Microfluidic Control of Dorsal-Ventral Patterning Within a Single Forebrain Organoid

ABSTRACT How distinct regional identities emerge within a single developing brain remains poorly understood. Current in vitro models address this by fusing independently generated organoids, but this introduces variability in size, maturation state, and connectivity, confounding the study of regionalization itself. Here, we present a microfluidic platform that supports the co-development of different tissue identities within a single, continuous 3D culture domain. The device integrates controlled microfluidic flow with real-time fluorescence imaging, providing stable perfusion and high-resolution tracking of molecular transport without the need for embedded sensors or disruptive sampling. By delivering SAG, a Sonic hedgehog pathway agonist, to one surface of mouse forebrain organoids, we induced spatially segregated ventral (Nkx2.1 + ) and dorsal (Pax6 + ) domains within a unified tissue architecture. Controlled morphogen delivery is sufficient to drive region-specific fate specification without organoid fusion, offering a practical, scalable alternative for studying tissue regionalization in vitro .