3D Nanoprinted Structures Promote Robust Integration of Brain Organoids into Topography-Guided Neuronal Networks.

Two-photon polymerization-based 3D nanoprinting (3DN) is a potent tool for the fabrication of highly specialized cultivation platforms that enable guided neuronal networks. However, despite the design freedom offered by 3DN, specialized cultivation platforms that actively direct brain organoid behavior in terms of positioning, substrate integration, and neurite outgrowth remain largely absent. Here, we address this by 3D nanoprinting slope, funnel, rod, step, and wall structures that facilitate precise and reliable organoid positioning, promote a tight organoid-substrate interface, and determine the direction of neurite outgrowth. Cultivation platforms featuring these structures are fabricated on coverslips to enable cultivation in standard 12-well cultivation plates. Human cortical organoids (hCOs) cultivated on top exhibit firing of action potentials, highlighting the biocompatibility of the 3DN structures. The slopes and funnels guide the hCO settling, the rods are engulfed by the hCOs, and the walls and steps direct neurite outgrowth and cell migration along their edges. Finally, a cultivation platform featuring all structures demonstrates the potential for the controlled co-culture of two brain organoids. The established 3DN structures pave the way for the implementation of brain organoids in guided neuronal networks and thus may increase the applicability of brain organoids for specialized and customizable brain models.