Nanosecond UV lasers stimulate transient Ca<sup>2+</sup>elevations in human hNT astrocytes

Raos, Brad; Graham, Euan; Unsworth, Charles P.

doi:10.1088/1741-2552/aa5f27

Cited by 12 publications

(23 citation statements)

References 46 publications

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“…hNTs have already been used for cell transplantation in stroke therapy [14], demonstrating functional astrocyte-neuron lactate shuttle [15] and developing a human neuronal cell model for human immunodeficiency Virus (HIV)-infected macrophage-induced neurotoxicity [16]. Our group has extensive experience of these cells, patterning both hNT neurons and astrocytes on parylene-C/SiO 2 substrates [7,9,[17][18][19] and demonstrating that hNT astrocytes elevate transient Ca 2+ response stimulated by nanosecond UV lasers [20]. In addition, hNT neurons and hNT astrocytes do not raise ethical concerns as they come from an immortalised stem cell line [21].…”

Section: Ntera2 Cell Line Derived Astrocytesmentioning

confidence: 99%

“…A 200 nm SiO 2 layer, verified with a Nanometrics NanoSpec/ AFT Microarea gauge, was produced on silicon wafers under a flow of 1.88 and 1.25 sccm of H 2 and O 2 , respectively, in a furnace at 950 °C for 40 min. Then, a 100 nm thick layer of parylene-C was deposited onto oxidised wafers via a speciality coating systems at room temperature [20].…”

Section: Manufacture Of Parylene-c/sio 2 Substratesmentioning

confidence: 99%

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

Simpson

Graham

et al. 2019

J. Neural Eng.

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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 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.

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Section: Ntera2 Cell Line Derived Astrocytesmentioning

confidence: 99%

Section: Manufacture Of Parylene-c/sio 2 Substratesmentioning

confidence: 99%

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

Simpson

Graham

et al. 2019

J. Neural Eng.

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“…Our group has previously demonstrated that nanosecond UV laser stimulation is a reliable and repeatable method to induce Ca 2+ transients in human NTERA2/D1 (hNT) astrocytes [45] in vitro and in small networks of hNT astrocytes [46,47]. Our aim in this article is to expand on this work to generate Ca 2+ elevations in three patient-derived GBM cell-lines.…”

Section: Introductionmentioning

confidence: 99%

Eliciting calcium transients with UV nanosecond laser stimulation in adult patient-derived glioblastoma brain cancer cells in vitro

Mellor,

Chung,

Graham

et al. 2023

J. Neural Eng.

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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 (Ca2+) is a fundamental cell molecular messenger employed in GBM being involved in a wide dynamic range of cellular processes. Understanding how the movement of Ca2+ 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 Ca2+ transients in three patient-derived GBM cell-lines (FPW1, RN1, and RKI1) such that Ca2+ 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 Ca2+ transients. Main results. We determine that 1.58 µJ µm−2 is the optimal UV nanosecond laser pulse energy density necessary to elicit a single Ca2+ transient in the GBM cell-lines whilst maintaining viability, functionality, the ability to be stimulated many times in an experiment, and to trigger further Ca2+ 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 Ca2+ communication in GBM brain cancer cells and their networks being more effectively studied.

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“…Our group is involved in understanding the calcium communication in networks of human hNT astrocytes (Raos et al, 2013(Raos et al, , 2017Jordan et al, 2016;Li et al, 2019). Astrocytes were commonly thought of as perfunctory cells, however, recent evidence has demonstrated that they possess far more functionality and complexity than was commonly thought.…”

Section: Introductionmentioning

confidence: 99%

Critical Spatial-Temporal Dynamics and Prominent Shape Collapse of Calcium Waves Observed in Human hNT Astrocytes in Vitro

Mellor

Graham²,

Unsworth³

2022

Front. Physiol.

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Networks of neurons are typically studied in the field of Criticality. However, the study of astrocyte networks in the brain has been recently lauded to be of equal importance to that of the neural networks. To date criticality assessments have only been performed on networks astrocytes from healthy rats, and astrocytes from cultured dissociated resections of intractable epilepsy. This work, for the first time, presents studies of the critical dynamics and shape collapse of calcium waves observed in cultures of healthy human astrocyte networks in vitro, derived from the human hNT cell line. In this article, we demonstrate that avalanches of spontaneous calcium waves display strong critical dynamics, including power-laws in both the size and duration distributions. In addition, the temporal profiles of avalanches displayed self-similarity, leading to shape collapse of the temporal profiles. These findings are significant as they suggest that cultured networks of healthy human hNT astrocytes self-organize to a critical point, implying that healthy astrocytic networks operate at a critical point to process and transmit information. Furthermore, this work can serve as a point of reference to which other astrocyte criticality studies can be compared.

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Nanosecond UV lasers stimulate transient Ca²⁺elevations in human hNT astrocytes

Cited by 12 publications

References 46 publications

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

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

Eliciting calcium transients with UV nanosecond laser stimulation in adult patient-derived glioblastoma brain cancer cells in vitro

Critical Spatial-Temporal Dynamics and Prominent Shape Collapse of Calcium Waves Observed in Human hNT Astrocytes in Vitro

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Nanosecond UV lasers stimulate transient Ca2+elevations in human hNT astrocytes

Cited by 12 publications

References 46 publications

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

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

Eliciting calcium transients with UV nanosecond laser stimulation in adult patient-derived glioblastoma brain cancer cells in vitro

Critical Spatial-Temporal Dynamics and Prominent Shape Collapse of Calcium Waves Observed in Human hNT Astrocytes in Vitro

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Nanosecond UV lasers stimulate transient Ca²⁺elevations in human hNT astrocytes

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

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