Large 10  ×  10 single cell grid networks of human hNT astrocytes on raised parylene-C/SiO 2 substrates.

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 SiO ₂ 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); SiO ₂ attraction index (SAI); node index (NI) and connectivity interval (χI), number of components (k) and fielder value (λ _ss ) 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, λ _ss   =  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.