MODELING NEURONAL DISEASES WITH AUTOMATICALLY GENERATED IPSC-DERIVED DORSAL FOREBRAIN 3D ORGANOIDS

Neuronal disorders remain difficult to model due to the complexity of the human brain and limitations of conventional in‑vitro systems. iPSC‑derived 3D neural organoids provide a physiologically relevant platform, yet organoid generation is labor‑intensive and variable. To overcome this, we developed an automated culture‑to‑analysis workflow integrating the CellXpress.ai Automated Cell Culture System with FLIPR and HCS.ai imaging technologies.
CellXpress.ai unifies liquid handling, incubation, live imaging, agitation, and analysis, enabling standardized long‑term organoid maintenance with automated media exchanges and developmental monitoring. Functional maturation was assessed via spontaneous calcium oscillations measured on FLIPR and HCS.ai systems following Calcium 6 dye loading, while structural features were evaluated by confocal imaging with neural markers.
After 80 days of automated culture, organoids exhibited multilayer neuroepithelial architecture and robust spontaneous activity, displaying synchronous or asynchronous oscillation modes. Synchronous networks were quantified with FLIPR PeakPro for metrics including peak count, amplitude, and width; asynchronous activity required cell‑resolved imaging analyses of intensity modulation and network synchronicity.
For high‑throughput pharmacology, organoids with synchronous oscillations were prioritized, showing expected responses to AMPA, GABA, 4‑AP, bicuculline, NMDA, and memantine. Dorsal forebrain organoids supported disease modeling: microfluidic pressure pulses induced TBI‑like suppression of oscillatory activity—partially reversible with 4‑AP—and modest viability reductions. Epileptiform activity triggered by 4‑AP was attenuated by valproic acid.
Together, automated iPSC‑derived neural organoids with detailed calcium‑oscillation analysis constitute a scalable, physiologically relevant tool for disease modeling and compound testing.