Patterning of functional human astrocytes onto parylene-C/SiO₂ substrates for the study of Ca²⁺ dynamics in astrocytic networks.

Abstract

Recent literature suggests that astrocytes form organized functional networks and communicate through transient changes in cytosolic Ca²⁺. Traditional techniques to investigate network activity, such as pharmacological blocking or genetic knockout, are difficult to restrict to individual cells. The objective of this work is to develop cell-patterning techniques to physically manipulate astrocytic interactions to enable the study of Ca²⁺ in astrocytic networks. We investigate how an in vitro cell-patterning platform that utilizes geometric patterns of parylene-C on SiO₂ can be used to physically isolate single astrocytes and small astrocytic networks. We report that single astrocytes are effectively isolated on 75  ×  75 µm square parylene nodes, whereas multi-cellular astrocytic networks are isolated on larger nodes, with the mean number of astrocytes per cluster increasing as a function of node size. Additionally, we report that astrocytes in small multi-cellular clusters exhibit spatio-temporal clustering of Ca²⁺ transients. Finally, we report that the frequency and regularity of Ca²⁺ transients was positively correlated with astrocyte connectivity. The significance of this work is to demonstrate how patterning hNT astrocytes replicates spatio-temporal clustering of Ca²⁺ signalling that is observed in vivo but not in dissociated in vitro cultures. We therefore highlight the importance of the structure of astrocytic networks in determining ensemble Ca²⁺ behaviour.