Comparative effects of hypoxia and serum deprivation on the morphological, structural, chemical, biological and functional characteristics of exosomes from adipose-derived mesenchymal stem cells

Background Adipose-derived mesenchymal stem cells (AdMSCs) are potent sources of therapeutic exosomes; however, their cargo and bioactivity are highly influenced by microenvironmental conditions. Although hypoxia and serum starvation have individually shown promise in enhancing exosome functionality, their combined effects remain poorly characterized. Methods This study systematically investigated the individual and synergistic effects of hypoxia and serum deprivation on AdMSCs and their secreted exosomes. AdMSCs were cultured under four distinct conditions: control, hypoxia (H), serum-free (SF), and hypoxia plus serum-free (HSF). Exosomes were isolated and characterized by electron microscopy, dynamic light scattering (DLS), zeta potential, Fourier-transform infrared spectroscopy (FTIR), and Western blotting for canonical exosome markers. Functional assessments included migration assays and cell viability effects on human dermal fibroblasts (HDFs). Results Despite minimal changes in AdMSC viability and immunophenotype, combined hypoxia and serum starvation (HSF) significantly enhanced cell migration, exosome yield, and marker expression. Exosomes derived under HSF conditions exhibited reduced size, improved homogeneity (PDI: 0.18), and highly negative surface charge (-61.8 mV), suggesting superior stability and cellular interaction potential. FTIR and Western blot analyses revealed distinct compositional shifts, particularly in lipid and protein profiles. Functionally, HSF-derived exosomes showed the greatest enhancement in HDF viability, outperforming exosomes from single-stress or control conditions. Conclusion Our findings demonstrate that the dual preconditioning of AdMSCs using hypoxia and serum deprivation synergistically optimizes exosome biogenesis and bioactivity. This combinatorial strategy holds translational promise for producing next-generation, cell-free therapeutics with enhanced regenerative efficacy.