MONITORING BRAIN ORGANOID DIFFERENTIATION AND DEVELOPMENT LEVERAGING LIVE-CELL IMAGING AND CYTOMETRY

hPSC-derived brain organoids are physiologically relevant 3D in vitro models that mimic human brain development and cellular organization. They are increasingly employed to study neurodevelopment, neuropathology, and drug screening but face challenges due to long protocols, sensitivity, large size, heterogeneity, and limited temporal methods for objective monitoring. This workflow incorporates live-cell analysis and HTS by cytometry to monitor cerebral organoid differentiation.

Human iPSCs were differentiated into cerebral organoids using a 40-day unguided protocol. Organoid formation and maturation were monitored with Brightfield imaging either continuously using the Incucyte® Live-Cell Analysis System (Day 0–7, 10x) or discretely every 3-4 days using the CellCelector Flex (Day 7+, 4x). Quantification revealed temporal changes in area, eccentricity, and brightness, with distinct stage-specific morphological features. For single-cell characterization, we used the iQue® HTS Platform to analyze viability, pluripotency and cortical marker expression. We observed stage-specific differences, and final timepoint analysis confirmed expected cortical markers and absence of pluripotency markers, indicating successful differentiation.

In a neurodevelopmental assay, organoids exposed to 50 mM ethanol (EtOH) from day 10 showed similar growth and morphology profiles, with EtOH-treated organoids slightly larger than control. On day 31, EtOH-exposure had reduced viability by 10% and significantly increased TUBB3 expression compared to control (74.3%±0.1 vs 41.3%±0.7, respectively), aligning with research demonstrating EtOH-induced cytotoxicity and premature neural differentiation.

The data exemplifies how integrating live-cell analysis with HTS by cytometry enables objective assessment of cerebral organoid differentiation by temporally monitoring growth, morphology, and marker expression, to support neurodevelopmental or toxicity studies.