Hybrid biofabrication of multilayered 3D neuronal networks with structural and functional interlayer connectivity

In vitro implementation of three-dimensional (3D) neuronal models that mimic the brain's structure, such as the modular organization, requires technologies that can precisely fabricate structural organization and functional connectivity. Conventional microextrusion bioprinting provides spatial control to impart structural features to neural tissue models but requires hydrogels of higher viscosity that compromise cellular activity and restrict neurite extension and network formation. In this study, we integrated a micromesh-based bioprinting platform with neuronal analysis techniques to fabricate and analyze the heterogeneous and multilayered modular neuronal constructs using fibrin with high printing resolution and cell viability. The platform was integrated with microelectrode arrays and calcium imaging to simultaneously measure neuronal activity at different layers within the modular organization. We found that each module layer exhibited spontaneous and synchronized activity with synapse formation via neurites connecting module layers. This functional connectivity was further validated by the propagation of electrical stimulation from the bottom layer to the top layer. This study provides a promising foundation for studying structure-function relationships in 3D neuronal networks and developing more physiologically relevant in vitro brain models.