Human induced pluripotent stem cell (iPSCs)-derived brain organoids could recapitulate the earliest phase of brain development, making them an effectivein vitrosystem for simulating both normal brain development and disorders. However, the current techniques for creating brain organoids are low throughput, suffer from high variability in organoid generation, and frequently involve lengthy processes of transferring and encapsulating the cells in hydrogels. These limitations hinder the application of brain organoids for high-throughput assessment of compounds in academic and industry settings. Here we demonstrate a simple method of generating multiple cerebral organoids from iPSCs on a unique 36PillarPlate with sidewalls and slits without tedious manual intervention that leads to the reproducible generation of cerebral organoids. Embryoid bodies (EBs) formed in an ultra-low attachment (ULA) 384-well plate were transferred to the 36PillarPlate with Matrigel on one-to-one basis by a simple sandwiching and inverting method. The success rate of spheroid transfer was 95 – 100%. Cerebral organoids were generated robustly with the coefficient of variation (CV) below 19% by differentiating EBs on the pillar plate, which demonstrate the robustness of our approach to support organoid culture. The spheroid transfer method on the pillar plate could overcome the potential limitation of organoid variability, reduce cell culture volume, and enhance assay throughput as compared to conventional organoid culture in petri dishes, spinner flasks, and 6-/24-well plates.
