Abstract
This protocol describes the biofabrication of 3D millimeter-scale human muscle units, embedding non-planar muscle fibers wrapped by fibroblasts-rich endomysium and intertwined with microvascular networks. Suspended muscle fibers are formed through the self-assembly of human myoblasts within cylindrical cavities generated in a sacrificial gelatin template cast in a 3D printed frame. Following myotube differentiation, muscle fibers are embedded in a 3D matrix containing endothelial cells and muscle-derived fibroblasts. The cellular complexity of the environment is instrumental to drive fibroblast migration towards muscle fibers and to induce the organ-specific differentiation of endothelial cells. This advanced 3D muscle model can be applied to analyze the biological mechanisms underlying specific muscle diseases which involve a complex remodeling of the muscle environment (e.g., muscular dystrophies and fibrosis) whereby the pathological interplay among different cell populations drives the onset and progression of the disease.
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