Os autores gostariam que agradecer Fedex Instituto de Tecnologia da Universidade de Memphis para seu apoio. Os autores gostariam de agradecer os alunos do grupo de projeto sênior de design no Departamento de Engenharia Mecânica da Universidade de Memphis (David Butler, Jackie Carter, Dominick Cleveland, Jacob Shaffer), Daniel Kohn do departamento de Universidade de Tecnologia de Engenharia de Memphis para controle de motor e Dr. Teng Bin e Ms. Charlean Luellen por sua ajuda em cultura de células. Este trabalho foi apoiado por K01 HL120912 (ER) e R01 HL123540 (CMW).

1. Construção de membrana com Bem Paredes de Retenção de celular Meios de Cultura (ver Figura 1D para o produto final) <ol><li> Usando o polidimetilsiloxano (PDMS) folhas revestidas com colagénio I, cortar o contorno da membrana flexível com um bisturi ou uma matriz. </li><li> Colocar cada membrana numa placa de 60 mm de Petri para o armazenamento. </li><li> Criação de paredes: <ol><li> Misture PDMS em uma proporção de 10: 1 em peso, de elastômero A a elastômero B (agente de cura). </li><li> Pour 5 ml de PDMS totalmente misturadas em tubos de 50 ml. </li><li> Coloque tubos de 50 ml com PDMS não curado horizontalmente num forno de hibridação. </li><li> Utilizar a função do rotor para revestir as paredes interiores dos tubos a 8 rpm durante o tempo de cura. À TA, o PDMS será totalmente curado em 2 dias. </li><li> Remover o PDMS do tubo em uma capa de cultura de células estéril. </li><li> Usando uma nova lâmina de barbear, particionar o cilindro de PDMS em seções 4 mm de altura. </li><li> Coloque as seções, que servirão de wa membranalls, para um recipiente de placa de Petri, sem permitir que as paredes se tornar vincado. </li></ol></li><li> Escolher e um centro da parede de PDMS em uma membrana, mantendo os dois na placa de Petri. </li><li> Suavemente colocar PDMS não curado (10: 1) de razão no perímetro exterior da parede a ser utilizados como cola. Evitar a formação de lacunas entre a parede ea membrana uma vez que é lá para reter líquido. </li><li> Colocar cada placa de Petri contendo a membrana e completou coberto num forno a 70 ° C durante 24 horas para curar. </li></ol> 2. Correlação de rotações do motor com braçadeira de deslocamento ou Crescimento Radial de Calibração (Figura 2) <ol><li> Inicie o software que controla o motor. </li><li> Deslocar os grampos do dispositivo usando a configuração manual no software do motor. </li><li> Medir a distância e registrar a posição do motor de contagem entre opostos grampos em ambos deslocamento mínimo e máximo dos grampos. </li><li> Calcule a variação percentual no distance entre os grampos em função da contagem do motor (~ 0-75k contagens). Isto irá indicar a tensão máxima atingida no potencial de membrana. </li></ol> 3. Aplicação de estiramento na linha celular epitelial de pulmão do rato (MLE12) <ol><li> Semente MLE12 células em 2,5 milhões de células sobre a membrana flexível (Passo 1) por cavidade a ser confluente dentro de dois dias. A densidade de sementeira pode variar para diferentes tipos de células. </li><li> Certifique-se o actuador mecânico está numa posição completamente relaxado. <ol><li> Remover o meio de cultura de células. </li><li> Usando 1,5 milímetros punções, dois furos em cada um dos seis braçadeira (ver Figura 1D) separadores de membrana. O posicionamento radial dos furos determina a quantidade de pré-tensão da membrana inicialmente as experiências. Perfurar os orifícios com um raio de 20,5 mm nas abas de membrana se sem pré-tensão é desejado. </li><li> Posicione a membrana na maca com os buracos perfurados se alinhando com os pinos dentro dos grampos.Coloque grampos superiores no lugar. Aperte os parafusos, um por vez alternando os lados. </li><li> Adicionar 1 ml de meio de cultura de células. </li></ol></li><li> Colocar o dispositivo na platina do microscópio de centragem meio da membrana com o percurso da luz. </li><li> Use fita ou ímãs (se possível) para fixar o dispositivo para o palco. Uma vez fixo, o controlo (em paralelo com a membrana) e no plano vertical (direcção z) do dispositivo mecânico com o controlador de fase do microscópio. </li><li> Aplicar estiramento com o software fornecido pelo fabricante por meio do controle manualmente a posição e velocidade de rotação do motor 20. </li></ol> 4. Medição da mitocondriais Espécies Reativas de Oxigênio (ROS) <ol><li> Uma vez que as células estão confluentes, adicionar 1-2 mL de MEMD com RT indicador superóxido mitocondrial (5 uM de concentração final) directamente nas células. </li><li> Incubar durante 10 min a 37 ° C. </li><li> Lavar as células suavemente três vezes com tampão aquecido num banho de água a37 ° C. </li><li> Imediatamente coloque a membrana flexível na maca (Passo 2.2) já em vigor sob um microscópio confocal vertical. </li><li> Adicionar 1 ml de DMEM com acesso médio de vermelho de fenol e 25 mM de HEPES. </li><li> Definir filtros de excitação / emissão de 510/580 nm. </li><li> Vários campos de imagem cada 15 min para criar o curso da produção de superóxido mitocondrial tempo desejado. </li><li> A partir de imagens capturadas, a intensidade de fluorescência registro histogramas a cada intervalo de tempo usando software capaz de quantificar a intensidade de fluorescência. </li></ol> 5. Aplicação de estiramento e Microscopia de Força Atômica (AFM) Observação: Essas etapas são fornecidas para um AFM específico e combinação microscópio óptico (Figura 4 e Lista de Materiais). <ol><li> Prepare o AFM para o experimento. <ol><li> Aumentar a altura da cabeça AFM para a sua posição máxima na direcção z. </li><li> Coloque extensores sobre as pernas da AFM paralevantar o avião no qual AFM cantilever contatos da amostra. O espécime e a cabeça AFM tem de ser levantada para acomodar a altura do dispositivo mecânico. </li></ol></li><li> Prepare o microscópio óptico (se disponível). <ol><li> Remova a placa de scanner de AFM. Remova o objetivo desejado. </li><li> Adicionar um espaçador para o objectivo. A altura do espaçador dependerá do objetivo eo AFM específico set-up, mas é necessário se imageamento óptico é desejada desde avião de observação será mudança na direção z de um montante igual à maca e altura adaptador (Figura 5A). Note-se que a AFM geralmente proporciona uma baixa ampliação de imagens a partir de um trajecto óptico de cima do dispositivo. </li><li> Monte o objectivo pretendido de volta em sua localização. Coloque o scanner de volta para a AFM. </li><li> Inicie o software AFM. Comece todas as fontes luminosas necessárias, incluindo a fonte de luz para medições de fluorescência. </li><li> Montar um chip com um cantilever queÉ conveniente que as medições desejadas. 200 pN / rigidez nm ou menos é preferida quando se mede o módulo de elasticidade de células vivas. </li><li> Alinhar o laser e calibrar a rigidez cantilever de acordo com as sugestões do fabricante em uma lamela de vidro montado sobre o dispositivo. </li></ol></li><li> Prepara-se uma membrana que no Passo 1, mas com as seguintes modificações. <ol><li> Imediatamente antes da montagem da membrana no dispositivo de alongamento, cortar as paredes de cerca de 1 mm de altura. Isto evita a interferência entre as paredes experimentado membrana e a célula de carga. </li><li> Montagem da membrana no dispositivo mecânico como descrito 3.2.1-3.2.3. </li><li> Remover o meio de cultura de células, para evitar danos AFM para o scanner, no caso de um derramamento. </li><li> Acoplar o dispositivo com um adaptador (Figura 5A). Coloque o dispositivo mecânico com o adaptador no scanner. </li><li> Distender a membrana para o nível de tensão de tracção desejada. </li></ol></li><li&gt; Nano-recuo de células esticadas. <ol><li> Adicionar uma limitada (&lt;0,5 mL) de volume da mídia sobre as células para evitar scanner de AFM ou danos microscópio devido a um derrame. </li><li> Envolver o cantilever com a membrana. </li><li> Seguir o protocolo do dispositivo de AFM em particular para verificar as áreas de interesse. </li></ol></li></ol>

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Os autores declaram que não têm interesses financeiros concorrentes.

Um dispositivo único para imagens de células vivas durante estiramento mecânico foi desenvolvido; e este dispositivo foi usado em um protocolo para estudar epiteliais do pulmão mechanobiology celular. Em estudos preliminares, verificou-se que uma única estiramento realizada estimulou a produção de superóxido mitocondrial em células epiteliais brônquicas. Além disso, foi demonstrado que o aumento dos níveis de tensão mecânica causou danos directos para a integridade de uma monocamada de células epiteliais ...

As células são submetidas a cargas mecânicas em muitos tecidos, e este estímulo mecânico foi mostrado para promover a alterações dos padrões de expressão de genes, a libertação de factores de crescimento, citoquinas, ou remodelação da matriz extracelular e do citoesqueleto 1-4. Os sinais intracelulares transduzidas de tais estímulos mecânicos ocorrer através do processo de mecanotransdutores 5-7. No sistema respiratório, um resultado de mecanotransdutores é o aumento de espécies reactivas de oxigénio (ROS) e 8,9 citocinas pró-inflamatórias 10 em células epiteliais pulmonares, na presença de cíclica esforço de tensão. Uma forte evidência também sugere que a tensão de tracção excessiva leva a dirigir a lesão do epitélio alveolar, para além das respostas bioquímicas das células 11-14. Embora o foco é principalmente na resposta de células pulmonares a deformação mecânica, as vias induzidas por mecanotransdutores desempenhar um papel-chave nos basic função de muitos tecidos no corpo humano, incluindo a regulação do tónus vascular e 15 o desenvolvimento da placa de crescimento 16. O interesse crescente em mecanotransdutores resultou no desenvolvimento de numerosos dispositivos para a aplicação de cargas mecânicas fisiologicamente relevantes para células cultivadas e tecidos. Em particular, dispositivos de aplicação de um esforço de tensão, que é uma forma comum de carregamento mecânico experimentado pelo tecido, são populares 11,17-19. No entanto, muitos dos dispositivos disponíveis ou são concebidos como um biorreactor para aplicações de engenharia de tecidos ou não são favoráveis ​​para imagiologia em tempo real com estiramento. Como tal, existe uma necessidade para desenvolver ferramentas e métodos que podem visualizar as células e tecidos em tensão para facilitar a investigação das vias de mecanotransdutores. Aqui, um dispositivo de estiramento mecânico no plano foi concebido e protocolos foram desenvolvidos para aplicar multiple formas de estirpe para os tecidos e células, enquanto permitindo imagiologia das respostas bioquímicas e mecânicas em tempo real (Figura 1A-D). O dispositivo utiliza seis grampos espaçados uniformemente dispostas circunferencialmente de compreender uma membrana flexível e aplicar uma no plano, a distensão radial até aproximadamente 20% (Figura 1B). O dispositivo de accionamento pode ser colocado em uma incubadora de cultura de células por um período prolongado de tempo, enquanto o motor (Figura 1C) está posicionado do lado de fora da incubadora e controlada por software proprietário fornecido pelo fornecedor do motor. O motor é ligado a um controlador linear, o qual roda um ressalto interno, conduzindo os seis grampos esticadores uniformemente em tensão e relaxamento. Em adição ao dispositivo mecânico, membranas flexíveis personalizadas foram criadas a partir de membranas de cultura de células imediata disponíveis comercialmente para serem utilizados no sistema mecânico. Em seguida, as paredes circulares (com um diâmetro de aproximadamente28 mm) foram feitos e ligados para a membrana flexível, de modo que as células podem ser cultivadas apenas na região do perfil estirpe bem descrito. A fim de determinar se o posicionamento destas membranas no interior do dispositivo de accionamento proporcionaria deformação uniforme e isotrópico no centro da membrana flexível, a análise de elementos finitos foi realizada usando o software comercialmente disponível (Figura 1E-F). A membrana flexível foi modelada com condições de contorno simétricas e utilizando todos os elementos quadrilaterais para a malha. Os anéis concêntricos visto no gráfico de contorno da estirpe principal máxima mostrada na Figura 1F indicam a distribuição isotrópica da estirpe. A tensão experimentado por a membrana foi medida por gravar imagens de marcações através de carga (Figura 2). A Figura 2D mostra que a tensão média de membrana medido em direcções radiais e axiais era aproximadamente linearno que diz respeito ao motor aplicado conta-se a uma estirpe linear máxima de 20%. Não houve diferença significativa entre os níveis de tensão medidos durante a distensão em comparação com os medidos durante a retracção de volta para a posição de repouso. Em seguida, o deslocamento de células epiteliais brônquicas humanas (16HBE) e seus núcleos cultivadas sobre a membrana flexível costume foram medidos. Marcado por fluorescência (DAPI) núcleos das células 16HBE foram visualizados utilizando uma objectiva de 20x num microscópio confocal, enquanto que o deslocamento de células inteiras foi medida com imagens de contraste de fase gravados com um microscópio digital. Como pode ser visto na Figura 3, a tensão medida pelo deslocamento de núcleos foi semelhante ao medido por deslocamento de marcações na membrana, até ~ 20% de deformação linear. Isto confirma que a estirpe aplicada às membranas foi transmitida para as células aderentes. Os protocolos que descrevem a utilização do dispositivo personalizado em um microscópio tradicional e uma força atómica microscope são fornecidos nas seguintes etapas.

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	<tbody>
		<tr>
			<td>SmartMotor NEMA 34: 3400 Series</td>
			<td>MOOG Animatics</td>
			<td>SM3416D</td>
			<td>Integrated motor, controller, amplifier, encoder and communications bus</td>
		</tr>
		<tr>
			<td>Flexcell Membrane (Collagen I coated)</td>
			<td>Flexcell International Corp</td>
			<td>SM2-1010C</td>
			<td>3.5x5.25x0.020&#34;</td>
		</tr>
		<tr>
			<td>Sylgard 184&#160;</td>
			<td>Dow Corning Corporation</td>
			<td></td>
			<td>10:1</td>
		</tr>
		<tr>
			<td>Hoechst 33342&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>H1399</td>
			<td>DAPI stain</td>
		</tr>
		<tr>
			<td>MitoSOX</td>
			<td>Sigma-Aldrich</td>
			<td>M36008</td>
			<td></td>
		</tr>
		<tr>
			<td>Tiron</td>
			<td>Sigma-Aldrich</td>
			<td>D7389&#160;</td>
			<td>mitochondrial superoxide label</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td></td>
			<td></td>
			<td>superoxide inhibitor</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml tubes</td>
			<td>Fisher Scientific</td>
			<td>06-443-19</td>
			<td>Any centriguge tube can be used to create an area for imaging.</td>
		</tr>
		<tr>
			<td>Hybridization oven</td>
			<td>Bellco Glass</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MLE12 Cells</td>
			<td>ATCC</td>
			<td>CRL-2110</td>
			<td>Mouse Lung Epithelial Cells&#160;</td>
		</tr>
		<tr>
			<td>16HBE cells</td>
			<td>ATCC</td>
			<td>CRL-2741</td>
			<td>Human Bronchial Epithelial Cells</td>
		</tr>
		<tr>
			<td>AFM Indentation Experiments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cantilever Beams for Nano-indentation</td>
			<td>Budget Sensors</td>
			<td>Si-Ni30</td>
			<td></td>
		</tr>
		<tr>
			<td>AFM&#160;</td>
			<td>Asylum Research</td>
			<td>MFP3D</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus microscope</td>
			<td>Olympus</td>
			<td>IX-71</td>
			<td>Inverted microscope with 20X and 40X objectives.</td>
		</tr>
		<tr>
			<td>AFM Leg Extenders</td>
			<td>Asylum Research</td>
			<td>Not available</td>
			<td>AFM microscope</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Finite Element Analyses</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ABAQUS</td>
			<td>Simulia</td>
			<td>6.12</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>NIH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscopes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Digital microscope</td>
			<td>Life Technologies</td>
			<td>EVOS XL Core</td>
			<td>Initially a self standing company, now owned by Life Technologies.</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Zeiss</td>
			<td>LSM 710</td>
			<td>2-photon upright microscope</td>
		</tr>
	</tbody>
</table>

live cell imaging during mechanical stretch

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in their capability for measuring responses in real time in live cells or viable tissue. A protocol was developed with the use of a cell actuator to distend live cells grown on or tissues attached to an elastic substrate while imaging with confocal and atomic force microscopy (AFM). Preliminary studies show that tonic stretching of human bronchial epithelial cells caused a significant increase in the production of mitochondrial superoxide. Moreover, using this protocol, alveolar epithelial cells were stretched and imaged, which showed direct damage to the epithelial cells by overdistention simulating one form of lung injury in vitro. A protocol to conduct AFM nano-indentation on stretched cells is also provided.

Espécies Reativas de Oxigênio e Deformação Estudos anteriores mostraram aumentos de espécies reativas de oxigênio (ROS) em vias aéreas e células epiteliais alveolares em resposta ao estiramento cíclico 21. Espécies reactivas de oxigénio incluem moléculas e radicais livres derivados de oxigénio molecular com alta reactividade com lípidos, proteínas, polissacáridos, ácidos nucleicos e 22-24. ROS servir como um sinal intracelular comum para regula...

Watch this Scientific Journal Video about Live Cell Imaging during Mechanical Stretch at JoVE.com

Live Cell Imaging during Mechanical Stretch

A novel imaging protocol was developed using a custom motor-driven mechanical actuator to allow the measurement of real time responses to mechanical strain in live cells. Relevant to mechanobiology, the system can apply strains up to 20% while allowing near real-time imaging with confocal or atomic force microscopy.

Imagens de células vivas durante Mecânica estiramento

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in ...

live-cell-imaging-during-mechanical-stretch

Research

JoVE Journal

Bioengineering

10.3K visualizações.  University of Tennessee Health Science Center. A novel imaging protocol was developed using a custom motor-driven mechanical actuator to allow the measurement of real time responses to mechanical strain in live cells. Relevant to mechanobiology, the system can apply strains up to 20% while allowing near real-time imaging with confocal or atomic force microscopy. 

Vídeo: Live Cell Imaging during Mechanical Stretch

Axon Stretch Growth: The Mechanotransduction of Neuronal Growth

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata are separated from their axon terminals due to skeletal growth of the enlarging organism (Weiss 1941; Gray, Hukkanen et al. 1992). This mechanotransduction induces a secondary mode of neuronal growth capable of accommodating continual elongation of the axon (Bray 1984; Heidemann and Buxbaum 1994; Heidemann, Lamoureux et al. 1995; Pfister, Iwata et al. 2004).
Axon Stretch Growth (ASG) is conceivably a central factor in the maturation of short embryonic processes into the long nerves and white matter tracts characteristic of the adult nervous system. To study ASG in vitro, we engineered bioreactors to apply tension to the short axonal processes of neuronal cultures (Loverde, Ozoka et al. 2011). Here, we detail the methods we use to prepare bioreactors and conduct ASG. First, within each stretching lane of the bioreactor, neurons are plated upon a micro-manipulated towing substrate. Next, neurons regenerate their axonal processes, via growth cone extension, onto a stationary substrate. Finally, stretch growth is performed by towing the plated cell bodies away from the axon terminals adhered to the stationary substrate; recapitulating skeletal growth after growth cone extension.
Previous work has shown that ASG of embryonic rat dorsal root ganglia neurons are capable of unprecedented growth rates up to 10mm/day, reaching lengths of up to 10cm; while concurrently resulting in increased axonal diameters (Smith, Wolf et al. 2001; Pfister, Iwata et al. 2004; Pfister, Bonislawski et al. 2006; Pfister, Iwata et al. 2006; Smith 2009). This is in dramatic contrast to regenerative growth cone extension (in absence of mechanical stimuli) where growth rates average 1mm/day with successful regeneration limited to lengths of less than 3cm (Fu and Gordon 1997; Pfister, Gordon et al. 2011). Accordingly, further study of ASG may help to reveal dysregulated growth mechanisms that limit regeneration in the absence of mechanical stimuli.

A unique tissue engineering method was developed to elongate numerous nerve fibers in culture by recapitulating axon stretch growth; a form of nervous system growth whereby nerves elongate in conjunction with growth of the enlarging body.

Axon Crescimento Stretch: O Mecanotransdução do Crescimento Neuronal

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata ...

Bioengenharia

Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound healing, tissue regeneration, and cancer metastasis, cells must navigate through complex, three-dimensional extracellular tissue. To better understand the mechanisms behind these biological processes, it is important to examine the roles of the proteins responsible for driving cell migration. Here, we outline a protocol to study the mechanisms of cell migration using the epithelial cell line (MDCK), and a three-dimensional, fibrous, self-polymerizing matrix as a model system. This optically clear extracellular matrix is easily amenable to live-cell imaging studies and better mimics the physiological, soft tissue environment. This report demonstrates a technique for directly visualizing protein localization and dynamics, and deformation of the surrounding three-dimensional matrix. 
Examination of protein localization and dynamics during cellular processes provides key insight into protein functions. Genetically encoded fluorescent tags provide a unique method for observing protein localization and dynamics. Using this technique, we can analyze the subcellular accumulation of key, force-generating cytoskeletal components in real-time as the cell maneuvers through the matrix. In addition, using multiple fluorescent tags with different wavelengths, we can examine the localization of multiple proteins simultaneously, thus allowing us to test, for example, whether different proteins have similar or divergent roles. Furthermore, the dynamics of fluorescently tagged proteins can be quantified using Fluorescent Recovery After Photobleaching (FRAP) analysis. This measurement assays the protein mobility and how stably bound the proteins are to the cytoskeletal network.
By combining live-cell imaging with the treatment of protein function inhibitors, we can examine in real-time the changes in the distribution of proteins and morphology of migrating cells. Furthermore, we also combine live-cell imaging with the use of fluorescent tracer particles embedded within the matrix to visualize the matrix deformation during cell migration. Thus, we can visualize how a migrating cell distributes force-generating proteins, and where the traction forces are exerted to the surrounding matrix. Through these techniques, we can gain valuable insight into the roles of specific proteins and their contributions to the mechanisms of cell migration.

Cellular processes such as cell migration have traditionally been studied on two-dimensional, stiff plastic surfaces. This report describes a technique for directly visualizing protein localization and analyzing protein dynamics in cells migrating in a more physiologically relevant, three-dimensional matrix.

Em células vivas, imagens de células que expressam proteínas fluorescentes Migrando-marcado em uma matriz tridimensional

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound ...

Monitoring the Wall Mechanics During Stent Deployment in a Vessel

Clinical trials have reported different restenosis rates for various stent designs1. It is speculated that stent-induced strain concentrations on the arterial wall lead to tissue injury, which initiates restenosis2-7. This hypothesis needs further investigations including better quantifications of non-uniform strain distribution on the artery following stent implantation. A non-contact surface strain measurement method for the stented artery is presented in this work. ARAMIS stereo optical surface strain measurement system uses two optical high speed cameras to capture the motion of each reference point, and resolve three dimensional strains over the deforming surface8,9. As a mesh stent is deployed into a latex vessel with a random contrasting pattern sprayed or drawn on its outer surface, the surface strain is recorded at every instant of the deformation. The calculated strain distributions can then be used to understand the local lesion response, validate the computational models, and formulate hypotheses for further in vivo study.

Stent-induced arterial strain distributions are characterized using an optical surface strain measurement system. This visualization technique is used to gain insights into the impact of stent implantation on the host vessel.

Monitorização da Mecânica de parede durante a implantação de stent em um vaso

Clinical trials have reported different restenosis rates for various stent designs1. It is speculated that stent-induced strain concentrations on the ...

High Throughput Single-cell and Multiple-cell Micro-encapsulation

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement from a bulk fluid environment with applications in high throughput screening, cytometry, and mass spectrometry. We describe a method to not only encapsulate single cells, but to repeatedly capture a set number of cells (here we demonstrate one- and two-cell encapsulation) to study both isolation and the interactions between cells in groups of controlled sizes. By combining drop generation techniques with cell and particle ordering, we demonstrate controlled encapsulation of cell-sized particles for efficient, continuous encapsulation. Using an aqueous particle suspension and immiscible fluorocarbon oil, we generate aqueous drops in oil with a flow focusing nozzle. The aqueous flow rate is sufficiently high to create ordering of particles which reach the nozzle at integer multiple frequencies of the drop generation frequency, encapsulating a controlled number of cells in each drop. For representative results, 9.9 &mu;m polystyrene particles are used as cell surrogates. This study shows a single-particle encapsulation efficiency Pk=1 of 83.7% and a double-particle encapsulation efficiency Pk=2 of 79.5% as compared to their respective Poisson efficiencies of 39.3% and 33.3%, respectively. The effect of consistent cell and particle concentration is demonstrated to be of major importance for efficient encapsulation, and dripping to jetting transitions are also addressed. 
Introduction 
Continuous media aqueous cell suspensions share a common fluid environment which allows cells to interact in parallel and also homogenizes the effects of specific cells in measurements from the media. High-throughput encapsulation of cells into picoliter-scale drops confines the samples to protect drops from cross-contamination, enable a measure of cellular diversity within samples, prevent dilution of reagents and expressed biomarkers, and amplify signals from bioreactor products. Drops also provide the ability to re-merge drops into larger aqueous samples or with other drops for intercellular signaling studies.1,2 The reduction in dilution implies stronger detection signals for higher accuracy measurements as well as the ability to reduce potentially costly sample and reagent volumes.3 Encapsulation of cells in drops has been utilized to improve detection of protein expression,4 antibodies,5,6 enzymes,7 and metabolic activity8 for high throughput screening, and could be used to improve high throughput cytometry.9 Additional studies present applications in bio-electrospraying of cell containing drops for mass spectrometry10 and targeted surface cell coatings.11 Some applications, however, have been limited by the lack of ability to control the number of cells encapsulated in drops. Here we present a method of ordered encapsulation12 which increases the demonstrated encapsulation efficiencies for one and two cells and may be extrapolated for encapsulation of a larger number of cells.
To achieve monodisperse drop generation, microfluidic "flow focusing" enables the creation of controllable-size drops of one fluid (an aqueous cell mixture) within another (a continuous oil phase) by using a nozzle at which the streams converge.13 For a given nozzle geometry, the drop generation frequency f and drop size can be altered by adjusting oil and aqueous flow rates Qoil and Qaq. As the flow rates increase, the flows may transition from drop generation to unstable jetting of aqueous fluid from the nozzle.14 
When the aqueous solution contains suspended particles, particles become encapsulated and isolated from one another at the nozzle. For drop generation using a randomly distributed aqueous cell suspension, the average fraction of drops Dk containing k cells is dictated by Poisson statistics, where Dk = &lambda;k exp(-&lambda;)/(k!) and &lambda; is the average number of cells per drop. The fraction of cells which end up in the "correctly" encapsulated drops is calculated using Pk = (k x Dk)/&Sigma;(k' x Dk'). The subtle difference between the two metrics is that Dk relates to the utilization of aqueous fluid and the amount of drop sorting that must be completed following encapsulation, and Pk relates to the utilization of the cell sample. As an example, one could use a dilute cell suspension (low &lambda;) to encapsulate drops where most drops containing cells would contain just one cell. While the efficiency metric Pk would be high, the majority of drops would be empty (low Dk), thus requiring a sorting mechanism to remove empty drops, also reducing throughput.15
Combining drop generation with inertial ordering provides the ability to encapsulate drops with more predictable numbers of cells per drop and higher throughputs than random encapsulation. Inertial focusing was first discovered by Segre and Silberberg16 and refers to the tendency of finite-sized particles to migrate to lateral equilibrium positions in channel flow. Inertial ordering refers to the tendency of the particles and cells to passively organize into equally spaced, staggered, constant velocity trains. Both focusing and ordering require sufficiently high flow rates (high Reynolds number) and particle sizes (high Particle Reynolds number).17,18 Here, the Reynolds number Re =uDh/&nu; and particle Reynolds number Rep =Re(a/Dh)2, where u is a characteristic flow velocity, Dh [=2wh/(w+h)] is the hydraulic diameter, &nu; is the kinematic viscosity, a is the particle diameter, w is the channel width, and h is the channel height. Empirically, the length required to achieve fully ordered trains decreases as Re and Rep increase. Note that the high Re and Rep requirements (for this study on the order of 5 and 0.5, respectively) may conflict with the need to keep aqueous flow rates low to avoid jetting at the drop generation nozzle. Additionally, high flow rates lead to higher shear stresses on cells, which are not addressed in this protocol. The previous ordered encapsulation study demonstrated that over 90% of singly encapsulated HL60 cells under similar flow conditions to those in this study maintained cell membrane integrity.12 However, the effect of the magnitude and time scales of shear stresses will need to be carefully considered when extrapolating to different cell types and flow parameters. The overlapping of the cell ordering, drop generation, and cell viability aqueous flow rate constraints provides an ideal operational regime for controlled encapsulation of single and multiple cells.
Because very few studies address inter-particle train spacing,19,20 determining the spacing is most easily done empirically and will depend on channel geometry, flow rate, particle size, and particle concentration. Nonetheless, the equal lateral spacing between trains implies that cells arrive at predictable, consistent time intervals. When drop generation occurs at the same rate at which ordered cells arrive at the nozzle, the cells become encapsulated within the drop in a controlled manner. This technique has been utilized to encapsulate single cells with throughputs on the order of 15 kHz,12 a significant improvement over previous studies reporting encapsulation rates on the order of 60-160 Hz.4,15 In the controlled encapsulation work, over 80% of drops contained one and only one cell, a significant efficiency improvement over Poisson (random) statistics, which predicts less than 40% efficiency on average.12
In previous controlled encapsulation work,12 the average number of particles per drop &lambda; was tuned to provide single-cell encapsulation. We hypothesize that through tuning of flow rates, we can efficiently encapsulate any number of cells per drop when &lambda; is equal or close to the number of desired cells per drop. While single-cell encapsulation is valuable in determining individual cell responses from stimuli, multiple-cell encapsulation provides information relating to the interaction of controlled numbers and types of cells. Here we present a protocol, representative results using polystyrene microspheres, and discussion for controlled encapsulation of multiple cells using a passive inertial ordering channel and drop generation nozzle.

Combining monodisperse drop generation with inertial ordering of cells and particles, we describe a method to encapsulate a desired number of cells or particles in a single drop at kHz rates. We demonstrate efficiencies twice exceeding those of unordered encapsulation for single- and double-particle drops.

Alta taxa de transferência de célula única e múltipla de células Micro-encapsulamento

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement ...

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the 'larva chip'. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3rd instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush.

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Drosophila larvae are an attractive model system for live imaging due to their translucent cuticle and powerful genetics. This protocol describes how to utilize a single-layer PDMS device, called the &#39;larva chip&#39; for live imaging of cellular processes within neurons of 3rd instar Drosophila larvae.

O uso de chips microfluídicos para Imagens ao vivo e estudo das respostas Lesão em<em&gt; Drosophila</em&gt; Larvas

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of ...

A Microfluidic Technique to Probe Cell Deformability

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an efficient manner. Typically, data for ~102 cells can be acquired within a 1 hr experiment. An automated image analysis program enables efficient post-experiment analysis of image data, enabling processing to be complete within a few hours. Our device geometry is unique in that cells must deform through a series of micron-scale constrictions, thereby enabling the initial deformation and time-dependent relaxation of individual cells to be assayed. The applicability of this method to human promyelocytic leukemia (HL-60) cells is demonstrated. Driving cells to deform through micron-scale constrictions using pressure-driven flow, we observe that human promyelocytic (HL-60) cells momentarily occlude the first constriction for a median time of 9.3 msec before passaging more quickly through the subsequent constrictions with a median transit time of 4.0 msec per constriction. By contrast, all-trans retinoic acid-treated (neutrophil-type) HL-60 cells occlude the first constriction for only 4.3 msec before passaging through the subsequent constrictions with a median transit time of 3.3 msec. This method can provide insight into the viscoelastic nature of cells, and ultimately reveal the molecular origins of this behavior.

We demonstrate a microfluidics-based assay to measure the timescale for cells to transit through a sequence of micron-scale constrictions.

A técnica de microfluídica para sondar Deformabilidade celular

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an ...

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn&#8217;t need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on &#181;m-sized membrane regions in the micro-to-milli-second time range.

Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups.

De fast imagens de fluorescência com a Lei difusão molecular em membranas de células vivas em um microscópio comercial

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

Isokinetic Robotic Device to Improve Test-Retest and Inter-Rater Reliability for Stretch Reflex Measurements in Stroke Patients with Spasticity

Measuring spasticity is important in treatment planning and determining efficacy after treatment. However, the current tool used in clinical settings has been shown to be limited in inter-rater reliability. One factor in this poor inter-rater reliability is the variability of passive motion while measuring the angle of catch (AoC) measurements. Therefore, an isokinetic device has been proposed to standardize the manual joint motion; however, the benefits of isokinetic motion for AoC measurements has not been tested in a standardized manner. This protocol investigates whether isokinetic motion itself can improve inter-rater reliability for AoC measurements. For this purpose, a robotic isokinetic device was developed that is combined with surface electromyography (EMG). Two conditions, manual and isokinetic motions, are compared with the standardized method to measure the angle and subjective feeling of catch. It is shown that in 17 stroke patients with mild elbow flexor spasticity, isokinetic motion improved the intraclass correlation coefficient (ICC) for inter-rater reliability of AoC measurements to 0.890 [95% confidence interval (CI): 0.685&#8211;0.961] by the EMG criteria, and 0.931 (95% CI: 0.791&#8211;0.978) by the torque criteria, from 0.788 (95% CI: 0.493&#8211;0.920) by manual motion. In conclusion, isokinetic motion itself can improve inter-rater reliability of AoC measurements in stroke patients with mild spasticity. Given that this system may provide greater standardized angle measurements and catch of feeling, it may be a good option for the evaluation of spasticity in a clinical setting.

Using a robotic isokinetic device with electromyography (EMG) measurements, this protocol illustrates that isokinetic motion itself can improve inter-rater reliability for the angle of catch measurements in stroke patients with mild elbow flexor spasticity.

Dispositivo robótico Isokinetic para melhorar a confiabilidade do teste-reteste e do Inter-rater para medidas do reflexo do estiramento em pacientes do curso com spasticity

Measuring spasticity is important in treatment planning and determining efficacy after treatment. However, the current tool used in clinical settings has ...

Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized in vitro stretching methods. Various available systems use 2D substrate-based stretchers, while the accessibility of 3D techniques to strain soft hydrogels, is more restricted. Here, we describe a method that allows external stretching of soft hydrogels from their circumference, using an elastic silicone strip as the sample carrier. The stretching system utilized in this protocol is constructed from 3D-printed parts and low-cost electronics, making it simple and easy to replicate in other labs. The experimental process begins with polymerizing thick (&#62;100 &#956;m) soft fibrin hydrogels (Elastic Modulus of ~100 Pa) in a cut-out at the center of a silicone strip. Silicone-gel constructs are then attached to the printed-stretching device and placed on the confocal microscope stage. Under live microscopy the stretching device is activated, and the gels are imaged at various stretch magnitudes. Image processing is then used to quantify the resulting gel deformations, demonstrating relatively homogenous strains and fiber alignment throughout the gel&#8217;s 3D thickness (Z-axis). Advantages of this method include the ability to strain extremely soft hydrogels in 3D while executing in situ microscopy, and the freedom to manipulate the geometry and size of the sample according to the user&#8217;s needs. Additionally, with proper adaptation, this method can be used to stretch other types of hydrogels (e.g., collagen, polyacrylamide or polyethylene glycol) and can allow for analysis of cells and tissue response to external forces under more biomimetic 3D conditions.

The presented method involves uniaxial stretching of 3D soft hydrogels embedded in silicone rubber while allowing live confocal microscopy. Characterization of the external and internal hydrogel strains as well as fiber alignment are demonstrated. The device and protocol developed can assess the response of cells to various strain regimes.

Tensão controlada de hidrogéis 3D sob imagem de microscopia ao vivo

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized ...

A Microphysiological System to Study Leukocyte-Endothelial Cell Interaction during Inflammation

Leukocyte-endothelial cell interactions play an important role in inflammatory diseases such as sepsis. During inflammation, excessive migration of activated leukocytes across the vascular endothelium into key organs can lead to organ failure. A physiologically relevant biomimetic microfluidic assay (bMFA) has been developed and validated using several experimental and computational techniques, which can reproduce the entire leukocyte rolling/adhesion/migration cascade to study leukocyte-endothelial cell interactions. Microvascular networks obtained from in vivo images in rodents were digitized using a Geographic Information System (GIS) approach and microfabricated with polydimethylsiloxane (PDMS) on a microscope slide. To study the effect of shear rate and vascular topology on leukocyte-endothelial cell interactions, a Computational Fluid Dynamics (CFD) model was developed to generate a corresponding map of shear rates and velocities throughout the network. The bMFA enables the quantification of leukocyte-endothelial cells interactions, including rolling velocity, number of adhered leukocytes in response to different shear rates, number of migrated leukocytes, endothelial cell permeability, adhesion molecule expression and other important variables. Furthermore, by using human-related samples, such as human endothelial cells and leukocytes, bMFA provides a tool for rapid screening of potential therapeutics to increase their clinical translatability.

In this protocol, a biomimetic microfluid assay, which can reproduce a physiologically relevant microvascular environment and reproduce the entire leukocyte adhesion/migration cascade, is employed to study leukocyte-endothelial cell interactions in inflammatory disease.

Um sistema microfisiológico para estudar a interação das células leucócitos-endoteliais durante a inflamação

Leukocyte-endothelial cell interactions play an important role in inflammatory diseases such as sepsis. During inflammation, excessive migration of ...