Speaker
Description
The neuromuscular junction (NMJ) mediates the transfer of neural signals to skeletal muscle fibers, enabling muscle contraction. The structural organization of the NMJ is critical for efficient signal conduction, and disruptions in its morphology are associated with neuromuscular disorders. However, conventional in vitro models, including 2D culture systems and animal models, offer limited ability to replicate the spatial complexity, structural fidelity, and dynamic interactions characteristic of native NMJs. Moreover, relatively few studies have systematically investigated how defined spatial separation between neural and muscle components affects NMJ formation in vitro, despite its critical role in synaptic development and intercellular communication. These limitations reduce the physiological relevance of mechanistic studies and disease modeling. To address this, a 3D in vitro platform was developed to provide defined spatial arrangement of neural and muscle components and to enable comparative evaluation of NMJ formation under static and interstitial flow conditions. A 3D in vitro platform was fabricated using polyethylene-vinyl acetate (PEVA) via 3D printing, designed to provide defined spatial separation between neural and muscle compartments. Platforms with different fixed neural-muscle distances were fabricated to evaluate the influence of spatial separation on NMJ formation. Neural spheroids were manually placed in the neural chamber, and 3D engineered skeletal muscle tissues were anchored to silicone-based mushroom-type pin structures in the muscle chamber. Culture conditions included static culture and interstitial flow, the latter generated passively by height differences in media reservoirs. Fluorescent tracer experiments were conducted to verify the generation of interstitial flow through the axonal guidance region. NMJ formation was assessed by immunostaining for axonal outgrowth and acetylcholine receptor (AChR) clustering following a defined culture period. This 3D in vitro platform offers a physiologically relevant model for investigating neuromuscular junction (NMJ) formation under defined spatial and mechanical conditions. By enabling a defined spatial relationship between neural and muscle components and applying interstitial flow, the system facilitates the study of morphological development and intercellular cross-talk critical for synaptic function. This approach provides a foundation for advancing fundamental research on NMJ physiology and for developing in vitro disease models with improved structural fidelity and experimental reproducibility.
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