Neuronal-glial populations form functional networks in a biocompatible 3D scaffold

•We examine mixed CNS cultures in a biocompatible 3D scaffold.•We compare 3D cultures with conventional monolayer cultures.•Glial morphology in 3D cultures is more consistent with that seen in vivo.•3D cultures form functional neuronal networks.•3D cultures exhibit spontaneous local field potentials. Monolayers of neurons and glia have been employed for decades as tools for the study of cellular physiology and as the basis for a variety of standard toxicological assays. A variety of three dimensional (3D) culture techniques have been developed with the aim to produce cultures that recapitulate desirable features of intact. In this study, we investigated the effect of preparing primary mouse mixed neuron and glial cultures in the inert 3D scaffold, Alvetex. Using planar multielectrode arrays, we compared the spontaneous bioelectrical activity exhibited by neuroglial networks grown in the scaffold with that seen in the same cells prepared as conventional monolayer cultures. Two dimensional (monolayer; 2D) cultures exhibited a significantly higher spike firing rate than that seen in 3D cultures although no difference was seen in total signal power (