Microfluidic Co-Culture System for Synaptically Segregated Neural Networks to Explore Astrocyte-Driven Neural Pathology

Investigating astrocyte–neuron communication in the absence of neuron-to-neuron signalling is not feasible using traditional cell culture, due to the complexity of synaptic networks. To overcome this limitation, we developed a three-compartment microfluidic co-culture device that fluidically isolates two neuronal populations while allowing astrocyte growth throughout. This design enables investigation of astrocyte-specific contributions to neuropathology between synaptically isolated neurons. The device features 10 banks forming maze-like structures that physically and fluidically isolate two primary cortical neuron populations by restricting neurite extension and fluid exchange, while enabling an astrocyte monolayer to infiltrate all compartments. Using this platform, we treated one neuron–astrocyte population with the excitotoxin kainic acid (KA) and observed neurite degeneration in the adjacent, fluidically isolated neuronal population, connected via astrocytes. Pre-treatment of the astrocyte-only compartment with the membrane-permeable calcium chelator BAPTA-AM significantly reduced neurotoxicity in the isolated neurons. These findings indicate a calcium-dependent role for astrocytes in mediating excitotoxic pathology between physically segregated neuronal populations. This is the first platform to construct a neuronal network with directional connectivity between different populations of neurons and astrocytes and enables us to uncouple and independently investigate the role of astrocytes in the temporal and spatial events of neuronal network activity.