Development of a 3D Vascularized Skeletal Muscle Model Using Bi-Layered Seeding Methods

Abstract Skeletal muscle is essential for locomotion and metabolic regulation. However, existing in vitro and in vivo models have significant limitations in recapitulating physiologic complexity, particularly regarding vascularization. The absence of a vascular network in conventional models has been demonstrated to affect muscle survival and recovery, as well as to impair myogenic differentiation, because blood vessels supply energy and oxygen to living tissues and are involved in muscle regeneration. To overcome these constraints, we engineered a microfluidic-based 3D vascularized skeletal muscle model by integrating endothelial and myogenic cells using a bi-layered seeding approach. A novel double-coating technique was implemented to mitigate vascular gel shrinkage, ensuring stable vessel formation. We confirmed the construction of vasculature and connection with the muscle tissue through immunofluorescence staining. Collectively, muscle functional assessments, including electrical stimulation-induced contraction force and myotube morphology analysis, demonstrated enhanced muscle differentiation and maturation in the vascularized model in comparison to the non-vascularized model. This bioengineered platform advances the accuracy of in vitro muscle models, thereby enabling the simulation of complex diseases and the large-scale fabrication of tissue.