Upcyte® hepatocyte spheroids were grown simultaneously in Gri3D® (SUN bioscience) and U-bottom 96WP to compare ease of use, throughput, assay sensitivity, and scalability for multi-assays. After exposure, drug effects on viability and enzymatic activities were assessed using Promega assays (CellTiter-Glo® 3D, CellToxTM Green, LDH-GloTM, and P450-GloTM) and Tecan’s Spark® Cyto multi-mode imaging plate reader, with readouts encompassing fluorescence and luminescence intensity measurements, and imaging. The assays were performed directly on-plate, without having to disturb the microtissues. Our 3D multiplexing approach, combining Gri3D® and compatible cell-based kits, provides valuable insights into drug impact on CYP activity and microtissue viability, all within a single experiment. This innovative method advances our understanding of liver spheroid responses to drug exposure and holds promise for larger-scale compound assessments and drug screening using 3D cell
cultures.