Engineering a skeletal muscle model to study extracellular vesicle dynamics

Skeletal muscle (SM) functions both mechanically and as a secretory organ, releasing myokines and extracellular vesicles (EVs) involved in myogenic regulation and inter-tissue communication. While 3D bioengineered SM models are widely used for studying muscle physiology, few have been applied to investigate EV dynamics. This study optimised a 3D SM model to support mature myotube formation and evaluated its utility for SM-EV analysis. Myosin heavy chain expression was reduced at higher Matrigel^® concentrations (40%-60% v/v), highlighting the importance of matrix composition in model design. EVs were successfully isolated using size-exclusion chromatography and ultrafiltration, with yield influenced by cellular differentiation status. Common EV markers (Alix, CD9, CD63) were consistently expressed. Importantly, sarcoplasmic reticulum markers α- and β-sarcoglycan were identified in SM-EV preparations. These findings validate our SM model as a defined platform for studying SM-EV biology and defining molecular cargo.