In vitro characterization and angiogenic potential of quality-and-quantity mononuclear cell-derived extracellular vesicles

Background This study evaluated the presence, production rate, and in vitro angiogenic properties of quality-and-quantity culture mononuclear cells-derived extracellular vesicles (QQMNC-EVs). Methods Peripheral blood from three healthy volunteers was processed into five groups: QQMNC-EVs, QQMNCs, peripheral blood mononuclear cell-derived EVs (PBMNC-EVs), CD34 + -EVs, and late endothelial progenitor cell-derived EVs (late EPC-EVs). EVs were identified and characterized using nanoparticle tracking analysis, flow cytometry, western blot analysis, and electron microscopy. EV production rates were quantified, and angiogenesis was evaluated via tube length formation assay. QQMNC-EVs were identified and met established EVs criteria. Results QQMNCs produced the highest EVs yield. This was significantly higher than CD34 + cells ( P = .044), late EPCs ( P = .004), and PBMNCs ( P = .046). At low concentration, QQMNC-EVs showed angiogenic effects comparable with CD34 + -EVs and late EPC-EVs, significantly greater than PBMNC-EVs ( P = .018). At high concentration, QQMNC-EVs produced the highest tube length formation, demonstrating a dose-dependent effect. This was significantly higher than CD34 + -EVs ( P = .018), late EPC-EVs ( P = .011), and PBMNC-EVs ( P = .002). Conclusions QQMNC-EVs were successfully identified and showed in vitro angiogenic efficacy, highlighting their potential as a novel therapeutic strategy for patients with no-option chronic limb-threatening ischemia. Clinical Relevance Extracellular vesicles (EVs) were successfully identified and characterized from QQMNCs and had the highest production rate among the four cell groups: QQMNCs, standard peripheral blood mononuclear cells, CD34 + cells, and late endothelial progenitor cells. Moreover, QQMNC-EVs produced markedly higher tube length formation than standard mononuclear cell-derived EVs. These findings in angiogenic efficacy in vitro highlight their potential as a novel optional therapeutic strategy. Further investigation may advance treatment options for patients with no-option chronic limb-threatening ischemia.