Microfluidic device to study spatial and temporal response of astrocyte networks in response to changes in the...

Astrocytes are the most abundant cell type in the central nervous system and are major players in brain homeostasis and inflammation. Astrocytes form a syncytial three-dimensional (3D) network across the brain tissue. Establishing a 3D network allows astrocytes to communicate with each other by a variety of mechanisms, including Ca²⁺ transients. Conversely, spatial and temporal disruption of the astrocyte network can have detrimental effects on brain biology. However, traditional in vitro models have struggled to generate astrocyte networks that can be spatially and temporally perturbed, which limits our capacity to understand astrocyte group dynamics. To address this, we developed a microphysiological platform to investigate both the ways in which 3D astrocyte networks form over time and how they are affected by localized perturbations in the biochemical milieu. We observed that extracellular matrix composition played a critical role in the development of astrocyte network structure, leading to fully interconnected networks within 48 h in optimal conditions. Furthermore, we observed that transient exposure to reactive oxygen species led to long-term disruption of the astrocyte network. This network collapse was accompanied by a decrease in astrocyte redox potential and loss of mitochondrial architecture, which transitioned from an organized filamentous pattern to small and fragmented mitochondria. Additionally, exposure to reactive oxygen species immediately led to disruption of Ca²⁺ transients. Interestingly, even following transient exposure, astrocytes exhibited persistent disruption of the network architecture, with individual cells still exhibiting fragmented mitochondria and Ca²⁺ signaling impairment. These findings highlight how temporary perturbations of the biochemical milieu can result in long-term changes in astrocyte behavior.