Automated expansion of iPSCs enables scalable extracellular vesicle production with anti-senescence bioactivity.

BACKGROUND: Induced pluripotent stem cell (iPSC)-based therapies are rapidly advancing, requiring scalable and standardized cell manufacturing strategies. We established an automated workflow for the expansion of iPSCs and iPSC-derived mesenchymal stromal cells (iMSCs). We evaluated the bioactivities of iPSC- and iMSC-derived extracellular vesicle (EV). METHODS: iPSCs and iMSCs were expanded by the automated culture system through iACE2. EVs were isolated from conditioned media by tangential flow filtration and evaluated for the bioactivities including immunomodulatory phenotypes and anti-senescence effects. RESULTS: Comparable yields of iPSC- and iMSC-derived EVs were isolated from the conditioned medium between automated and manual culture systems. Nanoparticle tracking analysis, transmission electron microscopy, and EV surface marker profiling detected no evident differences in physicochemical properties and bioactivities of EVs between the culture systems. In a radiation-induced fibroblast senescence model, iPSC-derived EVs suppressed a senescent marker CDKN2A/p16INK4A expression, reduced the proportion of senescence-associated β-galactosidase-positive cells, attenuated elevated mitochondrial respiration toward baseline levels, and reduced mitochondrial reactive oxygen species (ROS). We also found that iMSC-derived EVs enhanced mitochondrial respiration in fibroblasts. CONCLUSIONS: Automated culture system through iACE2 supports scalable EV production from iPSCs and iMSCs while preserving EV bioactivity relative to manual procedures. Automated expansion of iPSCs and iMSCs is a practical approach for standardized generation of therapeutically relevant EVs for future translational applications.