Esta investigación fue financiada por el Instituto Nacional de Salud de subvención números R01NS050452 y R01EB009429. La fabricación de los chips en voladizo se realizó externamente por los colaboradores en el Centro de nanofabricación situado en la Universidad de Cornell. Todos los equipos utilizados en el proceso de fabricación en voladizo se encuentra en esta instalación. Un agradecimiento especial a Mandy Esch y Jean-Matthieu Prot por su colaboración en voladizo microfabricación. Vídeo de animación de la funcionalidad voladizo fue generada por Charles Hughes, Alex y Eric Zelenin Imperiale desde el Laboratorio de Realidad sintética en la UCF.

1. Cantilever viruta Fabrication Detalles ilustrados de los pasos de fabricación descritos se proporcionan en la Figura 1. <ol><li> Coloque las obleas de silicio sobre aislante (SOI) en un horno y hornear a 125 ° C durante 20 minutos para deshidratar ellos. </li><li> Depositar una gruesa capa de óxido de silicio 1,5 m sobre la capa de mango de la oblea SOI deshidratado usando un Plasma Enhanced Chemical Vapor Deposition (PECVD) de la herramienta. </li><li> Coloque las tabletas sobre el mandril recubridora de rotación con la capa de dispositivo hacia arriba. Asegúrese de oblea se centra, dispensan 2 ml de cebador P20 en el centro de la oblea, y centrifugado a 3.000 rpm durante 60 segundos a una aceleración de 1.000 rpm / seg. P20 imprimación promueve la adhesión de resina fotosensible a la capa de dispositivo. </li><li> Mientras que la oblea todavía está en el revestidor de giro del mandril, prescindir de 2 ml de S1818 fotorresistente en el centro de la oblea, y centrifugado a 3.000 rpm durante 60 segundos a una aceleración de 1000 rpm / sec a Obtain una gruesa capa de resina fotosensible 1,5 m. </li><li> Coloque las tabletas sobre un plato caliente y realizar una cocción suave a 115 ° C durante 1 min. </li><li> Realizar un disco exposición por contacto UV utilizando un alineador de máscara de fotolitografía de contacto con el fin de transferir el patrón de la máscara en voladizo a la resina fotosensible sobre la capa de dispositivo. Calcular el tiempo de exposición basado en el valor energético de la exposición de 125 mJ / cm 2 para S1818 fotoprotector. </li><li> Desarrollar la fotoprotección mediante la inmersión de la oblea en un MIF promotoras de fotorresistencia 726 para 1 min mientras se agita suavemente la oblea de ida y vuelta. </li><li> Enjuague la oblea con (DI) de agua desionizada y se seca, preferiblemente usando una herramienta de secador de enjuague de centrifugado (SRD). </li><li> Retire la fotoprotección desde el borde de la oblea (hasta 5 mm del borde) con un bastoncillo de algodón humedecido en acetona. </li><li> Cargar la oblea en una herramienta de Deep Reactive Ion Etch (DRIE), con la cara de resina fotosensible, y ejecutar una receta para grabar la capa de silicio modelada a través de la Devicapa de ce. Las funciones de la capa de óxido enterrada como una parada de grabado, de modo realizar un grabado con 50% a 100% más ciclos que se espera que sea necesaria para garantizar un grabado completo. </li><li> Coloque las tabletas en un baño de solución fotoprotector caliente durante 20 minutos para quitar la fotoprotección. Transferir la oblea a un-dump-enjuagadora rápida (QDR) para enjuagar la oblea con agua DI. A raíz de la tira fotoprotector, cargue la oblea en una herramienta SRD para enjuagar y secar la oblea. Inspeccionar la oblea bajo un microscopio para asegurar el patrón voladizo aparece como se esperaba. </li><li> Depositar una gruesa capa de óxido de silicio dopado con un-1 m sobre la capa de dispositivo de la oblea usando una herramienta de PECVD. Esta capa de óxido funciones como una capa protectora para los voladizos durante etapas de procesamiento adicionales. </li><li> Coloque la oblea sobre el mandril recubridora de rotación con la capa de mango hacia arriba para comenzar el procesamiento de la parte trasera de la oblea. Asegúrese de oblea se centra, dispensan 2 ml de cebador P20 en el centro de la oblea, y centrifugado a 3000 rpmdurante 60 segundos con una aceleración de 1.000 rpm / seg. </li><li> Mientras que la oblea todavía está en el revestidor de giro del mandril, prescindir de 2 ml de resina fotosensible SPR220-4.5 sobre el centro de la oblea, y centrifugado a 2.000 rpm durante 45 segundos a una aceleración de 1.000 rpm / seg para obtener una capa gruesa de 6,5 micras fotoprotector. </li><li> Coloque la oblea en una placa caliente de proximidad para llevar a cabo una cocción suave a 115 ° C durante 2 min. </li><li> El uso de un alineador de contactos de fotolitografía capaz de delante / atrás alineación, alinear la máscara de la ventana trasera al patrón voladizo lado frontal y realizar un contacto dura la exposición UV con el fin de transferir el patrón de la máscara de la ventana trasera a la resina fotosensible sobre la capa de mango. Utilice un tiempo de exposición calculado con base en el valor de energía de exposición de 480 mJ / cm 2 para fotoprotector SPR220-4.5. </li><li> Después de la exposición UV, permiten las obleas permanecen en una zona oscura durante 30 min antes de la siguiente etapa de procesamiento. </li><li> Desarrollar la fotoprotección sumergiendo la oblea enun desarrollador 726 MIF fotoprotector durante 2 minutos mientras se agita suavemente la oblea de ida y vuelta. </li><li> Enjuague la oblea con (DI) de agua desionizada y se seca, preferiblemente usando una herramienta de secador de enjuague de centrifugado (SRD). </li><li> Coloque las obleas en un horno a 90 ° C durante 12 horas. </li><li> Etch la capa de óxido de silicio en la parte trasera de la oblea utilizando un sistema RIE con gases fluorados y una receta para el grabado de óxido. El óxido de modelado en la parte trasera de la oblea actúa como una máscara de grabado para su posterior procesamiento. Inspeccionar las obleas bajo un microscopio para asegurarse de que el óxido de silicio en la ventana expuesta está completamente grabado. </li><li> Retire la resina fotosensible en el borde de la oblea (hasta 5 mm del borde) usando un hisopo de sala limpia sumergido en acetona. </li><li> Cargar la oblea en una herramienta con DRIE y ejecutar una receta para grabar la capa modelada mango a una profundidad de 500 micras con el funcionamiento capa de óxido enterrada como un tope de grabado. Dividir esta grabado en múltiples carreras para evitar un calentamiento excesivo de laoblea, lo que hace que el grabado inconsistente del silicio. Este paso elimina por completo el ataque químico de silicio por debajo de los voladizos. </li><li> Realizar una etapa de ataque químico en húmedo utilizando 25% grabador HF diluido, para despojar a la capa de óxido enterrada debajo de los voladizos y la capa protectora de óxido en la parte superior de los voladizos. NOTA: Este paso libera la superficie inferior de las palancas de silicio y también abre una ventana debajo para proporcionar un acceso a sondear el voladizo con el láser. </li><li> Enjuague la oblea usando una serie de baños de agua DI y secar cuidadosamente con nitrógeno. Desde los voladizos se apoyan sólo en sus bases, no utilice aerosoles contundentes de agua desionizada o gas inerte directamente en los voladizos. </li><li> Escindir los chips individuales de la oblea a lo largo de las líneas escinden producidos durante el paso de capa de grabado mango. </li></ol> 2. Cell Culture <ol><li> Preparar 13F cubreobjetos de acuerdo con métodos publicados previamente 17. NOTA: Si 13F calarslips no están disponibles, cualquier superficie hidrofóbica con un ángulo de contacto por encima de 95 ° se puede utilizar. </li><li> Esterilizar fichas en voladizo y 13F cubreobjetos en una solución de etanol al 70% y dejar secar al aire en una campana de flujo. </li><li> Coloque fichas individuales en voladizo en la parte superior de 13F cubreobjetos interior una placa de estándar de 12. </li><li> Escudo los voladizos con la modificación de biopolímeros o superficie optimizado para el tipo de células que se utilizan de acuerdo con los protocolos estándar de cultivo celular. </li><li> Vuelva a suspender las células en su medio de crecimiento específico a la concentración deseada. NOTA: Este protocolo se traducirá en un número sustancial de células que caen a través de la ventana en voladizo y no se adhieren a la superficie en voladizo deseado. Por lo tanto, las preparaciones de células se deben hacer 3-4x más concentrada que para las preparaciones cubreobjetos estándar. Por ejemplo, la siembra de las células satélite de músculo esquelético de rata se lleva a cabo típicamente utilizando una densidad de siembra de 500 a 700 células / mm cuadrado 15,16, Y se usan con sustratos en voladizo a 2.000 células / mm cuadrado. Experimentos de optimización deben llevarse a cabo con nuevas fuentes de células para determinar una densidad de siembra apropiada. </li><li> Pipetear 200 l de la suspensión celular sobre la superficie del chip en voladizo, asegurando la burbuja de medio cubre las ventanas en voladizo por completo. Si el medio absorbe a través de la ventana y no forma una burbuja estática en la parte superior de las ménsulas reemplazar el cubreobjetos 13F y reintentar el forro. </li><li> Transferir la placa que contiene las fichas a una incubadora y permitir que las células se adhieran durante al menos 1 hora (preferiblemente 2-3 h). </li><li> Después de este período de chapado, el uso de fórceps estériles para transferir el chip a un pocillo limpio, sin un cubreobjetos 13F, y la parte superior de la cultura con 1 ml de medio de crecimiento. </li><li> Volver a la placa de la incubadora. </li><li> Mantener las células de acuerdo con su protocolo estándar para el mantenimiento in vitro en cubreobjetos. Para las células del músculo esquelético,un interruptor de la composición del medio para inducir la formación de miotubos una vez que las células se vuelven confluentes será necesario. </li></ol> 3. Configuración de Hardware y Software para el Análisis Cantilever deflexión <ol><li> Colocar una placa de cultivo calentado en la etapa de un microscopio de electrofisiología en posición vertical. </li><li> Añadir 3 ml del medio de alimentación de las células se suspenden en la actualidad (+ 10 mM HEPES) a la platina del microscopio climatizada. </li><li> Montar los electrodos de acero inoxidable en el interior de la placa de cultivo se calentó a una distancia de separación de 15 mm y conectarlos a un generador de impulsos, capaz de producir impulsos de estimulación campo de intensidad variable, frecuencia y forma de onda, para permitir que el sistema para producir la estimulación de campo de las células cuando sea apropiado. </li><li> Perno de un láser de helio-neón, montado en etapas de traslación XY, a la parte inferior de la mesa microscopio y ajustar de modo que el rayo láser se dirige a través de la base de la placa de cultivo se calentó a un ángulo de 30 ° relative al plano del voladizo. </li><li> Atornille un módulo fotodetector cuadrante, montado en etapas de traslación XY, en los bajos de la platina del microscopio y ajuste la posición para que las tierras rayo láser reflejado en el centro de los 4 cuadrantes. Figura 2 proporciona una visión general del hardware configurado necesarios para la aplicación del protocolo descrito. </li><li> Escriba un programa de software para controlar los actuadores lineales que exploran a través de los voladizos. Escribir el programa de software con referencia al diagrama de flujo proporcionado en la Figura 3. La interfaz gráfica programada para su uso con este sistema se proporciona en la Figura 4. </li></ol> 4. grabación Cantilever deflexión de datos <ol><li> Encienda el hardware análisis voladizo y el software asociado. </li><li> Inserte el termistor escenario caliente en el medio y esperar a leer 37 ° C. </li><li> Inserte el chip en voladizo en el escenario con el cantilevers orientados hacia el lado derecho del escenario. </li><li> Encienda la fuente de luz del microscopio. </li><li> Enfocar el microscopio para que los bordes de las ménsulas a la vista y utilizar el software de control de láser / fotodetector para posicionar el haz de láser en la punta del cantilever 1. NOTA: Suponiendo que los voladizos están orientados a la derecha del escenario, en voladizo 1 es la una situada en la parte superior izquierda de la matriz y los números se extienden hasta 16 en la parte inferior izquierda. Cantilever 17 está en la posición de arriba a la derecha y corre a 32 en la parte inferior derecha (Figura 5A). </li><li> Pulse el botón &quot;Play&quot; en el software de grabación. </li><li> Coloque el fotodetector para que la señal se lee &quot;0&quot; en ambos x y marcos Y ajustando los motores paso a paso que controlan el fotodetector. </li><li> Establecer voladizo 1 posición en el software de control de láser / foto-detector (Figura 4). </li><li> Mueva el láser a la punta del cantilever 16, repita el paso 4.7., Y establecer cantilever 16 posición en el software de control de láser / foto-detector. </li><li> Mueva el láser a la punta del cantilever 32, repita el paso 4.7., Y establecer en voladizo 32 posición en el software de control de láser / foto-detector. </li><li> Mueva el láser a la punta del cantilever 17, repita el paso 4.7., Y establecer en voladizo 17 posición en el software de control de láser / foto-detector. </li><li> Apague la fuente de luz del microscopio y la luz del techo en el laboratorio. </li><li> Pulse el botón &quot;Grabar&quot; en el software de grabación. </li><li> Configure el hardware generador de impulsos a 40 ms, 5 V pulsos a una frecuencia de 1 Hz, y encender la máquina. NOTA: De forma opcional, emplean protocolos de estimulación optimizados para fuentes de células específicas en este punto, los ajustes indicados son directrices basadas en datos recopilados utilizando humanos y celulares de roedores fuentes 12,13,15,16. </li><li> Usando el software de control de láser / foto-detector, establezca el hardware para explorar a través de la matriz 32 en voladizo, parando durante 5 segundos a cada uno ene. </li><li> Cuando el análisis de los 32 voladizos es completa, apague el estimulador, a continuación, detener el software de grabación y abrir el archivo de datos. </li><li> Examine la traza registrada de cada voladizo para la evidencia de la actividad contráctil. Tome nota de cada voladizo con respuestas positivas. Una contracción se define como un pico si la desviación es de al menos 0,1 V por encima de la línea de base. </li><li> Retire cualquier voladizos que no responden desde el protocolo de exploración en el software de control de láser / foto-detector. </li><li> Los voladizos activos pueden entonces ser analizados de nuevo sin estimulación con el fin de obtener una lectura de la actividad contráctil espontánea de la célula. </li><li> Ejecutar exploraciones con o sin un amplio campo de estimulación eléctrica, después de la adición de un compuesto terapéutico para el medio con el fin de observar su efecto sobre la salida funcional de las células cultivadas. </li><li> Llevar a cabo evaluaciones de la fatiga mediante la estimulación eléctrica de las células durante largos periodos y niveles de exploración de contr<html> actility para medir cuánto tiempo le toma a la fuerza máxima a caer por debajo de un umbral determinado. </li><li> En experimentos en los que las neuronas motoras se mantienen en co-cultivo con músculo, medir la formación de la unión neuromuscular a través del tratamiento de las neuronas motoras en voladizo myotube-co-cultivos con un estimulante neuronal (tales como glutamato) o inhibidor sináptica (por ejemplo, D-tubocurarina) y escaneo para aumentos y disminuciones en la actividad espontánea, respectivamente 16. </li><li> Realizar exploraciones específicas según lo dictado por las necesidades de los experimentos planeados. </li></ol> 5. Análisis de voladizo deflexión de datos <ol><li> Uso modificar las ecuaciones de Stoney 15 (detallados a continuación) para convertir datos de deflexión de voladizo en bruto (en voltios) en una lectura de la tensión en la capa de células o myotube (en Pascales), y una medición directa de la fuerza contráctil celular (en nano-Newtons): ad / 51866 / 51866eq1.jpg &quot;/&gt; la ecuación 1 <img alt="Ecuación 2" fo:content-width="2in" width="300" src="/files/ftp_upload/51866/51866eq2.jpg" /> Ecuación 2 </li><li> Donde δ = desviación de la punta en voladizo y el estrés producidos por el myotube, σ c = tensión producida por el myotube, suponiendo una película de espesor uniforme toda la anchura del voladizo, detector C = el coeficiente específico del sistema de tensión relativa a la posición del láser en la foto -detector, θ = el ángulo del láser y el detector con respecto al plano del voladizo, Si = E el módulo elástico de silicio, Si = t Los espesores de las voladizo, t = espesor myotube f, v = relación de Si de veneno de silicio, L = longitud en voladizo, longitud P = ruta de láser desde la punta en voladizo para detector, y w <sub&gt; Si = la anchura del voladizo. Un esquemática que explica los términos usados ​​en estas ecuaciones se proporciona en la Figura 6. </li><li> Suponiendo que el myotube es una película uniforme, la fuerza en la myotube es igual a la fuerza en la película, lo que lleva a la Ecuación 3, igualando el cálculo de la fuerza de la tensión y el área de sección transversal de células asumido que se utilizó para la aplicación de Stoney de ecuación. <img alt="Ecuación 3" fo:content-width="1.5in" src="files/ftp_upload/51866/51866eq3.jpg" /> Ecuación 3 </li><li> Tras la recogida de datos funcional, fijar los chips en voladizo para el análisis inmunocitoquímico o utilizar las células para análisis de proteína o ADN usando técnicas estándar. </li><li> Opcionalmente, devolver las células a la incubadora en lugar de prepararse para el análisis molecular con el fin de volver a evaluar el rendimiento funcional en un punto de tiempo después. </li></ol>

<ol>
	<li>Bischoff, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+liberation+of+myogenic+cells+from+adult+rat+muscle.">Enzymatic liberation of myogenic cells from adult rat muscle.</a> Anat. Rec. 180, 645-661 (1974).</li><li>Yasin, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quantitative+technique+for+growing+human+adult+skeletal+muscle+in+culture+starting+from+mononucleated+cells.">A quantitative technique for growing human adult skeletal muscle in culture starting from mononucleated cells.</a> J. Neurol. Sci. 32, 347-360 (1977).</li><li>Dennis, R. G., Kosnik, P. E., 2nd, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excitability+and+isometric+contractile+properties+of+mammalian+skeletal+muscle+constructs+engineered+in+vitro.">Excitability and isometric contractile properties of mammalian skeletal muscle constructs engineered in vitro.</a> In Vitro Cell Dev. Biol. Anim. 36, 327-335 (2000).</li><li>Khodabukus, A., Baar, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defined+electrical+stimulation+emphasizing+excitability+for+the+development+and+testing+of+engineered+skeletal+muscle.">Defined electrical stimulation emphasizing excitability for the development and testing of engineered skeletal muscle.</a> Tissue Eng Part C Methods. 18, 349-357 (2012).</li><li>Langelaan, M. L. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+maturation+by+electrical+stimulation:+Differences+in+response+between+C2C12+and+primary+muscle+progenitor+cells.">Advanced maturation by electrical stimulation: Differences in response between C2C12 and primary muscle progenitor cells.</a> Journal of tissue engineering and regenerative medicine. 5, 529-539 (2011).</li><li>Stephenson, G. M., O'Callaghan, A., Stephenson, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-fiber+study+of+contractile+and+biochemical+properties+of+skeletal+muscles+in+streptozotocin-induced+diabetic+rats.">Single-fiber study of contractile and biochemical properties of skeletal muscles in streptozotocin-induced diabetic rats.</a> Diabetes. 43, 622-628 (1994).</li><li>Harber, M., Trappe, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+muscle+fiber+contractile+properties+of+young+competitive+distance+runners.">Single muscle fiber contractile properties of young competitive distance runners.</a> Journal of applied physiology (Bethesda, Md: 1985). 105, 629-636 (2008).</li><li>Hvid, L. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Four+days+of+muscle+disuse+impairs+single+fiber+contractile+function+in+young+and+old+healthy+men.">Four days of muscle disuse impairs single fiber contractile function in young and old healthy men.</a> Experimental gerontology. 48, 154-161 (2013).</li><li>Edman, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contractile+performance+of+striated+muscle.">Contractile performance of striated muscle.</a> Advances in experimental medicine and biology. 682, 7-40 (2010).</li><li>Krivickas, L. S., Walsh, R., Amato, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+muscle+fiber+contractile+properties+in+adults+with+muscular+dystrophy+treated+with+MYO-029.">Single muscle fiber contractile properties in adults with muscular dystrophy treated with MYO-029.</a> Muscle Nerve. 39, 3-9 (2009).</li><li>Sung, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfabricated+mammalian+organ+systems+and+their+integration+into+models+of+whole+animals+and+humans.">Microfabricated mammalian organ systems and their integration into models of whole animals and humans.</a> Lab on a chip. 13, 1201-1212 (2013).</li><li>Wilson, K., Molnar, P., Hickman, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+functional+myotubes+with+a+Bio-MEMS+device+for+non-invasive+interrogation.">Integration of functional myotubes with a Bio-MEMS device for non-invasive interrogation.</a> Lab on a chip. 7, 920-922 (2007).</li><li>Wilson, K., Das, M., Wahl, K. J., Colton, R. J., Hickman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+contractile+stress+generated+by+cultured+rat+muscle+on+silicon+cantilevers+for+toxin+detection+and+muscle+performance+enhancement.">Measurement of contractile stress generated by cultured rat muscle on silicon cantilevers for toxin detection and muscle performance enhancement.</a> PLoS ONE. 5, (2010).</li><li>Binnig, G., Quate, C. F., Gerber, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+Force+Microscope.">Atomic Force Microscope.</a> Physical Review Letters. 56, 930-933 (1986).</li><li>Pirozzi, K. L., Long, C. J., McAleer, C. W., Smith, A. S., Hickman, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+of+embryonic+skeletal+muscle+myotube+physical+characteristics+with+contractile+force+generation+on+an+atomic+force+microscope-based+bio-microelectromechanical+systems+device.">Correlation of embryonic skeletal muscle myotube physical characteristics with contractile force generation on an atomic force microscope-based bio-microelectromechanical systems device.</a> Applied physics letters. 103, 83108 (2013).</li><li>Smith, A., Long, C., Pirozzi, K., Hickman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+functional+system+for+high-content+screening+of+neuromuscular+junctions+in+vitro.">A functional system for high-content screening of neuromuscular junctions in vitro.</a> Technology. 1, 37-48 (2013).</li><li>Stenger, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coplanar+molecular+assemblies+of+amino-+and+perfluorinated+alkylsilanes:+characterization+and+geometric+definition+of+mammalian+cell+adhesion+and+growth.">Coplanar molecular assemblies of amino- and perfluorinated alkylsilanes: characterization and geometric definition of mammalian cell adhesion and growth.</a> Journal of the American Chemical Society. 114, 8435-8442 (1992).</li><li>Das, M., Rumsey, J. W., Bhargava, N., Stancescu, M., Hickman, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+defined+long-term+in+vitro+tissue+engineered+model+of+neuromuscular+junctions.">A defined long-term in vitro tissue engineered model of neuromuscular junctions.</a> Biomaterials. 31, 4880-4888 (2010).</li><li>Rumsey, J. W., Das, M., Bhalkikar, A., Stancescu, M., Hickman, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+engineering+the+mechanosensory+circuit+of+the+stretch+reflex+arc:+Sensory+neuron+innervation+of+intrafusal+muscle+fibers.">Tissue engineering the mechanosensory circuit of the stretch reflex arc: Sensory neuron innervation of intrafusal muscle fibers.</a> Biomaterials. 31, 8218-8227 (2010).</li><li>Das, M., Rumsey, J. W., Bhargava, N., Stancescu, M., Hickman, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle+tissue+engineering:+A+maturation+model+promoting+long-term+survival+of+myotubes,+structural+development+of+the+excitation-contraction+coupling+apparatus+and+neonatal+myosin+heavy+chain+expression.">Skeletal muscle tissue engineering: A maturation model promoting long-term survival of myotubes, structural development of the excitation-contraction coupling apparatus and neonatal myosin heavy chain expression.</a> Biomaterials. 30, 5392-5402 (2009).</li><li>Das, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developing+a+novel+serum-free+cell+culture+model+of+skeletal+muscle+differentiation+by+systematically+studying+the+role+of+different+growth+factors+in+myotube+formation.">Developing a novel serum-free cell culture model of skeletal muscle differentiation by systematically studying the role of different growth factors in myotube formation.</a> In Vitro Cell Dev Biol Anim. 45, 378-387 (2009).</li><li>Stenger, D. A., Pike, C. J., Hickman, J. J., Cotman, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+determinants+of+neuronal+survival+and+growth+on+self-assembled+monolayers+in+culture.">Surface determinants of neuronal survival and growth on self-assembled monolayers in culture.</a> Brain research. 630, 136-147 (1993).</li><li>Hickman, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rational+pattern+design+for+in+vitro+cellular+networks+using+surface+photochemistry.">Rational pattern design for in vitro cellular networks using surface photochemistry.</a> Journal of Vacuum Scienc., &amp; Technology A: Vacuum, Surfaces, and Films. 12, 607-616 (1994).</li><li>Das, M., Molnar, P., Devaraj, H., Poeta, M., Hickman, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+and+morphological+characterization+of+rat+embryonic+motor+neurons+in+a+defined+system.">Electrophysiological and morphological characterization of rat embryonic motor neurons in a defined system.</a> Biotechnology progress. 19, 1756-1761 (2003).</li><li>Rumsey, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Node+of+Ranvier+formation+on+motor+neurons+in+vitro.">Node of Ranvier formation on motor neurons in vitro.</a> Biomaterials. 30, 3567-3572 (2009).</li><li>Murugan, R., Molnar, P., Rao, K. P., Hickman, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomaterial+Surface+patterning+of+self+assembled+monolayers+for+controlling+neuronal+cell+behavior.">Biomaterial Surface patterning of self assembled monolayers for controlling neuronal cell behavior.</a> International journal of biomedical engineering and technology. 2, 104-134 (2009).</li><li>Guo, X., Johe, K., Molnar, P., Davis, H., Hickman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+human+fetal+spinal+cord+stem+cell+line,+NSI-566RSC,+and+its+induction+to+functional+motoneurons.">Characterization of a human fetal spinal cord stem cell line, NSI-566RSC, and its induction to functional motoneurons.</a> Journal of Tissue Engineering and Regenerative Medicine. 4, 181-193 (2010).</li><li>Varghese, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+target+for+amyloid+beta+toxicity+validated+by+standard+and+high-throughput+electrophysiology.">A new target for amyloid beta toxicity validated by standard and high-throughput electrophysiology.</a> PLoS ONE. 5, e8643 (2010).</li><li>Natarajan, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterned+cardiomyocytes+on+microelectrode+arrays+as+a+functional,+high+information+content+drug+screening+platform.">Patterned cardiomyocytes on microelectrode arrays as a functional, high information content drug screening platform.</a> Biomaterials. 32, 4267-4274 (2011).</li><li>Davis, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+Cortical+Oligodendrocyte-Embryonic+Motoneuron+Co-Culture:+An+Axon-Oligodendrocyte+Interaction+Model.">Rat Cortical Oligodendrocyte-Embryonic Motoneuron Co-Culture: An Axon-Oligodendrocyte Interaction Model.</a> Journal of biomaterials and tissue. 2, 206-214 (2012).</li><li>Natarajan, A., DeMarse, T., Molnar, P., Hickman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+In+Vitro+Feed-Forward+Networks.">Engineered In Vitro Feed-Forward Networks.</a> J Biotechnol Biomater. 3, 2 (2013).</li></ol>

The authors have nothing to disclose.

Los pasos críticos en el análisis de voladizos microescala para la evidencia de la contracción celular son la colocación del chip en voladizo dentro de la platina del microscopio, y la posterior alineación del láser y foto-detector con la punta de los voladizos de las esquinas de la matriz. Si esto no se realiza con precisión, entonces el software no será capaz de extrapolar las posiciones de los voladizos restantes en la matriz, que puede conducir a la acumulación de falsos negativos durante la recogida de dat...

The in vitro culture of muscle cells from both human and rodent sources has been possible for decades1,2. However, while standard coverslip preparations are useful for biochemical assessment, they do not facilitate analysis of the cell&#8217;s primary functional output (contractility), and therefore are of somewhat limited value as a means to assess cellular maturation and performance. In order to maximize the amount of data obtainable from such in vitro cultures, it is necessary to advance the development of systems capable of housing such cells in configurations that permit the real-time assessment of their functional performance. The establishment of a multitude of three dimensional muscle models has made some progress toward fulfilling this need, and such systems have been used in a number of publications as a means to assess the contractile capacity of cultured muscle cells in vitro3-5. While such systems are invaluable for tissue modeling and reconstruction studies, they are limited in their applicability for studies of single cell responses. In such cases where single fiber studies are necessary, complex and labor intensive ex vivo methodologies remain the only option6-10. Furthermore, current movement toward the development of complex, multi-organ platforms for drug development and screening protocols requires the establishment of systems which are non-invasive, easily scalable and which integrate readily with supporting cells and tissue models11. 
Microscale cantilevers offer a simple method for assessing the functional contractile capacity of single cells/small populations of cells12,13. The technique is based on modified Atomic Force Microscopy (AFM) technology14, and uses a laser and photo-detector system to measure microscale cantilever deflection in response to cultured myotube contractile activity. Modified Stoney&#8217;s equations are then used to calculate stress in the myotube, and the force exerted by the myotube in order to generate the observed substrate deflection15. A scanning program has been written which enables simultaneous assessment of multiplexed cantilever arrays, offering potential moderate to high through-put applications for drug toxicity/efficacy studies15,16. Such technology may prove invaluable in the development of functional, pre-clinical assays for predicting drug efficacy in vivo. Furthermore, fabrication of cantilever chips in silicon does not impede post analysis processing of cells for standard biomolecular assays such as immunostaining, western blotting and PCR. 
This manuscript provides detailed instructions on the fabrication and preparation of microscale silicon cantilevers, the hardware and software set-up, and the operating guidelines for assessing the functionality of contractile cells cultured on these chips. Standard cell culture techniques can be implemented for plating and maintenance of cells on these surfaces, hence any contractile cell type for which reliable culture parameters exist should be able to integrate with this device with ease. The relatively simple 2D culture parameters utilized in this system makes integration of other cell models or addition of cell types that can interact with muscle (such as innervating neurons) straight-forward, greatly increasing the applicability of this model in the development of more complex functional in vitro assays and multi-organ models of mammalian systems.

<table border="0" cellpadding="0" cellspacing="0" width="690">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:299px;">Name of material/ equipment</td>
			<td style="width:128px;">Company</td>
			<td style="width:108px;">Catalog number</td>
			<td style="width:156px;">Comments/ Description</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Primary rat muscle growth medium</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Neurobasal medium</td>
			<td dir="LTR" style="width:128px;">Life Technologies</td>
			<td dir="LTR" style="width:108px;">21103-049&#160;</td>
			<td dir="LTR" style="width:156px;">N/A</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">B27 (50x)</td>
			<td dir="LTR" style="width:128px;">Life Technologies</td>
			<td dir="LTR" style="width:108px;">17504044</td>
			<td dir="LTR" style="width:156px;">1x</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Glutamax (100x)</td>
			<td dir="LTR" style="width:128px;">Life Technologies</td>
			<td dir="LTR" style="width:108px;">35050061</td>
			<td dir="LTR" style="width:156px;">1x</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">G5 supplement</td>
			<td dir="LTR" style="width:128px;">Life Technologies</td>
			<td dir="LTR" style="width:108px;">17503-012&#160;</td>
			<td dir="LTR" style="width:156px;">1x</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Glial-Derived Neurotrophic Factor</td>
			<td dir="LTR" style="width:128px;">Cell sciences</td>
			<td dir="LTR" style="width:108px;">CRG400B</td>
			<td dir="LTR" style="width:156px;">20 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Brain-Derived Neurotrophic Factor</td>
			<td dir="LTR" style="width:128px;">Cell sciences</td>
			<td dir="LTR" style="width:108px;">CRB600B</td>
			<td dir="LTR" style="width:156px;">20 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Ciliary Neurotrophic Factor</td>
			<td dir="LTR" style="width:128px;">Cell sciences</td>
			<td dir="LTR" style="width:108px;">CRC400A</td>
			<td dir="LTR" style="width:156px;">40 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Neurotrophin-3</td>
			<td dir="LTR" style="width:128px;">Cell sciences</td>
			<td dir="LTR" style="width:108px;">CRN500B</td>
			<td dir="LTR" style="width:156px;">20 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Neurotrophin-4</td>
			<td dir="LTR" style="width:128px;">Cell sciences</td>
			<td dir="LTR" style="width:108px;">CRN501B</td>
			<td dir="LTR" style="width:156px;">20 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Acidic Fibroblast Growth Factor</td>
			<td dir="LTR" style="width:128px;">Life Technologies</td>
			<td dir="LTR" style="width:108px;">13241-013&#160;</td>
			<td dir="LTR" style="width:156px;">25 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Vascular Endothelial Growth Factor</td>
			<td dir="LTR" style="width:128px;">Life Technologies</td>
			<td dir="LTR" style="width:108px;">P2654</td>
			<td dir="LTR" style="width:156px;">20 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Cardiotrophin-1</td>
			<td dir="LTR" style="width:128px;">Cell sciences</td>
			<td dir="LTR" style="width:108px;">CRC700B</td>
			<td dir="LTR" style="width:156px;">20 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Heparin Sulphate</td>
			<td dir="LTR" style="width:128px;">Sigma</td>
			<td dir="LTR" style="width:108px;">D9809&#160;</td>
			<td dir="LTR" style="width:156px;">100 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Leukemia Inhibitory Factor</td>
			<td dir="LTR" style="width:128px;">Sigma</td>
			<td dir="LTR" style="width:108px;">L5158&#160;</td>
			<td dir="LTR" style="width:156px;">20 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Vitronectin</td>
			<td dir="LTR" style="width:128px;">Sigma</td>
			<td dir="LTR" style="width:108px;">V0132</td>
			<td dir="LTR" style="width:156px;">100 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;"></td>
			<td dir="LTR" style="width:128px;"></td>
			<td dir="LTR" style="width:108px;"></td>
			<td dir="LTR" style="width:156px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Primary rat muscle differentiation medium</td>
			<td dir="LTR" style="width:128px;"></td>
			<td dir="LTR" style="width:108px;"></td>
			<td dir="LTR" style="width:156px;"></td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">NB Activ 4</td>
			<td dir="LTR" style="width:128px;">Brain Bits LLC</td>
			<td dir="LTR" style="width:108px;">NB4-500</td>
			<td dir="LTR" style="width:156px;">N/A</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;"></td>
			<td dir="LTR" style="width:128px;"></td>
			<td dir="LTR" style="width:108px;"></td>
			<td dir="LTR" style="width:156px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Class 2 red diode laser</td>
			<td>Newport</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Photo-detector</td>
			<td>Noah Industries</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Model 2100 Pulse stimulator</td>
			<td>A-M systems</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Multiclamp 700B Digitizer</td>
			<td>Axon Instruments</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Patch clamp microscope and stage</td>
			<td>Olympus</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Delta T4 culture dish controller</td>
			<td>Bioptechs</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Axoscope software</td>
			<td>Molecular Devices</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">LabVIEW software</td>
			<td>National Instruments</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">37oC, 5% CO2 incubator</td>
			<td>NAPCO</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Class 2 microbiological flow hood</td>
			<td>Labconco</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Pipettes and tips</td>
			<td>Eppendorf</td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

utilization of microscale silicon cantilevers to assess cellular contractile function in vitro

El desarrollo de ensayos in vitro más predictivos y biológicamente relevantes se basa en el avance de los sistemas de cultivo de células versátiles que facilitan la evaluación funcional de las células sembradas. Para ello, la tecnología voladizo microescala ofrece una plataforma con la que para medir la funcionalidad contráctil de una gama de tipos celulares, incluyendo esquelético, cardiaco, y las células de músculo liso, a través de la evaluación de la contracción inducida por sustrato flexión. Aplicación de las matrices multiplexadas en voladizo proporciona los medios para desarrollar protocolos de moderado a alto rendimiento para la evaluación de la eficacia del fármaco y la toxicidad, fenotipo de la enfermedad y la progresión, así como neuromusculares y otras interacciones célula-célula. Este manuscrito proporciona los detalles para la fabricación de matrices voladizo fiables para este fin, y los métodos necesarios para con éxito células de cultivo en estas superficies. Una descripción más detallada se proporciona en los pasos necesarios para realizar anal funcionalanalysis de tipos de células contráctiles mantuvo en tales matrices usando una novela láser y sistema de foto-detector. Los datos representativos proporcionados destaca la precisión y la naturaleza reproducible de análisis de la función contráctil posible utilizar este sistema, así como la amplia gama de estudios a los que tal tecnología se puede aplicar. Amplia adopción exitosa de este sistema podría proporcionar a los investigadores con los medios para llevar a cabo estudios funcionales rápidas y de bajo costo in vitro, lo que lleva a predicciones más exactas de rendimiento de tejidos, desarrollo de la enfermedad y la respuesta al nuevo tratamiento terapéutico.

Cultivo con éxito de células contráctiles en voladizos es un procedimiento relativamente sencillo, utilizando técnicas estándar de cultivo de células (Figura 5). El porcentaje de los voladizos soportan células contratantes variará dependiendo del tipo de célula que se examina y técnica de cultivo específica empleada. El uso de células embrionarias de rata primarios derivados de las extremidades posteriores, la actividad contráctil se detectó el 12% de los voladizos examinadas (n = 4). Aná...

Watch this Scientific Journal Video about Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro at JoVE.com

Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro

Este protocolo describe el uso de palancas de silicio microescala como superficies de cultivo flexibles para la medición de la contractilidad de las células musculares in vitro. La contracción celular hace que la flexión en voladizo, que se puede medir, grabada, y se convierte en lecturas de fuerza, proporcionando un sistema no invasivo y escalable para la medición de la función contráctil in vitro.

Utilización de microescala silicio voladizos para evaluar la función contráctil celular<em&gt; In Vitro</em

The development of more predictive and biologically relevant in vitro assays is predicated on the advancement of versatile cell culture systems which ...

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Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro

10.1K vistas.  University of Central Florida.  Este protocolo describe el uso de palancas de silicio microescala como superficies de cultivo flexibles para la medición de la contractilidad de las células musculares in vitro. La contracción celular hace que la flexión en voladizo, que se puede medir, grabada, y se convierte en lecturas de fuerza, proporcionando un sistema no invasivo y escalable para la medición de la función contráctil in vitro. 

Video: Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro

Utilization of Plasmonic and Photonic Crystal Nanostructures for Enhanced Micro- and Nanoparticle Manipulation

A method to manipulate the position and orientation of submicron particles nondestructively would be an incredibly useful tool for basic biological research. Perhaps the most widely used physical force to achieve noninvasive manipulation of small particles has been dielectrophoresis(DEP).1 However, DEP on its own lacks the versatility and precision that are desired when manipulating cells since it is traditionally done with stationary electrodes. Optical tweezers, which utilize a three dimensional electromagnetic field gradient to exert forces on small particles, achieve this desired versatility and precision.2 However, a major drawback of this approach is the high radiation intensity required to achieve the necessary force to trap a particle which can damage biological samples.3 A solution that allows trapping and sorting with lower optical intensities are optoelectronic tweezers (OET) but OET's have limitations with fine manipulation of small particles; being DEP-based technology also puts constraint on the property of the solution.4,5
This video article will describe two methods that decrease the intensity of the radiation needed for optical manipulation of living cells and also describe a method for orientation control. The first method is plasmonic tweezers which use a random gold nanoparticle (AuNP) array as a substrate for the sample as shown in Figure 1. The AuNP array converts the incident photons into localized surface plasmons (LSP) which consist of resonant dipole moments that radiate and generate a patterned radiation field with a large gradient in the cell solution. Initial work on surface plasmon enhanced trapping by Righini et al and our own modeling have shown the fields generated by the plasmonic substrate reduce the initial intensity required by enhancing the gradient field that traps the particle.6,7,8 The plasmonic approach allows for fine orientation control of ellipsoidal particles and cells with low optical intensities because of more efficient optical energy conversion into mechanical energy and a dipole-dependent radiation field. These fields are shown in figure 2 and the low trapping intensities are detailed in figures 4 and 5. The main problems with plasmonic tweezers are that the LSP's generate a considerable amount of heat and the trapping is only two dimensional. This heat generates convective flows and thermophoresis which can be powerful enough to expel submicron particles from the trap.9,10 The second approach that we will describe is utilizing periodic dielectric nanostructures to scatter incident light very efficiently into diffraction modes, as shown in figure 6.11 Ideally, one would make this structure out of a dielectric material to avoid the same heating problems experienced with the plasmonic tweezers but in our approach an aluminum-coated diffraction grating is used as a one-dimensional periodic dielectric nanostructure. Although it is not a semiconductor, it did not experience significant heating and effectively trapped small particles with low trapping intensities, as shown in figure 7. Alignment of particles with the grating substrate conceptually validates the proposition that a 2-D photonic crystal could allow precise rotation of non-spherical micron sized particles.10 The efficiencies of these optical traps are increased due to the enhanced fields produced by the nanostructures described in this paper.

Plasmonic tweezers and photonic crystal nanostructures are shown to produce useful enhancements in the efficiency and orientation control of optically trapping micro- and nano-particles.

Utilización de nanoestructuras fotónicas de cristal plasmónicos y para la manipulación mejorada de Micro y nanopartículas

A method to manipulate the position and orientation of submicron particles nondestructively would be an incredibly useful tool for basic biological ...

Investigación

Bioingeniería

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs 1-6. Yet the development of artificial molecular machines centers on devices capable of directional motion, i.e. molecular motors, and on their scaled-down mechanical parts (wheels, axels, pendants etc) 7-9. This imitates the macro-machines, even though the physical properties essential for these devices, such as inertia and momentum conservation, are not usable in the nanoworld environments 10. Alternative designs, which do not follow the mechanical macromachines schemes and use mechanisms developed in the evolution of biological molecules, can take advantage of the specific conditions of the nanoworld. Besides, adapting actual biological molecules for the purposes of nano-design reduces potential dangers the nanotechnology products may pose. Here we demonstrate the assembly and application of one such bio-enabled construct, a semi-artificial molecular device which combines a naturally-occurring molecular machine with artificial components. From the enzymology point of view, our construct is a designer fluorescent enzyme-substrate complex put together to perform a specific useful function. This assembly is by definition a molecular machine, as it contains one 12. Yet, its integration with the engineered part - fluorescent dual hairpin - re-directs it to a new task of labeling DNA damage12.
Our construct assembles out of a 32-mer DNA and an enzyme vaccinia topoisomerase I (VACC TOPO). The machine then uses its own material to fabricate two fluorescently labeled detector units (Figure 1). One of the units (green fluorescence) carries VACC TOPO covalently attached to its 3'end and another unit (red fluorescence) is a free hairpin with a terminal 3'OH. The units are short-lived and quickly reassemble back into the original construct, which subsequently recleaves. In the absence of DNA breaks these two units continuously separate and religate in a cyclic manner. In tissue sections with DNA damage, the topoisomerase-carrying detector unit selectively attaches to blunt-ended DNA breaks with 5'OH (DNase II-type breaks)11,12, fluorescently labeling them. The second, enzyme-free hairpin formed after oligonucleotide cleavage, will ligate to a 5'PO4 blunt-ended break (DNase I-type breaks)11,12, if T4 DNA ligase is present in the solution 13,14 . When T4 DNA ligase is added to a tissue section or a solution containing DNA with 5'PO4 blunt-ended breaks, the ligase reacts with 5'PO4 DNA ends, forming semi-stable enzyme-DNA complexes. The blunt ended hairpins will interact with these complexes releasing ligase and covalently linking hairpins to DNA, thus labeling 5'PO4 blunt-ended DNA breaks.
This development exemplifies a new practical approach to the design of molecular machines and provides a useful sensor for detection of apoptosis and DNA damage in fixed cells and tissues.

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

We demonstrate the assembly and application of a molecular-scale device powered by a topoisomerase protein. The construct is a bio-molecular sensor which labels two major types of DNA breaks in tissue sections by attaching two different fluorophores to their ends.

En la Asamblea in vitro de la semi-artificial de la máquina molecular y su utilización para la detección del daño del ADN

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs 1-6. Yet the development of artificial molecular ...

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised the only means available, the development of noninvasive imaging modalities (echocardiography, MRI, CT) having high temporal and spatial resolution provide a new window for quantitative diastolic function assessment. Echocardiography is the agreed upon standard for diastolic function assessment, but indexes in current clinical use merely utilize selected features of chamber dimension (M-mode) or blood/tissue motion (Doppler) waveforms without incorporating the physiologic causal determinants of the motion itself.&#160;The recognition that all left ventricles (LV) initiate filling by serving as mechanical suction pumps allows global diastolic function to be assessed based on laws of motion that apply to all chambers. What differentiates one heart from another are the parameters of the equation of motion that governs filling. Accordingly, development of the Parametrized Diastolic Filling (PDF) formalism&#160;has shown that the entire range of clinically observed early transmitral flow (Doppler E-wave) patterns are extremely well fit by the laws of damped oscillatory motion.&#160;This permits analysis of individual E-waves in accordance with a causal mechanism (recoil-initiated suction) that yields three (numerically) unique lumped parameters whose physiologic analogues are chamber stiffness (k), viscoelasticity/relaxation (c), and load (xo).&#160;The recording of transmitral flow (Doppler E-waves) is standard practice in clinical cardiology and, therefore, the echocardiographic recording method is only briefly reviewed. Our focus is on determination of the PDF parameters from routinely recorded E-wave data.&#160;As the highlighted results indicate, once the PDF parameters have been obtained from a suitable number of load varying E-waves, the investigator is free to use the parameters or construct indexes from the parameters (such as stored energy 1/2kxo2, maximum A-V pressure gradient kxo, load independent index of diastolic function, etc.) and select the aspect of physiology or pathophysiology to be quantified.

Accurate, causality-based quantification of global diastolic function has been achieved by kinematic modeling-based analysis of transmitral flow via the Parametrized Diastolic Filling (PDF) formalism. PDF generates unique stiffness, relaxation,&#160;and load parameters and elucidates &#39;new&#39; physiology while providing sensitive and specific indexes of dysfunction.

La cuantificación de la función diastólica por Global Análisis basado en Modelado cinemático de flujo transmitral mediante el formalismo de llenado diastólico parametrizada

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

Microscale Vortex-assisted Electroporator for Sequential Molecular Delivery

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant exogenous molecular probes into cells. This work reports a microfluidic electroporation platform capable of performing multiple molecule delivery to mammalian cells with precise and molecular-dependent parameter control. The system&#8217;s ability to isolate cells with uniform size distribution allows for less variation in electroporation efficiency per given electric field strength; hence enhanced sample viability. Moreover, its process visualization feature allows for observation of the fluorescent molecular uptake process in real-time, which permits prompt molecular delivery parameter adjustments in situ for efficiency enhancement. To show the vast capabilities of the reported platform, macromolecules with different sizes and electrical charges (e.g., Dextran with MW of 3,000 and 70,000 Da) were delivered to metastatic breast cancer cells with high delivery efficiencies (&#62;70%) for all tested molecules. The developed platform has proven its potential for use in the expansion of research fields where on-chip electroporation techniques can be beneficial.

A microfluidic vortex assisted electroporation platform was developed for sequential delivery of multiple molecules into identical cell populations with precise and independent dosage control. The system&#8217;s size based target cell purification step preceding electroporation aided to enhance molecular delivery efficiency and processed cell viability.

Vortex asistida Microscale Electroporador para entrega secuencial Molecular

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant ...

Analyzing Mixing Inhomogeneity in a Microfluidic Device by Microscale Schlieren Technique

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren system is constructed from a Hoffman modulation contrast microscope, which provides easy access to the rear focal plane of the objective lens, by removing the slit plate and replacing the modulator with a knife-edge. The working principle of microscale schlieren technique relies on detecting light deflection caused by variation of refractive index1-3. The deflected light either escapes or is obstructed by the knife-edge to produce a bright or a dark band, respectively. If the refractive index of the mixture varies linearly with its composition, the local change in light intensity in the image plane is proportional to the concentration gradient normal to the optical axis. The micro-schlieren image gives a two-dimensional projection of the disturbed light produced by three-dimensional inhomogeneity.
To accomplish quantitative analysis, we describe a calibration procedure that mixes two fluids in a T-microchannel. We carry out a numerical simulation to obtain the concentration gradient in the T-microchannel that correlates closely with the corresponding micro-schlieren image. By comparison, a relationship between the grayscale readouts of the micro-schlieren image and the concentration gradients presented in a microfluidic device is established. Using this relationship, we are able to analyze the mixing inhomogeneity from associate micro-schlieren image and demonstrate the capability of microscale schlieren technique with measurements in a microfluidic oscillator4. For optically transparent fluids, microscale schlieren technique is an attractive diagnostic tool to provide instantaneous full-field information that retains the three-dimensional features of the mixing process.

Herein, we describe a procedure that employs microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. Through calibration, distribution of concentration gradient can be derived from the micro-schlieren image.

Analizando Mezcla falta de homogeneidad en un dispositivo de microfluidos por Microscale Schlieren Técnica

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

Irritación de los ojos de prueba (IET) para la identificación del peligro de los ojos: Irrita los productos químicos que utilizan Reconstruido humana Córnea-como epitelial (RHCE) Modelo de Tejidos

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Techniques for the Evolution of Robust Pentose-fermenting Yeast for Bioconversion of Lignocellulose to Ethanol

Lignocellulosic biomass is an abundant, renewable feedstock useful for production of fuel-grade ethanol and other bio-products. Pretreatment and enzyme saccharification processes release sugars that can be fermented by yeast. Traditional industrial yeasts do not ferment xylose (comprising up to 40% of plant sugars) and are not able to function in concentrated hydrolyzates. Concentrated hydrolyzates are needed to support economical ethanol recovery, but they are laden with toxic byproducts generated during pretreatment. While detoxification methods can render hydrolyzates fermentable, they are costly and generate waste disposal liabilities. Here, adaptive evolution and isolation techniques are described and demonstrated to yield derivatives of the native Scheffersomyces stipitis strain NRRL Y-7124 that are able to efficiently convert hydrolyzates to economically recoverable ethanol despite adverse culture conditions. Improved individuals are enriched in an evolving population using multiple selection pressures reliant on natural genetic diversity of the S. stipitis population and mutations induced by exposures to two diverse hydrolyzates, ethanol or UV radiation. Final evolution cultures are dilution plated to harvest predominant isolates, while intermediate populations, frozen in glycerol at various stages of evolution, are enriched on selective media using appropriate stress gradients to recover most promising isolates through dilution plating. Isolates are screened on various hydrolyzate types and ranked using a novel procedure involving dimensionless relative performance index (RPI) transformations of the xylose uptake rate and ethanol yield data. Using the RPI statistical parameter, an overall relative performance average is calculated to rank isolates based on multiple factors, including culture conditions (varying in nutrients and inhibitors) and kinetic characteristics. Through application of these techniques, derivatives of the parent strain had the following improved features in enzyme saccharified hydrolyzates at pH 5-6: reduced initial lag phase preceding growth, reduced diauxic lag during glucose-xylose transition, significantly enhanced fermentation rates, improved ethanol tolerance and accumulation to 40 g/L.

Adaptive evolution and isolation techniques are described and demonstrated to yield derivatives of Scheffersomyces stipitis strain NRRL Y-7124 that are able to rapidly consume hexose and pentose mixed sugars in enzyme saccharified undetoxified hydrolyzates and to accumulate over 40 g/L ethanol.

Las técnicas para la Evolución de la robusta levaduras que fermentan la pentosa para la bioconversión de lignocelulosa en etanol

Lignocellulosic biomass is an abundant, renewable feedstock useful for production of fuel-grade ethanol and other bio-products. Pretreatment and enzyme ...

Measuring and Modeling Contractile Drying in Human Stratum Corneum

Stratum corneum (SC) is the most superficial skin layer. Its contact with the external environment means that this tissue layer is subjected to both cleansing agents and daily variations in ambient moisture; both of which can alter the water content of the tissue. Reductions in water content from severe barrier dysfunction or low humidity environments can alter SC stiffness and cause a build-up of drying stresses. In extreme conditions, these factors can cause mechanical rupture of the tissue. We have established a high throughput method of quantifying dynamic changes in the mechanical properties of SC upon drying. This technique can be employed to quantify changes in the drying behavior and mechanical properties of SC with cosmetic cleanser and moisturizer treatments. This is achieved by measuring dynamic variations in spatially resolved in-plane drying displacements of circular tissue samples adhered to an elastomer substrate. In-plane radial displacements acquired during drying are azimuthally averaged and fitted with a profile based on a linear elastic contractility model. Dynamic changes in drying stress and SC elastic modulus can then be extracted from the fitted model profiles.

This article describes a method of quantifying the dynamic drying behavior and mechanical properties of stratum corneum by measuring spatially resolved in-plane drying displacements of circular tissue samples adhered to an elastomer substrate. This technique can be used to measure how different chemical treatments alter drying and tissue mechanical properties.

La medición y Modelado contráctil que se secan en humano estrato córneo

Stratum corneum (SC) is the most superficial skin layer. Its contact with the external environment means that this tissue layer is subjected to both ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

Un<em&gt; In Vitro</em&gt; Órgano Cultura Modelo Murino del disco intervertebral

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (&#8804;14 days). These spheroid models (&#8805;500 &#181;m in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the &#8216;gold standard&#8217; micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

This procedure was established to be used for developing advanced 3D hepatic cultures in vitro, which can provide a more physiologically relevant assessment of the genotoxic hazards associated with nanomaterial exposures over both an acute or long-term, repeated dose regimes.

Modelos avanzados de hígado 3D para pruebas de genotoxicidad in vitro tras la exposición a nanomateriales a largo plazo

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...