Optimization of culture conditions for the differentiation of endothelial progenitor cells from mouse blood <i>in vitro</i>

Endothelial progenitor cells (EPCs) play a crucial role in neovascularization and tissue repair, with significant therapeutic potential in ischemic diseases, tumor therapy, and as gene carriers. However, the current methods for isolating and culturing EPCs are not standardized, leading to inconsistencies in cell numbers and functionality. This study aimed to optimize the in vitro culture conditions for EPCs using an orthogonal design, focusing on four main factors: cell density, culture medium, fetal bovine serum (FBS) concentration, and vascular endothelial growth factor (VEGF) concentration. The most effective conditions among those tested were a cell density of 1 × 10⁶/cm², EGM-2 medium, 10% FBS, and 20 ng/mL VEGF. Under these conditions, EPCs exhibited significantly enhanced proliferation, migration, and pro-angiogenic (paracrine) capacity. Immunohistochemistry and fluorescent staining confirmed high expression of EPC-specific markers, such as CD133 and KDR, and the ability to uptake DiI-ac-LDL and bind FITC-UEA-1. Angiogenesis assays showed that most effective conditions among those tested significantly increased the number of vessel-like structures. Additionally, the migration rate and proliferative activity of EPCs were significantly higher under the most effective conditions among those tested compared to conventional conditions. These findings provide a robust foundation for further refining in vitro EPC culture and pave the way for more effective clinical applications. Future studies should validate these optimized conditions in in vivo models to fully realize the therapeutic potential of EPCs.