Engineering migrasome-inspired extracellular vesicles through temperature stimuli on thermoresponsive substrate for retraction fiber formation and hypoosmotic vesiculation.

Migrasomes, recently identified extracellular vesicles, play critical roles in physiological processes such as tissue remodeling and intercellular communication. However, their generally low and cell-type-dependent biogenesis limits their broader applications. Here, a simple and scalable method for producing migrasome-inspired vesicles, termed osmotically-induced migrasome-like vesicles (OsMigs), through temperature stimuli-induced retraction fiber on thermoresponsive polymer-modified substrates and hypoosmotic vesiculation was developed. A two-step cooling treatment of 4 °C and 20 °C on the poly (N-isopropylacrylamide)-modified substrate coincidently led to the appearance of retraction fibers. This approach reproducibly achieved more than an order-of-magnitude increase in vesicle yield compared to migrasome biogenesis in normal human dermal fibroblasts, and is broadly applicable to multiple adherent cell types, including even those that do not naturally form migrasomes. OsMigs are enriched in migrasome-associated markers, including tetraspanin-4, integrin α5, and cholesterol, and encapsulate bioactive cytokines. Functionally, OsMigs significantly promote fibroblast migration and angiogenesis in vitro. Importantly, this entirely physical, enzyme- and chemical-treatment-free approach provides a scalable and reproducible platform for engineering migrasome-inspired EVs, opening new avenues for studying extracellular vesicle-mediated signaling and laying the foundation for future EV-based therapeutic applications.