Engineering Cortical Networks: An Open Platform for Controlled Human Circuit Formation and Synaptic Analysis In Vitro.

Neuronal circuits are organized by specific connections between neuron types across various brain regions. Understanding how these circuits form is crucial for uncovering the mechanisms behind circuit-related dysfunction in brain diseases. Human-induced pluripotent stem cell (iPSC) models enable the study of the molecular and cellular processes underlying neuronal networks, but their lack of precise architecture limits the investigation of specific neuronal interactions and activity-dependent processes. Microfluidic technologies offer structural control but are confined by closed systems that restrict 3D network integration, scalability, and cell retrieval. To overcome these challenges, we developed an open cortical network platform that integrates iPSC-derived cortical neurons with bioengineering techniques. Using a polydimethylsiloxane-based microgroove topography and a cell plating guide, we created "neuronal nodes" that facilitate flexible circuit construction in an open system. This design allows optogenetic control of neural activity and flexible network modifications, including cellular composition, neurite directionality, and synapse formation. The open, large-scale design allows neuronal material retrieval, supporting multi-level analyses of cortical circuits, such as proteomics. This platform represents a valuable tool for investigating neuronal network development and function, providing opportunities for study into both normal and pathological states, including molecular changes associated with connectivity loss in brain diseases.