Clinical-scale bioreactor production of hiPSC-derived extracellular vesicles modulates miRNA and protein cargo to...

Extracellular vesicles (EVs) derived from human induced pluripotent stem cells (hiPSC-EVs) hold great therapeutic promise, yet challenges in scalable production and the impact of 3D cellular architecture on EV content and function continue to hinder clinical translation. Here, we demonstrate a perfusion-based stirred-tank bioreactor (BR) system to scale up hiPSC-EV production, eliminating labor-intensive static cultures while preserving physiologically relevant cellular characteristics. The system was successfully scaled from 0.2 l BRs to 2 l BRs, yielding clinically relevant EV doses (10¹¹ EVs per 2 l BR). Notably, only EVs secreted under BR conditions elicited significant proangiogenic effects in vitro, promoting endothelial cell survival, proliferation, migration, and sprouting. Small RNA and proteomic profiling revealed enrichment of proangiogenic miRNA and proteins regulating endothelial function in BR-derived EVs, indicating a shift driven by the culture environment. These findings underscore the role of BR dynamics in shaping EV properties and supporting the scalable production of therapeutically potent hiPSC-EVs.