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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.

Cell patterning is becoming increasingly popular in neuroscience because it allows for the control in the location and connectivity of cells. A recently developed cell patterning technology uses patterns of an organic polymer, parylene-C, on a background of SiO2. When cells are cultured on the parylene-C/SiO2 substrate they conform to the underlying parylene-C geometry. Parylene-C is, however, just one member of a family of parylene polymers that have varying chemical and physical properties. In this work, we investigate whether two commercially available mainstream parylene derivatives, parylene-D, parylene-N and a more recent parylene derivative, parylene-HT to determine if they enable higher fidelity hNT astrocyte cell patterning compared to parylene-C. We demonstrate that all parylene derivatives are compatible with the existing laser fabrication method. We then demonstrate that parylene-HT, parylene-D and parylene-N are suitable for use as an hNT astrocyte cell attractive substrate and result in an equal quality of patterning compared to parylene-C. This work supports the use of alternative parylene derivatives for applications where their different physical and chemical properties are more suitable., Competing Interests: The authors have declared that no competing interests exist.

Objective: Recent literature suggests that astrocytes form organized functional networks and communicate through transient changes in cytosolic Ca 2+ . 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 Ca 2+ in astrocytic networks., Approach: We investigate how an in vitro cell-patterning platform that utilizes geometric patterns of parylene-C on SiO 2 can be used to physically isolate single astrocytes and small astrocytic networks., Main Results: 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 Ca 2+ transients. Finally, we report that the frequency and regularity of Ca 2+ transients was positively correlated with astrocyte connectivity., Significance: The significance of this work is to demonstrate how patterning hNT astrocytes replicates spatio-temporal clustering of Ca 2+ 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 Ca 2+ behaviour.

Recent literature suggests that glia, and in particular astrocytes, should be studied as organised networks which communicate through gap junctions. Astrocytes, however, adhere to most surfaces and are highly mobile cells. In order to study, such organised networks effectively in vitro it is necessary to influence them to pattern to certain substrates whilst being repelled from others and to immobilise the astrocytes sufficiently such that they do not continue to migrate further whilst under study. In this article, we demonstrate for the first time how it is possible to facilitate the study of organised patterned human astrocytic networks using hNT astrocytes in a SiO2 trench grid network that is inlayed with the biocompatible material, parylene-C. We demonstrate how the immobilisation of astrocytes lies in the depth of the SiO2 trench, determining an optimum trench depth and that the optimum patterning of astrocytes is a consequence of the parylene-C inlay and the grid node spacing. We demonstrate high fidelity of the astrocytic networks and demonstrate that functionality of the hNT astrocytes through ATP evoked calcium signalling is also dependent on the grid node spacing. Finally, we demonstrate that the location of the nuclei on the grid nodes is also a function of the grid node spacing. The significance of this work, is to describe a suitable platform to facilitate the study of hNT astrocytes from the single cell level to the network level to improve knowledge and understanding of how communication links to spatial organisation at these higher order scales and trigger in vitro research further in this area with clinical applications in the area of epilepsy, stroke and focal cerebral ischemia., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

We demonstrate, for the first time, how parylene-HT on SiO2 substrates can be used as a human cell patterning platform. We demonstrate this platform with hNT astrocytes, derived from the human NTera2.D1 cell line. We show how hNT astrocytes are attracted to Parylene-HT and repelled by the SiO2 and are shown to adopt a similar morphology as that attained on standard tissue culture polystyrene. Furthermore, parylene-HT was capable of patterning the astrocytes achieving a ratio of 8:1 for cells on parylene compared to SiO2. Thus, as parylene-HT has similar physical properties to parylene-C with the addition of UV and thermal resistance, parylene-HT represents a desirable alternative substrate for human cell patterning.

Cell patterning commonly employs photolithographic methods for the micro fabrication of structures on silicon chips. These require expensive photo-mask development and complex photolithographic processing. Laser based patterning of cells has been studied in vitro and laser ablation of polymers is an active area of research promising high aspect ratios. This paper disseminates how 800 nm femtosecond infrared (IR) laser radiation can be successfully used to perform laser ablative micromachining of parylene-C on SiO2 substrates for the patterning of human hNT astrocytes (derived from the human teratocarcinoma cell line (hNT)) whilst 248 nm nanosecond ultra-violet laser radiation produces photo-oxidization of the parylene-C and destroys cell patterning. In this work, we report the laser ablation methods used and the ablation characteristics of parylene-C for IR pulse fluences. Results follow that support the validity of using IR laser ablative micromachining for patterning human hNT astrocytes cells. We disseminate the variation in yield of patterned hNT astrocytes on parylene-C with laser pulse spacing, pulse number, pulse fluence and parylene-C strip width. The findings demonstrate how laser ablative micromachining of parylene-C on SiO2 substrates can offer an accessible alternative for rapid prototyping, high yield cell patterning with broad application to multi-electrode arrays, cellular micro-arrays and microfluidics.

This paper describes the use of 800nm femtosecond infrared (IR) and 248nm nanosecond ultraviolet (UV) laser radiation in performing ablative micromachining of parylene-C on SiO2 substrates for the patterning of human hNT astrocytes. Results are presented that support the validity of using IR laser ablative micromachining for patterning human hNT astrocytes cells while UV laser radiation produces photo-oxidation of the parylene-C and destroys cell patterning. The findings demonstrate how IR laser ablative micromachining of parylene-C on SiO2 substrates can offer a low cost, accessible alternative for rapid prototyping, high yield cell patterning.

It is estimated that the adult human brain contains 100 billion neurons with 5-10 times as many astrocytes. Although it has been generally considered that the astrocyte is a simple supportive cell to the neuron, recent research has revealed new functionality of the astrocyte in the form of information transfer to neurons of the brain. In our previous work we developed a protocol to pattern the hNT neuron (derived from the human teratocarcinoma cell line (hNT)) on parylene-C/SiO(2) substrates. In this work, we report how we have managed to pattern hNT astrocytes, on parylene-C/SiO(2) substrates to single cell resolution. This article disseminates the nanofabrication and cell culturing steps necessary for the patterning of such cells. In addition, it reports the necessary strip lengths and strip width dimensions of parylene-C that encourage high degrees of cellular coverage and single cell isolation for this cell type. The significance in patterning the hNT astrocyte on silicon chip is that it will help enable single cell and network studies into the undiscovered functionality of this interesting cell, thus, contributing to closer pathological studies of the human brain., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

In our previous work we developed a successful protocol to pattern the human hNT neuron (derived from the human teratocarcinoma cell line (hNT)) on parylene-C/SiO(2) substrates. This communication, reports how we have successfully managed to pattern the supportive cell to the neuron, the hNT astrocyte, on such substrates. Here we disseminate the nanofabrication, cell differentiation and cell culturing protocols necessary to successfully pattern the first human hNT astrocytes to single cell resolution on parylene-C/SiO(2) substrates. This is performed for varying parylene strip widths providing excellent contrast to the SiO(2) substrate and elegant single cell isolation at 10 μm strip widths. The breakthrough in patterning human cells on a silicon chip has widespread implications and is valuable as a platform technology as it enables a detailed study of the human brain at the cellular and network level.