Comparative Analysis of Matrigel and Tunable Collagen‐Fibrin Blends for in Vitro Skeletal Muscle Models

ABSTRACT In this study, we describe the gelation kinetics, cytocompatibility, and mechanical properties of interpenetrating networks of collagen (COL), fibrin (FIB), hyaluronan (HA), and laminin (LAM) to evaluate their potential to produce mature skeletal muscle tissue. Skeletal muscle is a dynamic tissue that relies on the fusion of myoblasts into multinucleated myofibers to maintain homeostasis. In progressively degenerative conditions, impaired myoblast fusion leads to skeletal muscle atrophy and significant mass loss. Three‐dimensional (3D) in vitro models for skeletal muscle disease have been developed to better understand disease mechanisms and facilitate drug screening. However, most rely on Matrigel, a tumor‐derived matrix that supports robust cell growth but has limited clinical relevance. To address this limitation, we focused on creating natural, multi‐component scaffolds specifically tailored for muscle applications with clinically relevant drug testing use. Using spectrophotometry and rheology, we characterized the gelation kinetics and viscoelastic properties of interpenetrating networks with varying mass ratios of COL to FIB, supplemented with fixed proportions of HA and LAM. Tunable gelation was achieved within a range of 10 to 16 min. Cytocompatibility studies with C2C12 murine myoblasts demonstrated favorable cell viability in 1:1 and 1:2 (w/w) COL:FIB blends incorporating HA and LAM. Immunostaining of differentiated C2C12 cells confirmed Myosin 4 Monoclonal Antibody (MF‐20) expression in these blends when seeded into polydimethylsiloxane (PDMS)‐anchored bundles. Notably, in cell‐laden 1:1 COL:FIB gels with a seeding density of 10 × 10 6 cells/mL, the compressive modulus increased three‐fold between days 4 and 7 of differentiation. These findings highlight the potential of COL:FIB interpenetrating networks, enhanced with HA and LAM, as promising scaffolds for developing clinically relevant models of skeletal muscle tissue.