Microfluidic co-culture system for synaptically segregated neural networks to explore astrocyte-driven neural pathology

Abstract Investigating astrocyte–neuron communication in the absence of neuron-to-neuron signalling is challenging using traditional culture systems due to the complexity of synaptic networks. To address this, we designed a three-compartment microfluidic co-culture device that fluidically isolates two neuronal populations while permitting astrocyte growth throughout. This design enables assessment of astrocyte-specific contributions to neuropathology between synaptically segregated neurons. The device incorporates ten microchannel banks forming maze-like structures that restrict neurite extension and fluid exchange, while allowing an astrocyte monolayer to infiltrate all compartments. Using this platform, we exposed one neuron–astrocyte population to the excitotoxin kainic acid (KA) and observed neurite degeneration in the adjacent, fluidically isolated neurons connected only via astrocytes. Pre-treatment of astrocytes with the membrane-permeable chelator BAPTA-AM markedly attenuated this effect, implicating calcium in astrocyte-mediated excitotoxicity. This microfluidic system provides a controllable in vitro model of neuron–astrocyte networks, enabling directional connectivity and mechanistic studies of circuit behaviour. Our findings highlight the utility of this platform for exploring intercellular signalling pathways relevant to neurodegenerative disease.