High‐Throughput Assessment of Metabolism‐Mediated Neurotoxicity by Co‐Culture of Neurospheres and Liver Spheroids

Abstract The liver's role in the biotransformation of chemicals is critical for both augmented toxicity and detoxification. However, there has been a significant lack of effort to integrate biotransformation into in vitro neurotoxicity testing. Traditional in vitro neurotoxicity testing systems are unable to assess the qualitative and quantitative differences between parent chemicals and their metabolites as they would occur in the human body. As a result, traditional in vitro toxicity screening systems cannot incorporate hepatic biotransformation to predict the neurotoxic potential of chemical metabolites. To bridge this gap, a high‐throughput, metabolism‐mediated neurotoxicity testing system has been developed, which combines metabolically competent HepaRG cell spheroids with a three‐dimensional (3D) culture of ReNcell VM human neural progenitor cell line. The article outlines protocols for generating HepaRG cell spheroids using an ultralow attachment (ULA) 384‐well plate and for cultivating ReNcell VM in 3D on a 384‐pillar plate with sidewalls and slits (384PillarPlate). Metabolically sensitive test compounds are introduced into the ULA 384‐well plate containing HepaRG spheroids and then tested with 3D‐cultured ReNcell VM on the 384PillarPlate. This configuration permits the in situ generation of metabolites by HepaRG cells and their subsequent testing on neurospheres. By analyzing cell viability data, researchers can determine the IC 50 values for each compound, thus evaluating metabolism‐mediated neurotoxicity. © 2024 Wiley Periodicals LLC. Basic Protocol 1 : HepaRG spheroid culture in an ultralow attachment (ULA) 384‐well plate and the assessment of drug‐metabolizing enzyme (DME) activities Basic Protocol 2 : 3D neural stem cell (NSC) culture on a 384PillarPlate and compound treatment for the assessment of metabolism‐mediated neurotoxicity Basic Protocol 3 : Image acquisition, processing, and data analysis