Disorders of the brain are often therapeutically managed rather than curative with the number of treatment modalities stalling in recent years. There is a lack of relevant drug screening models because current in vitro 2D cell culture systems fail to recapitulate adult neuronal network connectivity [1]. Here, we culture 3D neuronal networks displaying a level of functional connectivity and maturation which is not achieved using standard culture techniques.Polymer scaffolds were 3D printed using a digital light processor with a microstructure resolution of ~50μm and a maximum structure size of 10x10x9mm X, Y and Z respectively. These scaffolds were used to pattern a 3D gel network by providing support for a bio-instructive composite gel [2]. These materials supported viable (>80%) neuronal cells from both mouse primary and human stem cell sources.Polymer scaffolds showed patterning of 2D networks of seeded cells. When cells were cultured with scaffolds on multielectrode array chips, neuronal firing was observed that was consistent with the constraints of the printed scaffold structure.These findings present a novel system for the patterning and 3D culture neuronal cells. We show a method for complex patterning and functional guidance of in vitro neuronal networks. The materials used promote neurite outgrowth and maturation. Scaffold patterns guide the shape of these networks. These findings form a potential new generation of neuronal cell culture systems.1. Kang, S., et al. Scientific Reports, 2017. 7(1): p. 12233.2. Yan, Y., et al., 2024. 31(2): p. 260-274.e7.