Large 10 × 10 single cell grid networks of human hNT astrocytes on raised parylene-C/SiO2 substrates

Abstract

Objective. The ‘Astrocytic Network’ is an emerging research field for researchers in cell biology. Culturing astrocytes in organised networks is a novel method for permitting controlled studies and investigations into the calcium transients of such networks. Recent research has photolithographically patterned hNT astrocytes on parylene-C inlayed SiO2 trench grid networks. However, it was observed that the trench networks could not specifically immobilise the astrocyte cell bodies to the nodes of the networks. Approach. In this study, for the first time, we demonstrate how it is possible to establish grid networks of human hNT astrocytes on raised parylene-C structures where the cell bodies are specifically organised down to the single-cell level on nodes of the grid and connected throughout. Main results. Here, we report these to be the largest patterned single-cell grid network of astrocytes of their kind consisting of 100 cells in a 10 × 10 grid arrangement to an 80% efficiency. We quantify the level of patterning through six cell patterning assessment indices: the parylene adhesion index (PAI); SiO2 attraction index (SAI); node index (NI) and connectivity interval (χI), number of components (k) and fielder value (λss) and report that the best connected network is obtained with 65 µm node size, 90 µm node spacing, and 5 µm interconnecting track width (PAI = 0.77 ± 0.040, SAI = 0.12 ± 0.049, NI = 0.81 ± 0.066, χI = 0.25 ± 0.064, k = 2.33 ± 1.528, λss = 0.0249 ± 0.0018). We finally demonstrate, through delivery of ATP, that the networks are functional on the raised parylene-C grid structures. Significance. The significance of this study is that it determines the optimal dimensions to obtain highly organised, large, interlinked, single-cell networks which provide an effective platform to investigate calcium communication within astrocytic networks in an accurate, controlled and repeatable manner.