ORION: AN OPEN-SOURCE PIPELINE FOR REPRODUCIBLE BRAIN ORGANOID GENERATION AND MULTIMODAL ELECTROPHYSIOLOGICAL CHARACTERIZATION

Human brain organoids derived from induced pluripotent stem cells provide unique access to early human neurodevelopment and disease mechanisms. However, high inter-organoid variability and fragmented analytical practices limit cross-study comparisons and translational progress. We developed ORION (Organoid Integrated Open Network), an open-source computational framework that integrates standardized organoid generation with comprehensive electrophysiological characterization to enable end-to-end reproducibility.

ORION implements the single neural rosette (SNR) protocol, which yields telencephalic organoids with predictable cytoarchitecture and functionally mature neurons. The pipeline captures complete metadata from iPSC maintenance through SNR induction to mature organoids. For functional characterization, ORION supports three complementary modalities: high-density extracellular array recordings with automated spike sorting via SpikeInterface (Kilosort, SpykingCircus), local field potential analysis with standardized spectral decomposition, and whole-cell patch-clamp recordings with automated feature extraction.

We validated the pipeline using positive controls (acute mouse brain recordings) and negative controls (empty-well recordings), demonstrating accurate spike detection, physiologically consistent spectral signatures, and reliable quality control gating. Application to SNR-derived organoids revealed spontaneous network activity with pharmacologically modulated responses to 4-aminopyridine, bicuculline, and tetrodotoxin, confirming functional synaptic connectivity. Standardized metadata enabled cross-organoid comparisons, revealing inter-organoid variability in network dynamics and functional differences between brain organoids and chimeric preparations.

ORION provides the neuroscience community with a scalable, fully documented ecosystem that unifies organoid generation metadata, multimodal electrophysiology, and automated analysis. This infrastructure supports cross-laboratory data sharing and comparison, accelerating the use of human organoids as ethical, physiologically relevant models for studying neural development and neurological disorders.