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Objective. Glioblastoma (GBM) is the most common and lethal type of high-grade adult brain cancer. The World Health Organization have classed GBM as an incurable disease because standard treatments have yielded little improvement with life-expectancy being 6-15 months after diagnosis. Different approaches are now crucial to discover new knowledge about GBM communication/function in order to establish alternative therapies for such an aggressive adult brain cancer. Calcium (Ca 2+ ) is a fundamental cell molecular messenger employed in GBM being involved in a wide dynamic range of cellular processes. Understanding how the movement of Ca 2+ behaves and modulates activity in GBM at the single-cell level is relatively unexplored but holds the potential to yield opportunities for new therapeutic strategies and approaches for cancer treatment. Approach. In this article we establish a spatially and temporally precise method for stimulating Ca 2+ transients in three patient-derived GBM cell-lines (FPW1, RN1, and RKI1) such that Ca 2+ communication can be studied from single-cell to larger network scales. We demonstrate that this is possible by administering a single optimized ultra-violet (UV) nanosecond laser pulse to trigger GBM Ca 2+ transients. Main results. We determine that 1.58 µ J µ m -2 is the optimal UV nanosecond laser pulse energy density necessary to elicit a single Ca 2+ transient in the GBM cell-lines whilst maintaining viability, functionality, the ability to be stimulated many times in an experiment, and to trigger further Ca 2+ communication in a larger network of GBM cells. Significance. Using adult patient-derived mesenchymal GBM brain cancer cell-lines, the most aggressive form of GBM cancer, this work is the first of its kind as it provides a new effective modality of which to stimulate GBM cells at the single-cell level in an accurate, repeatable, and reliable manner; and is a first step toward Ca 2+ communication in GBM brain cancer cells and their networks being more effectively studied., (Creative Commons Attribution license.)

Objective. Platinum nanograss (Ptng) has been demonstrated as an excellent coating to increase the electrode roughness and reduce the impedance of microelectrodes for neural recording. However, the optimisation of the original potentiostatic electrochemical deposition (PSED) method has been performed by the original group only and no in vitro validation of functionality was reported. Approach. This study firstly reinvestigates the use of the PSED method for Ptng coating at different charge densities which highlights non-uniformities in the edges of the microelectrodes for increasing deposition charge densities, leading to a decreased impedance which is in fact an artefact. We then introduce a novel Ptng fabrication method of galvanostatic electrochemical deposition (GSED). Main results. We demonstrate that the GSED deposition method also significantly reduces the electrode impedance, raises the charge storage capacity and provides a significantly more planar electrode surface in comparison to the PSED method with negligible edge effects. In addition, we demonstrate how high-quality neural recordings were performed, for the first time, using the Ptng GSED deposition microelectrodes from human hNT neurons and how spiking and bursting were observed. Significance. Thus, the GSED Ptng deposition method presented here provides an alternative method of microelectrode fabrication for neural applications with excellent impedance and planarity of surface., (© 2021 IOP Publishing Ltd.)

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 SiO 2 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., (© 2021 IOP Publishing Ltd.)

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.

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.

Objective: Astrocytes respond to various stimuli resulting in intracellular Ca 2+ signals that can propagate through organized functional networks. Recent literature calls for the development of techniques that can stimulate astrocytes in a fast and highly localized manner to emulate more closely the characteristics of astrocytic Ca 2+ signals in vivo., Approach: In this article we demonstrate, for the first time, how nanosecond UV lasers are capable of reproducibly stimulating Ca 2+ transients in human hNT astrocytes., Main Results: We report that laser pulses with a beam energy of 4-29 µJ generate transient increases in cytosolic Ca 2+ . These Ca 2+ transients then propagate to adjacent astrocytes as intercellular Ca 2+ waves., Significance: We propose that nanosecond laser stimulation provides a valuable tool for enabling the study of Ca 2+ dynamics in human astrocytes at both a single cell and network level. Compared to previously developed techniques nanosecond laser stimulation has the advantage of not requiring loading of photo-caged or -sensitising agents, is non-contact, enables stimulation with a high spatiotemporal resolution and is comparatively cost effective.