"Smart" Nerves Sprout and Assemble in an Extracellular Matrix‐Based 3D Nerve‐in‐a‐Chip Microfluidic Model

Abstract Controlling directional neurite outgrowth and Schwann cell (SC) migration remains critical for nerve regeneration. While 2D anisotropic topographies guide nerve growth via contact guidance, these models fail to recapitulate the 3D extracellular matrix microenvironment of native nerves. Herein, a microfluidic “nerve‐in‐a‐chip” platform is customized and filled with decellularized nerve matrix hydrogel (DNM‐G) to investigate how microchannel dimensions regulate 3D neurite extension and SC dynamics. Mid‐sized microchannels optimally enhanced longitudinal neurite growth and fasciculation, whereas excessively narrow microchannels impeded both neurite/SC penetration. Crucially, a self‐directing mechanism is identified wherein fasciculated neurite bundles (>3 µm diameter) followed trajectories dictated by highly dynamic branches (“pathfinder neurites” <1 µm diameter). These tiny neurites exhibited cytoskeletal plasticity to enable exploratory branching and directional shifts. Larger‐sized channels induced excessive tiny neurites branching, resulting in directional instability and prolonged pausing intervals that impeded efficient longitudinal neurite extension. The findings revealed a 3D growth paradigm in which microtubule‐driven pathfinder neurites guide collective axonal navigation, which is distinct from 2D contact guidance. This study establishes a biomimetic model for deciphering microenvironmental regulation of nerve regeneration and provides de novo design principles for neural scaffolds, emphasizing the interplay between channel geometry and axonal dynamics.