High-throughput screening of human induced pluripotent stem cell-derived brain organoids

Background: The need for scalable high-throughput screening (HTS) approaches for 3D human stem cell platforms remains a central challenge for disease modeling and drug discovery. We have developed a workflow to screen cortical organoids across platforms. New method: We used serum-free embryoid bodies (SFEBs) derived from human induced pluripotent stem cells (hiPSCs) and employed high-content imaging (HCI) to assess neurite outgrowth and cellular composition within SFEBs. We multiplexed this screening assay with both multi-electrode arrays (MEAs) and single-cell calcium imaging. Results: HCI was used to assess the number of excitatory neurons (VGlut(+)) in experimental replicates of hiPSC-derived SFEBs, demonstrating experiment-to-experiment consistency. Neurite detection using HCI was applied to assess neurite morphology. MEA analysis showed that firing and burst rates in SFEBs decreased with blockade of NMDARs and AMPARs and increased with GABAR blockade. We also demonstrate effective combination of both MEA and HCI to analyze VGlut(+) populations surrounding electrodes within MEAs. HCI-based (Ca2+) transient analysis revealed firing in individual cells surrounding active MEA electrodes. Comparison with existing methods: Current methods to generate neural organoids show high degrees of variability, and often require sectioning or special handling for analysis. The protocol outlined in this manuscript generates SFEBs with high degree of consistency making them amenable to complex assays combining HTS and electrophysiology allowing for an in-depth, unbiased analysis. Conclusions: SFEBs can be used in combination with HTS to compensate for experimental variability common in 3D cultures, while significantly decreasing processing speed, making this an efficient starting point for phenotypic drug screening.