Abstract
Skeletal muscle tissue engineering aims to create bioartificial muscle (BAM) constructs that recapitulate key structural and functional features of native skeletal muscle for applications including regenerative medicine, disease modeling, and drug screening. While most BAM models are generated using myoblasts, multicellular building blocks such as myogenic spheroids may better mimic native cell-cell interactions and microarchitectural organization. In this study, we compared the use of primary human myogenic spheroids with single cells for skeletal muscle tissue engineering. Myogenic spheroids composed of primary human skeletal myoblasts were generated in a high-throughput manner using nonadhesive micropatterned agarose chips. Single cell myoblasts and myogenic spheroids were subsequently embedded in fibrin hydrogels and cast into silicone molds between two metal pins serving as anchor points. Results showed that maturation stage of the spheroids prior to encapsulation significantly influenced BAM morphology after 7 days. Early maturation stage-spheroids promoted enhanced cellular outgrowth, myoblast fusion, and increased myotube diameter compared with more mature spheroids, while the spheroid size did not significantly affect construct formation. Direct comparison between spheroid-based and single-cell BAMs revealed distinct phenotypic differences. Spheroid-based constructs generated fewer but larger multinucleated myotubes with higher fusion indices, whereas single-cell constructs produced a greater number of smaller myotubes. Despite these differences in morphology, no significant changes were observed in the expression of myosin heavy chain isoforms, indicating comparable myogenic differentiation under the tested conditions. Together, these findings demonstrate that primary human myogenic spheroids represent an effective alternative building block for skeletal muscle tissue engineering and provide insight into how cell assembly strategies influence muscle tissue formation.