Patterning of functional human astrocytes onto parylene-C/SiO<sub>2</sub>substrates for the study of Ca<sup>2+</sup>dynamics in astrocytic networks

Raos, Brad; Simpson, M. Cather; Doyle, Carol; Murray, AF; Graham, Euan; Unsworth, Charles P.

doi:10.1088/1741-2552/aaae1d

Cited by 16 publications

(26 citation statements)

References 37 publications

(59 reference statements)

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“…Visualisation of the astrocyte Ca 2+ signalling was evaluated using time-lapse microscopy and a fluorescent Ca 2+ probe using a method we developed previously (see Raos et al for a detailed description of the methods used [11]). Briefly, astrocytes were labelled with Fluo-4 (1 μm in 1% FBS/Fluorobrite DMEM) for 30 min at 37°C and 5% CO 2 .…”

Section: Methodsmentioning

confidence: 99%

“…Similarly, Hughes et al demonstrated patterning of LUHMES neuronal networks by first patterning a template of glial-like stem cells on parylene-C/SiO 2 substrates [10]. More recently, human neurons and astrocytes, which were derived from the NTera2.D1 (hNT/NT2) cell line, have been patterned by Unsworth et al, Jordan et al, and Raos et al [7, 9, 11, 12]. The NT2 (hNT) cell line provides a readily available source of neurons and astrocytes that are more applicable to human physiology than primary cells of non-human origin [13].…”

Section: Introductionmentioning

confidence: 99%

“…That work was expanded on by Raos et al who demonstrated that the parylene-C/SiO 2 platform can be used to selectively control the patterning of hNT astrocytes in vitro . Raos et al demonstrated that patterned clusters of hNT astrocytes exhibited spatio-temporally clustered Ca 2+ transients that were not observed in non-patterned cultures [11]. Similarly, Jordan et al used parylene-C that was deposited into grids of photolithographically defined SiO 2 trenches to demonstrate that functionality of the hNT astrocytes through ATP evoked calcium signalling was dependent on the grid spacing [12].…”

Section: Introductionmentioning

confidence: 99%

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Evaluation of parylene derivatives for use as biomaterials for human astrocyte cell patterning

et al. 2019

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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 SiO 2 . When cells are cultured on the parylene-C/SiO 2 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.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Evaluation of parylene derivatives for use as biomaterials for human astrocyte cell patterning

et al. 2019

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“…NT2-Astrocytes (NT2A). NT2A cells were generated as described previously (19) and were cultured on uncoated 25 cm 2 culture flasks in DMEM/F12 at 37 ˚C in 20% O2, 5% CO2.…”

Section: For Experimental Conditions Requiring Fetal Bovine Serum (Fbmentioning

confidence: 99%

Expression of inhibitory checkpoint ligands by Glioblastoma Multiforme cells and the implications of an enhanced stem cell-like phenotype

Joseph

Finlay

et al. 2019

Preprint

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Glioblastoma Multiforme is a highly aggressive brain malignancy commonly refractory to classical and novel chemo-, radio-and immuno-therapies, with median survival times of ~15 months following diagnosis. Poor immunological responses exemplified by the downregulation of T-cell activity, and upregulation of immunosuppressive cells within the tumour micro-environment have limited the effectiveness of immunotherapy in GBM to date. Here we show that GBM cells express a large repertoire of inhibitory checkpoint ligands. Furthermore, GBM cells with an enhanced stem cell-like phenotype exhibit heightened levels of inhibitory checkpoint ligands, compared to non-stem cell-like GBM cells. Understanding how GBM modulates an extensive repertoire of immune checkpoint ligands and the functional consequence on immune evasion are necessary to develop effective immuno-therapeutics.

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“…Therefore, the efficient derivation of functional astrocytes from differentiating stem cells is rapidly becoming the focus of many research labs, as it is seen as a crucial step in understanding brain function in health and disease. Furthermore, from a neuroengineering perspective, facilitation of astrocyte procurement enables multiple studies, investigating neural circuit patterning (Delivopoulos et al, 2009;Delivopoulos and Murray, 2011;Unsworth et al, 2011;Raos et al, 2018), and development (Clarke and Barres, 2013) both in vitro and in vivo.…”

Section: Introductionmentioning

confidence: 99%

Fast and Efficient Differentiation of Mouse Embryonic Stem Cells Into ATP-Responsive Astrocytes

Juneja

Nasuto

Delivopoulos

2020

Front. Cell. Neurosci.

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Astrocytes are multifunctional cells in the CNS, involved in the regulation of neurovascular coupling, the modulation of electrolytes, and the cycling of neurotransmitters at synapses. Induction of astrocytes from stem cells remains a largely underdeveloped area, as current protocols are time consuming, lack granularity in astrocytic subtype generation, and often are not as efficient as neural induction methods. In this paper we present an efficient method to differentiate astrocytes from mouse embryonic stem cells. Our technique uses a cell suspension protocol to produce embryoid bodies (EBs) that are neurally inducted and seeded onto laminin coated surfaces. Plated EBs attach to the surface and release migrating cells to their surrounding environment, which are further inducted into the astrocytic lineage, through an optimized, heparin-based media. Characterization and functional assessment of the cells consists of immunofluorescent labeling for specific astrocytic proteins and sensitivity to adenosine triphosphate (ATP) stimulation. Our experimental results show that even at the earliest stages of the protocol, cells are positive for astrocytic markers (GFAP, ALDH1L1, S100β, and GLAST) with variant expression patterns and purinergic receptors (P2Y). Generated astrocytes also exhibit differential Ca 2+ transients upon stimulation with ATP, which evolve over the differentiation period. Metabotropic purinoceptors P2Y 1 R are expressed and we offer preliminary evidence that metabotropic purinoceptors contribute to Ca 2+ transients. Our protocol is simple, efficient and fast, facilitating its use in multiple investigations, particularly in vitro studies of engineered neural networks.

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Patterning of functional human astrocytes onto parylene-C/SiO₂substrates for the study of Ca²⁺dynamics in astrocytic networks

Cited by 16 publications

References 37 publications

Evaluation of parylene derivatives for use as biomaterials for human astrocyte cell patterning

Evaluation of parylene derivatives for use as biomaterials for human astrocyte cell patterning

Expression of inhibitory checkpoint ligands by Glioblastoma Multiforme cells and the implications of an enhanced stem cell-like phenotype

Fast and Efficient Differentiation of Mouse Embryonic Stem Cells Into ATP-Responsive Astrocytes

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Patterning of functional human astrocytes onto parylene-C/SiO2substrates for the study of Ca2+dynamics in astrocytic networks

Cited by 16 publications

References 37 publications

Evaluation of parylene derivatives for use as biomaterials for human astrocyte cell patterning

Evaluation of parylene derivatives for use as biomaterials for human astrocyte cell patterning

Expression of inhibitory checkpoint ligands by Glioblastoma Multiforme cells and the implications of an enhanced stem cell-like phenotype

Fast and Efficient Differentiation of Mouse Embryonic Stem Cells Into ATP-Responsive Astrocytes

Contact Info

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Patterning of functional human astrocytes onto parylene-C/SiO₂substrates for the study of Ca²⁺dynamics in astrocytic networks