Liver organoid-mediated cyclophosphamide neurotoxicity in CNS organoids in a multi-organ microphysiological system

Abstract Cyclophosphamide (CP) is a widely used alkylating agent whose cytotoxic activity depends on hepatic CYP450-mediated bioactivation. While CP-associated neurotoxicity and cognitive impairment are recognized clinically, the mechanisms of secondary organ damage through metabolic cross-talk remain poorly understood due to limitations of conventional monoculture models. Here we employ a multi-organ microphysiological system (MPS) connecting stem cell derived liver and CNS organoids via microfluidic channels to model inter-organ drug metabolism and secondary toxicity. Liver organoids were treated with CP (0-200 µM) for 48 hours, and connected CNS organoids were assessed for secondary damage by confocal Z-stack imaging of DNA damage (γH2AX), neuronal identity (NeuN), and nuclear content (DAPI). We observe dose-dependent reduction in NeuN expression and γH2AX signal in connected CNS organoids, consistent with neurotoxic metabolite transfer from liver. Critically, CNS-to-CNS control connections show no comparable damage at equivalent CP concentrations, confirming that hepatic metabolism is required for CNS toxicity. These findings validate the MPS platform for modelling multi-organ drug toxicity and provide direct evidence that liver-derived CP metabolites drive secondary neurotoxicity through inter-organ metabolic communication.