Abstract
Reconstructing networks of neurons in vitro is essential for advancing our understanding of functional mechanisms and disease pathogenesis. However, neuronal culture methods including organoids are limited in network structure complexity required for their functionality and dynamics. In this study, we present modular organoid network tissues – loop connectoids – in which multiple cerebral organoids are connected via axon bundles using microfluidic devices. We compared network activity of three- and four-membered loop cerebral connectoids, two reciprocally connected organoids, and single organoids. We observed a significant trend in larger organoid networks exhibiting more complex activity, showing longer activity periods, more bursts, and richer temporal patterns. Additionally, the activity in connectoids shifts closer to a critical state, a hallmark of efficient information processing in the brain, as more organoids are connected. Pharmacological perturbation reveals prominent excitatory and inhibitory responses, supporting the physiological relevance of the observed dynamics. Furthermore, optogenetic stimulation of organoids in a specific sequence can influence their spontaneous activity propagation pattern within the network. This work represents a foundational step toward constructing more complex and physiologically relevant neural networks in vitro, offering a platform for studying neuronal network function and therapeutic intervention.
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Data availability
All source data of graphs and charts of the main manuscript are included in Supplementary Data 1. Any additional information about the data reported in this paper is available from the lead contact upon request.
Code availability
Source codes for processing MEA data are available on GitHub here https://github.com/utokyoIkeuchilab/loop-connectoid-activity-MEA-analysis. Calculations of network connectivity metrics using previously published32 functions can be found here http://www.brain-connectivity-toolbox.net/. The analysis on criticality metrics using previously published35 functions can be found here http://www.nicholastimme.com/software.html.
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Acknowledgements
We thank all Ikeuchi lab members for discussions and comments. We also thank past members, especially Tatsuya Osaki and Yasuhiro Ikegami, for discussions. This work was supported in part by a Grant-in-Aid for Challenging Research (Pioneering) from the JSPS (20K20643); a Grant-in-Aid for Transformative Research Areas (20H05786, 24H02307, 25H02596); AMED-CREST (JP20gm1410001, 24wm0625318, 25wm0625323); JSPS Core-to-Core Program (JPJSCCA 20190006); HFSP (RGP012/2024) and the Institute for AI and Beyond. The study was also supported by JST SPRING (JPMJSP2108), the ANRI fellowship, and a Grant-in-Aid for Research Activity Start-up from JSPS (25K23557).
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T.D. and Y.I. designed the experiments and wrote the manuscript. T.D performed the experiments and analyzed the data under the supervision of Y.I.
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Communications Biology thanks Itzy E. Morales Pantoja, Francesca Ciarpella and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Benjamin Bessieres. A peer review file is available.
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Duenki, T., Ikeuchi, Y. Multi-organoid loop cerebral connectoids exhibit enhanced neuronal network dynamics and sequence-specific entrainment. Commun Biol (2026). https://doi.org/10.1038/s42003-026-09589-9
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DOI: https://doi.org/10.1038/s42003-026-09589-9
