The 'Astrocyte Network' and the understanding of its communication has been posed as a new grand challenge to be investigated by contemporary science. However, communication studies in astrocyte networks have investigated traditional petri-dish in vitro culture models where cells are closely packed and can deviate from the stellate form observed in the brain. Using novel cell patterning approaches, highly organised, regular grid networks of astrocytes on chip, to single-cell fidelity are constructed, permitting a stellate-like in vitro network model to be realised. By stimulating the central cell with a single UV nanosecond laser pulse, the initiation/propagation pathways of stellate-like networks are re-explored. The authors investigate the mechanisms of intercellular Ca2+ communication and discover that stellate-like networks of adult human astrocytes in vitro actually exploit extracellular ATP release as their dominant propagation pathway to cells in the network locally; being observed even down to the nearest neighbour and next nearest neighbouring cells-contrary to the reported gap junction. This discovery has significant ramifications to many neurological conditions such as epilepsy, stroke and aggressive astrocytomas where gap junctions can be targeted. In cases where such gap junction targeting has failed, this new finding suggests that these conditions should be re-visited and the ATP transmission pathway targeted instead.

Objective. Cell patterning approaches commonly employed to direct the cytoplasmic outgrowth from cell bodies have been via chemical cues or biomaterial tracks. However, complex network designs using these approaches create problems where multiple tracks lead to manifold obstructions in design. A less common but alternative cell patterning modality is to geometrically design the nodes to project the cytoplasmic processes into a specific direction, thus, removing the need for tracks. Jang et al performed an in-depth study of how rodent neuron primaries could be directed accurately using geometric micro-shapes. In parallel and in contrast, to the work of Jang et al we investigate, for the first time, the effect that micro-shape geometry has on the cytoplasmic process outgrowth of human cells of astrocyte origin using the biomaterial parylene-C. Approach. We investigated eight different types of parylene-C micro-shape on SiO2 substrates consisting of the: circle, square, pentagon, hexagon, equilateral triangle and three isosceles triangles with top vertex angles of 14.2°, 28.8°, and 97.6°, respectively. We quantified how each micro-shape influenced the: cell patterning, the directionality of the cytoplasmic process outgrowth and the functionality for human astrocyte. Main results. Human astrocytes became equally well patterned on all different micro-shapes. Human astrocytes could discriminate the underlying micro-shape geometry and preferentially extended processes from the vertices of equilateral triangles and isosceles triangles where the vertex angle equal to 28.8° in a repeatable manner whilst remaining functional. Significance. We demonstrate how human astrocytes are extremely effective at directing their cytoplasmic process outgrowth from the vertices of geometric micro-shapes, in particular the top vertex of triangular shapes. The significance of this work is that it demonstrates that geometric micro-shapes offer an alternative patterning modality to direct cytoplasmic process outgrowth for human astrocytes, which can serve to simplify complex network design, thus, removing the need for tracks.

Recently, the study of communication in an 'Astrocyte Network' has been suggested to be of equal importance to that of the traditional 'Neural Network'. In this paper, for the first time, we use nanosecond laser stimulation to stimulate the central cell in an organized grid network of connected human astrocytes in order to observe calcium wave propagation at the single-cell level. We show that the calcium waves indeed propagate from the central astrocyte to the outer periphery of the organized astrocyte network. We observe also, like astrocytes in standard in vitro petri dishes, that the calcium wave propagates through specific connections to the outer periphery of cells rather than in a uniform radial manner predicted by mathematical theory. The results show that such a platform provides an excellent environment to perform repeatable, controlled studies of calcium wave signal propagation through an organized grid network of human astrocytes at single-cell resolution.

Astrocytes are a non-homogeneous cell type, highly mobile which constantly extend and retract their cytoplasmic processes in what would seem random in direction. In this paper, we investigate how simple geometric microshapes can be used to control the outgrowth of human astrocytes cytoplasmic processes. We investigate the effect of how five regular microshapes: the circle, triangle, square, pentagon and hexagon control astrocyte cytoplasmic process outgrowth. For all the different microshape types, we observe that it is the corners of the shapes that that cause the astrocyte to produce spontaneous outgrowth except for the circle where the outgrowth occurs at a random radial position. This work suggests that the geometry of cell adhesive regions effects the outgrowth of hNT astrocytes.

To further investigate the communication in astrocytic networks, in vitro astrocytes were patterned in simple networks by using parylene-C/SiO2 platform. We designed a 2×2 small network on parylene-C/SiO2 platform and demonstrated, for the first time, that the intracellular calcium response of a single human hNT astrocyte stimulated by UV laser pulses can be transmitted to neighboring astrocytes in this 2×2 small network. The calcium responses of the astrocytes network were recorded and analyzed.

In this paper, we demonstrate, for the first time, how we can pattern grid networks of human hNT astrocytes on parylene-C/SiO2 substrates down to the single cell level. We demonstrate that the functionality of the astrocyte networks by calcium release on the introduction of ATP and show that this is similar to that of a control sample. Thus, we demonstrate that the parylene-C/SiO2 platform is a viable way to investigate glial networks at the single cell level.

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.