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1.
Sci Rep ; 8(1): 2754, 2018 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-29426929

RESUMO

Controlling the spatial distribution of glia and neurons in in vitro culture offers the opportunity to study how cellular interactions contribute to large scale network behaviour. A recently developed approach to cell-patterning uses differential adsorption of animal-serum protein on parylene-C and SiO2 surfaces to enable patterning of neurons and glia. Serum, however, is typically poorly defined and generates reproducibility challenges. Alternative activation methods are highly desirable to enable patterning without relying on animal serum. We take advantage of the innate contrasting surface chemistries of parylene-C and SiO2 to enable selective bonding of polyethylene glycol SiO2 surfaces, i.e. PEGylation, rendering them almost completely repulsive to cell adhesion. As the reagents used in the PEGylation protocol are chemically defined, the reproducibility and batch-to-batch variability complications associated with the used of animal serum are avoided. We report that PEGylated parylene-C/SiO2 substrates achieve a contrast in astrocyte density of 65:1 whereas the standard serum-immersion protocol results in a contrast of 5.6:1. Furthermore, single-cell isolation was significantly improved on PEGylated substrates when astrocytes were grown on close-proximity parylene-C nodes, whereas isolation was limited on serum-activated substrates due tolerance for cell adhesion on serum-adsorbed SiO2 surfaces.


Assuntos
Astrócitos/citologia , Adesão Celular , Polietilenoglicóis/química , Animais , Células Cultivadas , Humanos , Microtecnologia , Polímeros/química , Reprodutibilidade dos Testes , Soro/química , Dióxido de Silício/química , Xilenos/química
2.
J Neural Eng ; 15(3): 036015, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29424361

RESUMO

OBJECTIVE: Recent literature suggests that astrocytes form organized functional networks and communicate through transient changes in cytosolic Ca2+. 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 Ca2+ in astrocytic networks. APPROACH: We investigate how an in vitro cell-patterning platform that utilizes geometric patterns of parylene-C on SiO2 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 Ca2+ transients. Finally, we report that the frequency and regularity of Ca2+ 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 Ca2+ 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 Ca2+ behaviour.


Assuntos
Astrócitos/fisiologia , Sinalização do Cálcio/fisiologia , Diferenciação Celular/fisiologia , Rede Nervosa/fisiologia , Polímeros , Dióxido de Silício , Xilenos , Astrócitos/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Humanos , Rede Nervosa/citologia , Rede Nervosa/efeitos dos fármacos , Polímeros/administração & dosagem , Polímeros/química , Dióxido de Silício/administração & dosagem , Dióxido de Silício/química , Xilenos/administração & dosagem , Xilenos/química
3.
J Neural Eng ; 14(3): 035001, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28291741

RESUMO

OBJECTIVE: Astrocytes respond to various stimuli resulting in intracellular Ca2+ 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 Ca2+ signals in vivo. APPROACH: In this article we demonstrate, for the first time, how nanosecond UV lasers are capable of reproducibly stimulating Ca2+ 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 Ca2+. These Ca2+ transients then propagate to adjacent astrocytes as intercellular Ca2+ waves. SIGNIFICANCE: We propose that nanosecond laser stimulation provides a valuable tool for enabling the study of Ca2+ 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.


Assuntos
Astrócitos/fisiologia , Astrócitos/efeitos da radiação , Sinalização do Cálcio/fisiologia , Sinalização do Cálcio/efeitos da radiação , Lasers , Raios Ultravioleta , Cálcio/metabolismo , Linhagem Celular , Relação Dose-Resposta à Radiação , Humanos , Estimulação Luminosa/métodos , Doses de Radiação
4.
Biomaterials ; 105: 117-126, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27521614

RESUMO

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.


Assuntos
Astrócitos/citologia , Técnicas de Cultura Celular por Lotes/instrumentação , Rede Nervosa/citologia , Polímeros/química , Dióxido de Silício/química , Engenharia Tecidual/instrumentação , Alicerces Teciduais , Xilenos/química , Astrócitos/fisiologia , Técnicas de Cultura Celular por Lotes/métodos , Materiais Biocompatíveis/química , Adesão Celular/fisiologia , Linhagem Celular , Proliferação de Células/fisiologia , Células Cultivadas , Desenho de Equipamento , Análise de Falha de Equipamento , Humanos , Teste de Materiais , Rede Nervosa/fisiologia , Propriedades de Superfície , Engenharia Tecidual/métodos
5.
Biofabrication ; 5(2): 025006, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23466346

RESUMO

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.


Assuntos
Raios Infravermelhos , Polímeros/química , Dióxido de Silício/química , Xilenos/química , Astrócitos/citologia , Linhagem Celular , Eletrodos , Humanos , Técnicas Analíticas Microfluídicas , Microtecnologia , Neurônios/citologia , Oxirredução , Análise Serial de Tecidos
6.
Artigo em Inglês | MEDLINE | ID: mdl-24109824

RESUMO

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.


Assuntos
Astrócitos/citologia , Encéfalo/citologia , Raios Infravermelhos , Lasers , Microtecnologia/métodos , Polímeros/farmacologia , Raios Ultravioleta , Xilenos/farmacologia , Astrócitos/efeitos dos fármacos , Astrócitos/efeitos da radiação , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Núcleo Celular/efeitos da radiação , Custos e Análise de Custo , Humanos
7.
J Neurosci Methods ; 194(1): 154-7, 2010 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-20933004

RESUMO

In this communication, we describe a new method which has enabled the first patterning of human neurons (derived from the human teratocarcinoma cell line (hNT)) on parylene-C/silicon dioxide substrates. We reveal the details of the nanofabrication processes, cell differentiation and culturing protocols necessary to successfully pattern hNT neurons which are each key aspects of this new method. The benefits in patterning human neurons on silicon chip using an accessible cell line and robust patterning technology are of widespread value. Thus, using a combined technology such as this will facilitate the detailed study of the pathological human brain at both the single cell and network level.


Assuntos
Encéfalo/patologia , Microcomputadores , Neurônios/patologia , Animais , Neoplasias Encefálicas/patologia , Diferenciação Celular , Linhagem Celular Tumoral , Técnicas Citológicas , Humanos , Dispositivos Lab-On-A-Chip , Microtecnologia , Polímeros , Ratos , Ratos Sprague-Dawley , Dióxido de Silício , Teratoma/patologia , Xilenos
8.
Neural Comput ; 18(9): 2122-45, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16846389

RESUMO

In this letter, we demonstrate that the generalization properties of a neural network (NN) can be extended to encompass objects that obscure or segment the original image in its foreground or background. We achieve this by piloting an extension of the noise injection training technique, which we term excessive noise injection (ENI), on a simple feedforward multilayer perceptron (MLP) network with vanilla backward error propagation to achieve this aim. Six tests are reported that show the ability of an NN to distinguish six similar states of motion of a simplified human figure that has become obscured by moving vertical and horizontal bars and random blocks for different levels of obscuration. Four more extensive tests are then reported to determine the bounds of the technique. The results from the ENI network were compared to results from the same NN trained on clean states only. The results pilot strong evidence that it is possible to track a human subject behind objects using this technique, and thus this technique lends itself to a real-time markerless tracking system from a single video stream.


Assuntos
Meio Ambiente , Redes Neurais de Computação , Estimulação Luminosa/métodos , Eletricidade
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