Los autores desean que gracias Fedex Instituto de Tecnología de la Universidad de Memphis por su apoyo. Los autores desean agradecer a los estudiantes del grupo de proyecto de diseño de alto nivel en el Departamento de Ingeniería Mecánica de la Universidad de Memphis (David Butler, Jackie Carter, Dominick Cleveland, Jacob Shaffer), Daniel Kohn desde el departamento de la Universidad de Memphis Tecnología de Ingeniería para el control del motor y el Dr. Bin Teng y la Sra Charlean Luellen por su ayuda en el cultivo celular. Este trabajo fue apoyado por K01 HL120912 (ER) y R01 HL123540 (CMW).

1. Construcción de membrana con paredes bien para Retención de Cultivo Celular Medios (ver Figura 1D para el producto final) <ol><li> El uso de polidimetilsiloxano (PDMS) láminas revestidas con colágeno I, cortar el contorno de la membrana flexible con un bisturí o un troquel. </li><li> Coloque cada membrana en una placa de Petri de 60 mm para el almacenamiento. </li><li> Creación de paredes: <ol><li> Mezclar PDMS en una proporción de 10: 1 en peso de elastómero de A a elastómero B (endurecedor). </li><li> Vierta 5 ml de PDMS completamente mezclados en tubos de 50 ml. </li><li> Colocar 50 ml tubos con PDMS sin curar horizontalmente en un horno de hibridación. </li><li> Utilice la función de rotor para revestir las paredes interiores de los tubos a 8 rpm durante el tiempo de curado. A TA, la PDMS se curarán completamente en 2 días. </li><li> Retire los PDMS de tubo en una campana de cultivo celular estéril. </li><li> Usando una nueva hoja de afeitar, particionar el cilindro de PDMS en secciones de 4 mm de altura. </li><li> Coloque las secciones, las cuales servirán de wa membranaLLS, en un recipiente placa de Petri sin permitir que las paredes se conviertan arrugado. </li></ol></li><li> Seleccionar y el centro de una pared de PDMS en una membrana, mientras que el mantenimiento de los dos en la placa de Petri. </li><li> Colocar suavemente PDMS sin curar (10: 1 ratio) en el perímetro exterior de la pared para ser utilizados como pegamento. Evitar la formación de huecos entre la pared y la membrana, ya que es allí para retener líquido. </li><li> Coloque cada placa de Petri que contiene la membrana completado y cubierto en un horno a 70 ° C durante 24 horas para curar. </li></ol> 2. Correlación de Rotaciones de motor con abrazadera Desplazamiento o Crecimiento radial de calibración (Figura 2) <ol><li> Inicie el software de control del motor. </li><li> Plegar las abrazaderas del dispositivo utilizando la configuración manual en el software del motor. </li><li> Medir la distancia y registrar la posición de conteo del motor entre las abrazaderas opuestas tanto en el desplazamiento mínimo y máximo de las abrazaderas. </li><li> Calcular el porcentaje de cambio en el dIstance entre las abrazaderas como una función del recuento de motor (~ 0-75k recuentos). Esto indicará la cepa potencial máximo alcanzado en la membrana. </li></ol> 3. Aplicación de estiramiento en la línea celular epitelial de pulmón de ratón (MLE12) <ol><li> Semilla MLE12 células en 2,5 millones de células en la membrana flexible (Paso 1) por pocillo a confluentes en dos días. La densidad de siembra puede variar para diferentes tipos de células. </li><li> Asegúrese de que el accionador mecánico está en una posición completamente relajada. <ol><li> Retire el medio de cultivo celular. </li><li> El uso de 1,5 mm golpes de biopsia, sacador dos agujeros en cada uno de los seis abrazadera (véase la Figura 1D) lengüetas de la membrana. La colocación radial de los orificios determina la cantidad de pre-tensión las experiencias de membrana inicialmente. Perforar los agujeros en un radio de 20,5 mm en las fichas de membrana si no se desea pre-tensión. </li><li> Coloque la membrana en la camilla con los agujeros perforados alineando con los pasadores dentro de las abrazaderas.Coloca los mejores abrazaderas en su lugar. Apriete los tornillos de uno a la vez alternando los lados. </li><li> Añadir 1 ml de medio de cultivo celular. </li></ol></li><li> Coloque el dispositivo en la platina del microscopio de centrado el centro de la membrana con la trayectoria de la luz. </li><li> Use cinta adhesiva o imanes (si es posible) para fijar el dispositivo a la etapa. Una vez fijo, controlar el en el plano (paralelo a la membrana) y vertical (dirección z) del dispositivo mecánico con el controlador de platina del microscopio. </li><li> Aplicar tramo con el software proporcionado por el fabricante por controlar manualmente la posición y la velocidad de rotación del motor 20. </li></ol> 4. Medición de la mitocondriales especies reactivas del oxígeno (ROS) <ol><li> Una vez que las células son confluentes, añadir 1-2 ml de DMEM RT con indicador de superóxido mitocondrial (5 mM concentración final) directamente sobre las células. </li><li> Incubar durante 10 minutos a 37 ° C. </li><li> Lavar las células suavemente tres veces con tampón calentado en un baño de agua para37 ° C. </li><li> Coloque inmediatamente la membrana flexible en la camilla (Paso 2.2) ya en el lugar con un microscopio confocal vertical. </li><li> Añadir 1 ml de DMEM con medio libre de rojo de fenol y 25 mM de HEPES. </li><li> Establecer filtros de excitación / emisión de 510/580 nm. </li><li> Imagen de varios campos de cada 15 min para crear el curso de tiempo deseado de la producción de superóxido mitocondrial. </li><li> A partir de las imágenes capturadas, la intensidad de fluorescencia registro histogramas en cada intervalo de tiempo utilizando un software capaz de cuantificar la intensidad de fluorescencia. </li></ol> 5. Aplicación de Estiramiento y Fuerza Atómica Microscopía (AFM) NOTA: Estos pasos se proporcionan para un AFM específica y la combinación de microscopio óptico (Figura 4 y Lista de Materiales). <ol><li> Preparar el AFM para el experimento. <ol><li> Aumentar la altura de la cabeza AFM a su posición máxima en la dirección z. </li><li> Ponga extensores en las patas de la AFM alevantar el plano en el que AFM en voladizo en contacto con la muestra. El espécimen y la cabeza AFM necesita ser levantado para dar cabida a la altura del dispositivo mecánico. </li></ol></li><li> Preparar el microscopio óptico (si está disponible). <ol><li> Retire la placa de escáner AFM. Retire el objetivo deseado. </li><li> Añadir un espaciador para el objetivo. La altura del espaciador dependería de lo objetivo y lo AFM específica puesta a punto, pero es necesario si se desea imágenes ópticas desde plano de observación será cambio en la dirección z en una cantidad igual a la camilla y la altura del adaptador (Figura 5A). Tenga en cuenta que la AFM por lo general proporciona imágenes bajo aumento de un camino óptico por encima del dispositivo. </li><li> Montar la parte trasera objetivo deseado en su ubicación. Coloque el escáner de nuevo en el AFM. </li><li> Inicie el software de AFM. Iniciar todas las fuentes luminosas necesarias, como la fuente de luz para mediciones de fluorescencia. </li><li> Montar un chip con una viga en voladizo quees apropiado para las mediciones deseadas. Se prefiere 200 pN / rigidez nm o menos cuando se mide el módulo de elasticidad de células vivas. </li><li> Alinear el láser y calibrar la rigidez en voladizo de acuerdo con las sugerencias del fabricante sobre un cubreobjetos de vidrio montado en el dispositivo. </li></ol></li><li> Preparar una membrana como en el Paso 1, pero con las siguientes modificaciones. <ol><li> Inmediatamente antes de montar la membrana en el dispositivo de estiramiento, cortar las paredes a aproximadamente 1 mm de altura. Esto evita la interferencia experimentada entre las paredes de la membrana y la célula de carga. </li><li> Montar la membrana en el dispositivo mecánico tal como se describe 3.2.1-3.2.3. </li><li> Retire el medio de cultivo de células para evitar daños en el escáner AFM en caso de un derrame. </li><li> Pareja el dispositivo con un adaptador (Figura 5A). Coloque el dispositivo mecánico con el adaptador en el escáner. </li><li> Estire la membrana al nivel de deformación a la tracción deseada. </li></ol></li><li&gt; Nano-sangría de células estirados. <ol><li> Añadir un (&lt;0,5 ml) Volumen limitado de los medios en las células para evitar escáner AFM o daños microscopio debido a un derrame. </li><li> Involucrar a la viga en voladizo con la membrana. </li><li> Siga el protocolo del dispositivo de AFM en particular a analizar áreas de interés. </li></ol></li></ol>

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Los autores declaran que no tienen intereses financieros en competencia.

Un dispositivo único para imágenes de células vivas durante el estiramiento mecánico fue desarrollado; y este dispositivo se utilizó en un protocolo para estudiar mecanobiología células epiteliales de pulmón. En estudios preliminares, se encontró que un solo tramo celebrada estimuló la producción de superóxido mitocondrial en las células epiteliales bronquiales. Además, se demostró que el aumento de los niveles de tensión mecánica causaron daño directo a la integridad de una monocapa de células epitel...

Las células se someten a cargas mecánicas en muchos tejidos, y esta estimulación mecánica se ha demostrado para promover cambios en los patrones de la expresión génica, la liberación de factores de crecimiento, citoquinas, o remodelación de la matriz extracelular y el citoesqueleto 1-4. Las señales intracelulares transducidas a partir de tales estímulos mecánicos se producen a través del proceso de mechanotransduction 5-7. En el sistema respiratorio, uno de los resultados de mechanotransduction es el aumento de especies reactivas de oxígeno (ROS) 8,9 y citoquinas pro-inflamatorias 10 en las células epiteliales pulmonares en la presencia de deformación por tracción cíclica. Una fuerte evidencia también sugiere que la deformación por tracción excesiva conduce a dirigir lesión en el epitelio alveolar, además de las respuestas bioquímicas de las células 11-14. Aunque el enfoque aquí es principalmente sobre la respuesta de las células pulmonares a la deformación mecánica, las vías inducidas por mechanotransduction juegan un papel clave en los basic función de muchos tejidos en el cuerpo humano, incluyendo la regulación del tono vascular 15 y el desarrollo de la placa de crecimiento 16. El creciente interés en mechanotransduction se ha traducido en el desarrollo de numerosos dispositivos para la aplicación de cargas mecánicas fisiológicamente relevantes a las células cultivadas y el tejido. En particular, los dispositivos que aplican una tensión de tracción, que es una forma común de la carga mecánica experimentada por el tejido, son populares 11,17-19. Sin embargo, muchos de los dispositivos disponibles están diseñados ya sea como un biorreactor para aplicaciones de ingeniería de tejidos o no son propicias para la formación de imágenes en tiempo real con estiramiento. Por lo tanto, existe la necesidad de desarrollar herramientas y métodos que se pueden visualizar las células y tejidos en tensión para facilitar la investigación de las vías de mecanotransducción. En este documento, un dispositivo de estiramiento mecánico en el plano fue diseñado y protocolos fueron desarrollados para aplicar mformas ultiple de la cepa a los tejidos y células, mientras que permiten imágenes de las respuestas bioquímicas y mecánicas en tiempo real (Figura 1A-D). El dispositivo utiliza seis pinzas uniformemente espaciadas dispuestas circunferencialmente para captar una membrana flexible y aplicar una, distensión radial en el plano a aproximadamente 20% (Figura 1B). El dispositivo de accionamiento puede ser colocado en una incubadora de cultivo celular durante un período prolongado de tiempo, mientras que el motor (Figura 1C) se coloca fuera de la incubadora y controlado por software propietario proporcionada por el proveedor del motor. El motor está conectado a un conductor lineal, que gira una leva interna, conduciendo las seis abrazaderas de camilla de manera uniforme en la tensión y la relajación. Además del dispositivo mecánico, membranas flexibles personalizados se crean a partir de cultivo de células de las membranas disponibles comercialmente listos para ser utilizado en el sistema mecánico. Entonces paredes circulares (con un diámetro de aproximadamente28 mm) se hicieron y se une a la membrana flexible para que las células podrían ser cultivadas sólo en esta región del perfil de deformación bien descrito. Con el fin de determinar si la colocación de estas membranas dentro del dispositivo de accionamiento proporcionaría cepa uniforme e isotrópico en el centro de la membrana flexible, se realizó análisis de elementos finitos utilizando el software disponible comercialmente (Figura 1E-F). La membrana flexible se modeló con condiciones de contorno simétricas y utilizando todos los elementos cuadriláteros para la malla. Los anillos concéntricos visto en el gráfico de contorno de máxima deformación principal se muestra en la Figura 1F indican la distribución isotrópica de la cepa. La tensión experimentada por la membrana se midió mediante la grabación de imágenes de marcas a través de la carga (Figura 2). La figura 2D muestra que la cepa promedio membrana medido en direcciones radiales y axiales era aproximadamente linealcon respecto al motor aplicado cuenta hasta una deformación máxima lineal de 20%. No hubo diferencia significativa entre los niveles de deformación medidos durante la distensión en comparación con los medidos durante la retracción de nuevo a la posición de reposo. A continuación, se midió el desplazamiento de las células humanas bronquiales epiteliales (16HBE) y sus núcleos cultivadas sobre la membrana de encargo flexible. Marcados con fluorescencia (DAPI) núcleos de las células 16HBE se obtuvieron imágenes utilizando un objetivo 20X en un microscopio confocal, mientras que el desplazamiento de célula entera se midió con imágenes de contraste de fase grabadas con un microscopio digital. Como se ve en la Figura 3, la cepa medido por el desplazamiento de los núcleos fue similar al medido por el desplazamiento de las marcas en la membrana, hasta ~ 20% de deformación lineal. Esto confirma que la deformación aplicada a las membranas se transmite a las células adherentes. Los protocolos que describen el uso del dispositivo de encargo en un microscopio tradicional y una fuerza atómica microscópicase se proporcionan en los siguientes pasos.

<table border="0" cellpadding="0" cellspacing="0">
	<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.

Especies reactivas del oxígeno y Deformación Estudios anteriores han demostrado aumentos en especies reactivas de oxígeno (ROS) en las vías respiratorias y las células epiteliales alveolares en respuesta al estiramiento cíclico 21. Especies reactivas de oxígeno incluyen moléculas y radicales libres derivados del oxígeno molecular con alta reactividad a los lípidos, proteínas, polisacáridos, y ácidos nucleicos 22-24. ROS servir como una señal intra...

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.

Imágenes de células vivas durante el estiramiento mecánico

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 vistas.  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. 

Video: 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.

El crecimiento del axón Tramo: El Mecanotransducción de crecimiento neuronal

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

Investigación

Bioingeniería

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.

Células vivas imágenes de la migración de las células que expresan proteínas de fluorescencia etiquetado en una 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.

El seguimiento de los mecánica de la pared durante la implementación del stent en un buque

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.

De alto rendimiento de una sola célula y múltiple las células de micro-encapsulación

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.

El uso de chips de microfluídica para imágenes en vivo y de estudio de las respuestas de lesiones en<em&gt; Drosophila</em&gt; Las 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.

Una técnica de microfluidos investigará Deformabilidad Cell

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 Fluorescencia Fast Imaging a la Ley de Difusión Molecular en membranas de células vivas en un microscopio 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 isocinético para mejorar la fiabilidad de la prueba y el intertasador para las mediciones de reflejo de estiramiento en pacientes con accidente cerebrovascular con espasticidad

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.

Cepa controlada de hidrogeles 3D bajo imágenes de microscopía en 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.

Un sistema microfisiológico para estudiar la interacción leucocitario-endotelial durante la inflamación

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