A vascularized 3D muscle microphysiological system for identifying anti-geronic factors in heterochronic parabiosis

Introduction: Skeletal muscle possesses a remarkable regenerative capacity due to the presence of dedicated stem cells, also known as muscle satellite cells (MuSCs), but this ability declines with age. Recent studies utilizing heterochronic parabiosis, in which two animals are surgically joined to share a common blood circulation, have demonstrated remarkable rejuvenation effects across various aged tissues. Despite these findings, the complexity of in vivo parabiosis experiments has made it challenging to identify the underlying mechanisms and key rejuvenation factors. Here, we developed a vascularized 3D muscle microphysiological system (MPS) that replicates essential features of the native MuSC niche. We hypothesized that this engineered platform would enable detailed mechanistic studies of cellular interactions and molecular dynamics relevant to aging in a parabiosis-like environment. Methods: We designed a dual layer microphysiological device separated by a porous membrane with 8 µm pore size. The MuSC vascular network was recreated by co-culturing muscle-relevant cells (myotube-like fibers, endothelial cells, and MuSCs) in an ECM-mimetic hydrogel within the device. Quiescent MuSCs (Pax7 + ) and serum were isolated from the hindlimb muscles of GFP + mice using flow cytometry. Experiments focused on investigating the effects of (1) systemic aging and oxidative stress, (2) vascular microenvironments, and (3) parabiotic circulation on myogenic differentiation of MuSCs. Cytokine profiling during young-old chip interconnection was conducted using Luminex immunoassays to identify rejuvenating factors. RESULTS AND DISCUSSION: We successfully established a vascularized 3D MuSC niche in engineered MPS, where young (3–4 months) and aged (20–24 months) MuSCs were cultured in myofibrillar-like structures embedded in hydrogels covered by an endothelial cell monolayer. This platform enabled real-time monitoring of sequential MuSC myogenic events and sensitive detection of systemic factors. Notably, blood circulation between young and aged MPS systems significantly enhanced myogenic differentiation of aged MuSCs, recapitulating the rejuvenating effects observed in heterochronic parabiosis. After cytokine screening, we identified vascular endothelial growth factor (VEGF) as a key factor contributing to aged MuSC rejuvenation. SIGNIFICANCE/CLINICAL RELEVANCE: Using the 3D muscle MPS integrating young and aged muscle models, we investigated the myogenic activities of MuSCs and systemic cytokine profiles during heterochronic parabiotic circulation. Our 3D MPS model offers significant potential as a preclinical tool to identify anti-geronic factors that promote muscle rejuvenation. References: Lee et al., Adv. Funct. Mater. (2020). National Institute of Health under award number (R01AG072309, R01AR080169, R21AR072287) and Department of Defense under award number W81XWH-20-1-0336. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.