High-content interrogation of human induced pluripotent stem cell-derived cortical organoid platforms.

The need for scalable and high-throughput approaches to screening using 3D human stem cell models remains a central challenge in using stem cell disease models for drug discovery. It is imperative to develop standardized systems for phenotypic screening, yet most researchers screen cells across different platforms using a multitude of assays. In this study, we have developed a workflow centered on a small array of assays that can be employed to screen 3D stem cell cultures across a set of platforms. This workflow can be used as a starting point for a standardized approach to phenotypic screening. In this manuscript we hope to provide a roadmap for groups looking to start high-content screening using 3D organoid systems. To do this, we employ serum-free embryoid bodies (SFEBs) created from human induced pluripotent stem cells (hiPSCs). SFEBs are used in this study because they do not display the same level of heterogeneity observed in other neural organoid systems and they are amenable to high content imaging without cryosectioning. They contain populations of excitatory and inhibitory neurons that form synaptically active networks and medium- to high-throughput electrophysiology can be performed using SFEBs via the multielectrode array (MEA). The assays outlined in this study allow SFEBs to be scanned for neurite outgrowth, cell number and electrophysiological activity. SFEBs derived from control and disease hiPSCs can be used in combination with high-throughput screening assays to generate sufficient statistical power to compensate for the biological and experimental variability common in 3D cultures, while significantly decreasing processing speed, thus making this an efficient starting point for phenotypic drug screening.