Skeletal muscle is one of the largest organs in the human body, playing a central role in mobility, metabolism, and endocrine regulation. The aging global population underscores the need to understand skeletal muscle aging, which is bottlenecked by the lack of in vitro models that recapitulate biological and functional features of aged human muscle as well as discrepancies between humans and animal models. Here, we present a 3D biomimetic aged skeletal muscle model using human primary skeletal muscle cells (SkMCs) embedded in a skeletal muscle-derived decellularized extracellular matrix (dECM) scaffold. Constructs fabricated from aged and young SkMCs were systematically evaluated across structural, molecular, mitochondrial, calcium-handling, and contractile readouts. Compared with young constructs, aged constructs recapitulated several aging-associated biological and functional phenotypes, including smaller myotubes, altered myogenic and inflammatory marker expression, mitochondrial alterations, delayed calcium responses, and weakened contractile forces. This human-relevant platform enables simultaneous assessment of biological and functional aspects of muscle aging and may serve as a translational tool to study mechanisms and screen therapies for age-associated muscle disorders. This aligns with the FDA Modernization Act 2.0-which recognizes in vitro human systems as alternatives to animal testing-underscoring the practical relevance of this model.
