This study provides a comparative analysis of various components of mesenchymal stem cells (MSC) conditioned media (CM) obtained using serum-containing and serum-free culture methods, revealing significant differences in their composition and potential clinical applicability. Serum-containing CM exhibits significantly higher levels of total protein, non-vesicular RNA, exosomes, and nanoparticles compared to serum-free CM, reflecting the contribution of both the MSC secretome and residual fetal bovine serum components. Ultrafiltration-based fractionation (0.2 µm–50 kDa) allows the isolation of fraction enriched in exosomes and proteins, preserving the functionally significant components of the MSC secretome. This strategy effectively captures small vesicles and mid-sized proteins while excluding larger or smaller biomolecules, enhancing utility for targeted analyses. The presented data underscore the need for context-driven CM selection and provide information for choosing the optimal strategy for obtaining the MSC secretome balancing yield, purity, and regulatory demands in MSC research and therapy.
