MODULARITY EFFECT ON 3D NEURONAL NETWORKS DERIVED FROM HUMAN INDUCED PLURIPOTENT STEM CELLS COUPLED TO HIGH-DENSITY DEVICES

The modular organization of the human brain maintains a critical balance between the segregation and integration of information, leading to a remarkable functional complexity. Replicating this organization in vitro represents a major challenge for developing models that capture human-specific neural dynamics and mechanisms. In this work, three-dimensional neuronal networks derived from human induced pluripotent stem cells (hiPSCs) were used to investigate how modularity shapes network behaviour. Spheroids with two distinct cellular compositions, i.e., with only excitatory or excitatory and inhibitory neurons, were assembled into structures of increasing complexity, from single spheroids to double and triple assembloids. Spontaneous electrophysiological activity was monitored at 63 Days In Vitro with high density Micro-Electrode Arrays (MEAs, 3Brain, 2304 electrodes), providing high-resolution spatiotemporal data. The analysis focused on two fundamental properties: network burst fragmentation, describing how synchronous activity divides into multiple smaller fragments, and dynamical richness, reflecting the variability and diversity of spatiotemporal patterns. We observed that as modularity increased, the networks displayed enhanced complexity, bringing these in vitro models closer to human brain dynamics. Furthermore, the balance between excitatory and inhibitory populations emerged as a key factor, reflecting the impact of inhibitory neurons on cell–cell adhesion and growth dynamics, emphasizing the combined role of both structural and functional modularity. Overall, this study demonstrates that introducing modularity into hiPSCs-derived 3D neuronal networks enhances their functional complexity. These findings highlight that modular assembloids provide an advanced in vitro platform for studying fundamental principles of neural dynamics, with promising applications in disease modeling and personalized medicine.