Scaffold-free assembly of cortical–hippocampal circuit from modular neurospheroids: a high-throughput platform to investigate network development and dynamics

Three-dimensional<i>in vitro</i>models are critical for recapitulating key aspects of neural network development and interregional interactions. We present a scaffold-free modular system based on primary cortical and hippocampal neurospheroids (NSs), which are subsequently coupled to self-assemble into reproducible assembloid-like structures (ASs). Through a multimodal approach, we characterized their morphological, mechanical, metabolic, and functional properties. NSs displayed progressive growth, viability surpassing 2D cultures, and stiffness approaching physiological brain ranges. Immunostaining verified proper neuronal and astrocytic ratios and confirmed a physiologically relevant GABAergic component. Upon coupling, ASs exhibited robust structural integration while maintaining functional modularity. Calcium imaging enabled the investigation of synchronization patterns at modules' interface, while electrophysiology revealed maturation-dependent and configuration-specific emergence of rhythms, a type of activity typically found<i>in vivo</i>. Functional excitation-inhibition balance remained constant throughout development and was pharmacologically modulated successfully. Our platform balances biological relevance and experimental tractability, offering a versatile tool for investigating neural circuit development, network dynamics, and region-specific perturbations in a reproducible and scalable<i>in vitro</i>environment.