Multi-organoid loop cerebral connectoids exhibit enhanced neuronal network dynamics and sequence-specific entrainment.

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.