Preserved functions with profound morphological reorganization in human organotypic cultures

Abstract Human organotypic slice cultures provide experimental access to adult human neuronal circuits ex vivo, yet it remains unclear whether these networks preserve function or undergo fundamental reorganization following the profound perturbation of slice preparation. Here, we combined extracellular population and single-unit electrophysiology, longitudinal calcium imaging, and quantitative histology to track the changes of human cortical slice cultures over several weeks in vitro. Early phases were marked by pronounced variability and instability, with reduced firing rates, increased burst propensity in principal cells, elevated discharge irregularity, and heterogeneous recruitment during population activity. These functional changes coincided with substantial structural remodeling, including considerable neuronal loss, disruption of laminar architecture, reactive gliosis, and selective vulnerability of inhibitory interneurons. Strikingly, despite this progressive structural degradation, neuronal activity did not diverge but instead converged. By the fourth week in culture, electrophysiological properties, cell-type-specific firing patterns, and population-event recruitment became stable and highly consistent across patients. Calcium imaging revealed persistent, spatially confined regions of synchronous activity, indicating the preservation of structured network dynamics. These events remained within physiological regimes and lacked features of epileptiform discharges. Thus, human neuronal circuits exhibit a robust capacity for self-organization, transitioning from heterogeneous, injury-driven dynamics to stable and homogeneous functional states. This dissociation between structural deterioration and functional convergence establishes human organotypic slice cultures as a reproducible and translationally relevant platform for studying human brain network dynamics ex vivo. Competing Interest Statement The authors have declared no competing interest.