High‐Yield Bioproduction of Extracellular Vesicles from Stem Cell Spheroids via Millifluidic Vortex Transport

Abstract Extracellular vesicles (EVs) are emerging as novel therapeutics, particularly in cancer and degenerative diseases. Nevertheless, from both market and clinical viewpoints, high‐yield production methods using minimal cell materials are still needed. Herein, a millifluidic cross‐slot chip is proposed to induce high‐yield release of biologically active EVs from less than three million cells. Depending on the flow rate, a single vortex forms in the outlet channels, exposing transported cellular material to high viscous stresses. Importantly, the chip accommodates producer cells within their physiological environment, such as human mesenchymal stem cells (hMSCs) spheroids, while facilitating their visualization and individual tracking within the vortex. This precise control of viscous stresses at the spheroid level allows for the release of up to 30000 EVs per cell at a Reynolds number of ≈400, without compromising cellular integrity. Additionally, it reveals a threshold initiating EV production, providing evidence for a stress‐dependent mechanism governing vesicle secretion. EVs mass‐produced at high Reynolds displayed pro‐angiogenic and wound healing capabilities, as confirmed by proteomic and cytometric analysis of their cargo. These distinct molecular signatures of these EVs, compared to those derived from monolayers, underscore the critical roles of the production method and the 3D cellular environment in EV generation.