Profiling paracrine interactions between hypoxic and normoxic skeletal muscle tissue in a microphysiological system fabricated from 3D printed components.

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Disrupted blood flow in conditions such as peripheral artery disease and critical limb ischemia leads to variations in oxygen supply within skeletal muscle tissue, creating regions of poorly perfused, hypoxic skeletal muscle surrounded by regions of adequately perfused, normoxic muscle tissue. These oxygen gradients may have significant implications for muscle injury or disease, as mediated by the exchange of paracrine factors between differentially oxygenated tissue. However, creating and maintaining heterogeneous oxygen landscapes within a controlled experimental setup to ensure continuous paracrine signaling is a technological challenge. Here, we engineer oxygen-controlled microphysiological systems to investigate paracrine interactions between differentially oxygenated engineered muscle tissue. We fabricated microphysiological systems with dual oxygen landscapes that also had engineered control over paracrine interactions between hypoxic and normoxic skeletal muscle tissues, which were differentiated from C2C12 myoblasts cultured on micromolded gelatin hydrogels. The microphysiological systems interfaced with a new 3D-printed oxygen control well plate insert, which we designed to distribute flow to multiple microphysiological systems and minimize evaporation for longer timepoints. With our system, we demonstrated that amphiregulin, a myokine associated with skeletal muscle injury, exhibits unique upregulation in both gene expression and secretion after 24 hours due to paracrine interactions between hypoxic and normoxic skeletal muscle tissue. Our platform can be extended to investigate other impacts of paracrine interactions between hypoxic and normoxic skeletal muscle and can more broadly be used to elucidate many forms of oxygen-dependent crosstalk in other organ systems.