Functional human-derived neurospheroids capture cortical-like network complexity

Three-dimensional in vitro neuronal cultures have emerged as promising platforms for modeling human brain function and disease under controlled conditions. However, their ability to recapitulate in vivo-like complexity and rich dynamics remains unexplored. In this study, we developed and characterized neurospheroids derived from human-induced pluripotent stem cells to investigate how key features, such as three-dimensionality, cellular heterogeneity, and modular organization, contribute to replicating brain-like network dynamics. We engineered neurospheroids with varying excitatory/inhibitory ratios and assembled them into modular constructs (assembloids), evaluating their electrophysiological activity with high-density micro-electrode arrays capturing the basal layer of the 3D cultures. We assessed spontaneous and evoked activity through recent metrics of dynamical richness and perturbational complexity. Our results show that three-dimensionality significantly enhances the richness and complexity of network activity, reaching values that can be qualitatively contextualized with in vivo recordings, while cellular heterogeneity contributes to functional aspects of the network, such as nuanced activity patterns, dynamical variability, and modular coordination. Furthermore, the modular constructs demonstrate the scalability of this platform, opening the way to more complex network configurations. These findings highlight the critical role of spatial organization in reproducing cortical-like activity and lay the groundwork for future studies using patient-derived neurospheroids to model disease-specific dynamics.