Human-derived cortical neurospheroids coupled to passive, high-density and 3D MEAs: a valid platform for functional tests

With the advent of human-induced pluripotent stem cells (hiPSCs) differentiation protocols, different methods to create in-vitro human-derived neuronal networks have been proposed. Although monolayer cultures represent a valid model, adding three-dimensionality would make them more representative of an in-vivo environment. Thus, human-derived neurospheroids and brain-organoids are becoming increasingly used for in-vitro disease modeling. Achieving control over the final cell composition and investigating the exhibited electrophysiological activity is still a challenge. Thence, platforms capable of measuring and characterizing the functional aspects of these samples are needed. Here, we propose a method to rapid generate neurospheroids of human origin with control over cell composition that can be used for functional investigations. We show a characterization of the electrophysiological activity exhibited by the neurospheroids by presenting for the first-time results from the main micro-electrodes arrays (MEAs) types available on the market (passive electrode, C-MOS electrodes, 3D electrodes). Neurospheroids grown in free culture and transferred on MEA exhibited functional activity that can be chemically and electrically modulated. Our results indicates that this model holds great potential for in-depth study of signal transmission to drug screening, disease modeling and offers a reproducible and stable platform for in-vitro functional testing. ### Competing Interest Statement Conflict of interest statement: Alessandro Maccione is co-founder and employee of 3Brain AG, Matteo Falappa was a former employee of 3Brain AG.