markers (CD73, CD90, CD105) and negative expression of hematopoietic markers (CD34, CD45), confirming their stem cell identity. Colony-forming assays demonstrated robust osteogenic differentiation potential, supporting their application in bone regeneration. Conclusions This study highlights the properties of r-AdSVF and r-AdMSCs while emphasizing the need for improved characterization and standardization to advance their use in mesodermal tissue engineering. By establishing a foundational framework, this research contributes to developing safe, effective, and accessible regenerative therapies for mesodermal tissue repair and regeneration.