Abstract

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This study demonstrates the development of a high-throughput screening platform using 3D human induced pluripotent stem cell (iPSC)-derived cortical organoids to identify compounds that modulate neural network activity. These organoids exhibit spontaneous and synchronized calcium oscillations, providing a functional readout for central nervous system (CNS) drug discovery. A screen of 1,280 pharmacologically active compounds identified 111 hits that altered neural network behavior, showcasing the platform’s relevance and robustness for phenotypic screening.

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Key Learnings

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• 3D iPSC-derived neurospheroids produce consistent, spontaneous neural activity detectable via calcium oscillations, ideal for CNS drug screening.
• The platform enables detection of excitatory and inhibitory responses to reference compounds, confirming the presence of functionally integrated neuronal circuits.
• A screen of the LOPAC®1280 library revealed 111 active compounds, with 16 of 17 follow-up hits confirmed via dose-response.
• This assay provides an innovative and scalable approach to human-relevant neuroactive compound discovery.

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Methods

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• Model System: Human iPSC-derived cortical neurospheroids composed of neurons and astrocytes, cultured in 384-well formats.
• Assay Readout: Calcium oscillation detection using FLIPR Tetra® to monitor spontaneous network activity.
• Screening: LOPAC®1280 compound library tested at 1 μM in duplicate; hits defined as activity changes exceeding twice the standard deviation of controls.
• Analysis: Activity measured at 30 min, 2 hr, and 4 hr post-treatment. Peak frequency and amplitude were evaluated.
• Confirmation: 17 compounds were selected for dose-response testing; 16 confirmed to modulate network activity reproducibly.

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Conclusions

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The 3D cortical spheroid model enables robust and reproducible assessment of spontaneous neural network activity. It overcomes limitations in 2D models and rodent systems by offering a human-relevant platform with consistent activity across wells and strong biological relevance. The system reliably identifies neuroactive compounds, supporting its use in CNS phenotypic drug discovery, disease modeling, and functional validation efforts.

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Reference

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Woodruff G, Phillips N, Carromeu C, et al. Screening for modulators of neural network activity in 3D human iPSC-derived cortical spheroids. PLoS One. 2020;15(10):e0240991. doi:10.1371/journal.pone.0240991