Hydrodynamically assembled multicellular system toward structural self-organization and functional crosstalk

The physiological relevance of dorsal root ganglion cultures is limited by monolayer formats that disrupt native multicellular organization. We present an in vitro model in which sensory neurons and glial cells are rapidly assembled into defined geometries using a sound-driven hydrodynamic process that requires no scaffold. This initial physical organization enables spontaneous self-assembly that restores essential features of native tissue. Glial cells envelop neurons with reduced gliosis, neurons regain an upright soma morphology, and axons extend in three dimensions. Mechanosensitive signaling shifts, with decreased nuclear Yes-associated protein (YAP) in glia and increased levels in neurons. The assembled in vitro system exhibits elevated neuronal calcium activity, enhanced cross-neuron correlation, and cluster-dependent activation of extracellular signal-regulated kinase (ERK) and calcitonin gene-related peptide (CGRP). Proteomic analysis shows reduced inflammatory, adhesion, and migratory pathways. This hydrodynamically assembled in vitro platform reveals how multicellular architecture governs neuron-glia communication and provides a scalable tool for studying sensory neurobiology.