Extracellular Vesicles with Unique Proteomic Signatures from Self-Organized Cardiac Spheroids under Hydrodynamic Stimulation

Cardiac extracellular vesicles (EVs) hold promise as cell-free therapeutics for heart repair. However, robust methods to produce scalable, functional and cardiac-specific EVs at high yield remain a limiting factor in their exploitation. Here, we report an engineered platform that combines 3D cardiac microtissues with hydrodynamic stimulation to address these hurdles. We exploited differential cell mechanics, quantified via surface tension measurements, to establish a unique 3D spheroid architecture showing cardiomyocytes preferentially localizing at the spheroid periphery. A controlled-flow bioreactor then enabled high-yield EV production, at a 10-fold increase compared to non-stimulated spheroids, all while maintaining cell viability comparable to standard 2D production conditions while preserving vesicle structural integrity. Proteomic profiling revealed that EVs generated under these conditions carry a cardiac-specific signature, enriched in sarcomeric, mitochondrial, ribosomal and heat shock proteins, all while retaining core EV markers. Functionally, these EVs enhanced wound closure and reduced fibroblast activation more effectively than EVs derived from standard 2D fibroblast cultures, at 1.3 and 1.5-fold, respectively. Our findings establish a scalable, physiologically relevant strategy for generating cardiac EVs and demonstrate that combining 3D microenvironment engineering with hydrodynamic cues can yield therapeutically potent vesicles suitable for regenerative medicine.