This work was funded by start-up funds provided to Dr. Silviya Zustiak by Saint Louis University as well as by a President&#8217;s Research Fund (PRF) grant awarded to Dr. Silviya Zustiak by Saint Louis University. We thank Naveed Ahmed and Keval Shah for technical assistance.

1. Preparación de soluciones y alícuotas asociados hidrogel <ol><li> Preparación de la solución de poliacrilamida precursor del gel. <ol><li> Preparar la solución de precursor de gel de poliacrilamida mediante la mezcla de acrilamida (A) (40% w / v, r M 71,08 g / mol), el bisacrilamida reticulante (B) (2% w / v, r M 154,17 g / mol), y de- agua ionizada en los porcentajes en volumen especificados en la Tabla 1. NOTA: Estas soluciones se pueden preparar en grandes lotes y se almacenaron a 4 ° C durante hasta varios meses. <ol><li> PRECAUCIÓN: La acrilamida es tóxico por inhalación o ingestión, en particular, cuando en forma de polvo: por lo tanto, utilizar preferentemente 40% w solución / v para reducir los riesgos de toxicidad. Manipular sólo con ropa de protección, como guantes, gafas y una bata de laboratorio. Almacenar en un recipiente resistente a la luz, bien cerrado en la nevera (&lt;23 ° C, zona bien ventilada). </li><li> PRECAUCIÓN: Bis-acrilamida es tóxico por inhalación o ingestión, en particular,cuando está en forma de polvo: por lo tanto, utilizar preferentemente 2% w solución / v para reducir los riesgos de toxicidad. Manipular sólo con ropa de protección, como guantes, gafas y una bata de laboratorio. Almacenar en un recipiente resistente a la luz, bien cerrado en la nevera (&lt;4 ° C, zona bien ventilada). </li></ol></li></ol></li><li> Preparación y uso de N -Sulfosuccinimidyl-6- (4 &#39;-azido-2&#39; nitrofenilamino) hexanoato (sulfo-SANPAH, M r 492,40 g / mol) alícuotas. PRECAUCIÓN: sulfo-SANPAH provoca irritación ocular grave; manejar con guantes y adecuado de los ojos o la cara. Almacenar a -20 ° C al recibirla y antes de alícuotas. <ol><li> Para almacenar sulfo-SANPAH: disolver sulfo-SANPAH en dimethylsulfoxane (DMSO) a 50 mg / ml. Alícuota de la solución madre en 50 tubos de centrífuga micro de 20 l por tubo. Congelación de Flash en hielo seco o en nitrógeno líquido (un paso opcional, pero preferido para preservar la máxima eficiencia reticulante) y almacenar a -80 ° C. NOTA: Las alícuotas can almacenarse durante varios meses. </li><li> Para utilizar sulfo-SANPAH: descongelar brevemente alícuota y se diluye en 480 l de agua desionizada. Utilizar inmediatamente. Sulfo-SANPAH hidroliza rápidamente en agua: por lo tanto, tomar la precaución de realizar todos los pasos anteriores rápidamente. </li></ol></li><li> Preparación de persulfato de amonio (M r 228,18 g / mol) alícuotas. NOTA: persulfato de amonio provoca ojos, piel y vías respiratorias. Llevar equipo de protección personal adecuado al manipular. Manejar polvo persulfato de amonio en una campana de extracción química. Conserve el polvo en un lugar seco y bien ventilado. Una vez en alícuotas, la solución diluida se podría utilizar en el banco de trabajo. <ol><li> Disolver persulfato de amonio en agua desionizada para lograr una concentración final de persulfato de amonio del 10% w / v. Alícuota y almacenar a -20 ° C. Descongelar inmediatamente antes del uso. </li></ol></li><li> Preparación de solución de colágeno de tipo I. <ol><li> Preparar 0,2 mg solución de colágeno / ml por dilución de la acción de modolución en 1x tampón fosfato salino (PBS) de pH 7,4. Almacene en breve hielo o utilizar inmediatamente después de la dilución. </li></ol></li></ol> 2. Preparación de hidrogel (Ver Figura 1) <ol><li> Preparación de portaobjetos de vidrio hidrófobos. <ol><li> Coloque unas pocas gotas de una solución hidrófoba sobre una placa de vidrio y el uso de papel de seda para propagarse a través de la superficie. Deje que se seque al aire y limpie con un pañuelo de papel de nuevo para igualar el recubrimiento hidrófobo. NOTA: Guarde la solución hidrófoba en un armario inflamable a temperatura ambiente. Llevar equipo de protección personal durante la manipulación. Trabajar en un lugar bien ventilado. </li></ol></li><li> Preparación de soporte de plástico flexible. <ol><li> Cortar el soporte de plástico flexible para que coincida con el tamaño de la placa de vidrio hidrófobo revestido. </li><li> Marcar el lado hidrófobo del soporte de plástico flexible por arañar ligeramente la superficie con una herramienta afilada tal como un bisturí. NOTA: Una vez que el gel - que es totalmente transparente - se seca, Las marcas de arañazos ayudarán a distinguir qué lado flexibles de soporte de plástico contiene el gel. NOTA: El soporte de plástico flexible tiene una hidrofóbico y un lado hidrofílico. Una vez depositado, de poliacrilamida se adhiere permanentemente a la parte hidrófila del soporte de plástico que facilita un fácil manejo de hidrogel posterior. </li></ol></li><li> Preparación Gel (Ejemplo volúmenes se dan para soluciones precursoras gel 5 ml). NOTA: 5 ml sería suficiente para producir ~ 40 geles cuando el espesor inicial gel es 0,5 mm. Más geles se pueden producir si el grosor del gel se reduce. <ol><li> Coloque 4972,5 l de solución de precursor de poliacrilamida de concentración final deseada (Tabla 1) en un tubo cónico de 50 ml. Colocar en una cámara de desgasificación durante 30 minutos con la tapa abierta. NOTA: oxígeno actúa como una trampa de radicales libres y cuando está presente, se inhibe la polimerización. </li><li> Añadir 25 l de 10% w / v de persulfato de amonio (véase el punto 1.3) para lograr una finalconcentración de persulfato de amonio de 0,05%. </li><li> Añadir 2,5 l de N, N, N &#39;, N&#39;-tetrametiletilendiamina (TEMED, M r 116,24 g / mol) a la solución de gel de desgasificado para lograr una concentración final de 0,5 TEMED%. NOTA: Tienda TEMED en un armario inflamable a temperatura ambiente. Manejar en una campana de humos químicos al usar el equipo de protección personal adecuado. Mantener bien cerrado bajo gas inerte, ya que es altamente sensible a la humedad y aire. </li><li> Mezclar la solución suavemente pipeteando arriba y abajo de 3 - 5 veces. No vortex para evitar la difusión de oxígeno en la solución de precursor de gel. </li><li> Pipetear la solución de gel en el lado hidrófila del soporte de plástico flexible y sándwich con portaobjetos de vidrio hidrófobo revestido. Separar las dos correderas con espaciadores de silicona de espesor deseado (por ejemplo, 0,5 mm). Dejar una pequeña cantidad (~ 100 l) de solución de precursor de polímero en el tubo cónico de 50 ml para usar como un indicador de la gelificación. NOTA:Cualquier espaciador podría ser utilizado. Por ejemplo, una sola tira de Parafilm da un espesor final de gel hinchado de 100 a 120 micras. </li><li> Coloque otra placa de vidrio encima del sándwich de soporte / gel de plástico flexible para determinar una superficie uniforme de hidrogel en la polimerización. </li><li> Deje que el gel de polimerizar durante 45 min, aunque menos tiempo se puede utilizar para geles de mayor% en peso si se desea. Para cerciorarse de que el gel se haya polimerizado, observar la solución restante en el tubo cónico de 50 ml; Si la solución restante ha gelificado, entonces es probable que la gelificación en la placa de vidrio se ha iniciado también. Sin embargo, tenga en cuenta que la apertura prematura del molde evitará polimerización completa. </li><li> Una vez formado el gel, retire el soporte de plástico flexible con el gel de poliacrilamida unido covalentemente en la parte superior, y establecer gel de lado hasta que se seque al aire. NOTA: convección o secado por calor también se pueden utilizar para acelerar este paso. Una vez seco sobre el soporte de plástico flexible, el gel puede ser tiendad indefinidamente. <ol><li> Marque las áreas descubiertas del soporte de plástico flexible, donde los geles de poliacrilamida no se formaron debido a las burbujas. Marcos mientras que el gel se sigue hidratada ya que esto se mezclan con el apoyo de plástico flexible una vez que el gel se seque. </li></ol></li></ol></li></ol> 3. Asamblea Multiwell Plate, colágeno, Revestimientos, y Esterilización <ol><li> Conjunto de la placa de múltiples pocillos. <ol><li> Una vez seco, cortar geles PA en las formas deseadas. <ol><li> Para una placa de 96 pocillos, utilice una perforadora de alta resistencia con un diámetro de ~ 6 mm. Utilice un cortador de papel de alta resistencia para cortar geles en formas cuadradas o rectangulares. Alternativamente, use tijeras. </li></ol></li><li> Preparar aproximadamente 500 l de PDMS por placa de 96 pocillos de acuerdo con las instrucciones del fabricante. Para pegar los geles a la parte inferior de una placa de múltiples pocillos, colocar una pequeña gota (~ 5 l) de polidimetilsiloxano (PDMS) en el centro de la cada pocillo. Con unas pinzas, coloque una poliacrilamidagel de correo en cada lado del soporte de plástico bien, flexible hacia abajo. Para permitir PDMS para curar, deje la placa montada a 37 ° C durante un mínimo de 4 horas. </li></ol></li><li> Recubrimiento de colágeno de geles de poliacrilamida. <ol><li> Con una pipeta de transferencia, coloque una pequeña cantidad (7-8 l) de sulfo-SANPAH (véase el punto 1.2.2) en cada pocillo y agitar de lado a lado para cubrir la superficie del gel uniformemente. Trabajar con prontitud desde sulfo-SANPAH no es estable en agua. </li><li> Coloque la placa de pocillos bajo una lámpara de UV de alta intensidad (intensidad = 37 mW, λ = 302 a 365 nm) durante 5 min. Enjuagar los geles con PBS para eliminar el exceso de sulfo-SANPAH. </li><li> Yo solución de la pipeta 50 l de 0,2 mg / ml de colágeno tipo (Véase el punto 1.4) en cada pocillo. Deje el plato cubierto a temperatura ambiente durante al menos 2 horas o O / N a 4 ° C. NOTA: Para acelerar el proceso de recubrimiento de colágeno, una pipeta de transferencia se puede utilizar para añadir la solución de colágeno. Típicamente, 1 - 2 gotas de solución debe ser suficiente para completarLy cubre la superficie del gel. </li><li> Deje la placa de pocillos a temperatura ambiente durante al menos 2 horas para permitir la unión de colágeno. </li><li> Enjuagar con PBS para eliminar el exceso de solución de colágeno y esterilizar bajo UV (λ = 200 nm) en una campana de cultivo de tejidos durante 2 h. </li><li> Remoje los geles en medio completo (Consulte la Sección 4.1 para medianas composición) O / N para hidratar y equilibrar. El uso para la siembra de células inmediatamente o almacenar en el refrigerador hasta por 2 días. </li></ol></li></ol> 4. siembra de células en Rigidez Ensayo PA NOTA: Aunque típico para líneas celulares de mamíferos comunes, el protocolo descrito en esta sección se utiliza específicamente con el cáncer de mama línea celular MDA-MB-231 (ver figuras 4 y 5). <ol><li> Recoger células del matraz de cultivo de tejido por la exposición a 5% de tripsina / EDTA durante 5 min a 37 ° C. Utilice ~ 80 l de tripsina / EDTA por cada cm 2 de área frasco de cultivo; por ejemplo, usar 2 ml de tripsina / EDTA fora T25 matraz de cultivo celular. Vuelva a suspender las células recogidas en medio completo suplementado con 10% de suero bovino fetal (FBS) y 1% de penicilina / estreptomicina a una concentración celular final deseada. Teniendo en cuenta tiempo de duplicación celular, así como la longitud de tiempo, las células se sembraron en el ensayo, elegir una concentración apropiada de células sub-confluente. Asegúrese de que el medio añadido es suficiente para sumergir completamente los hidrogeles. NOTA: Debido a que los hidrogeles han sido previamente equilibrada en los medios de comunicación (consulte el paso 3.2.6) un volumen de 100 l debería ser suficiente. Volúmenes medios típicos utilizados para las placas de múltiples pocillos deben ser suficientes ya que los geles han sido previamente equilibrada en los medios de comunicación en el paso anterior. NOTA: El ensayo sería apropiado para cualquier tipo de célula apego dependiente. <ol><li> Contar el número de células bajo un microscopio invertido usando un hemocitómetro. Carga 10 l de suspensión celular en cada puerto hemocitómetro y promediar el recuento de células de al menos 8 cuadrantes. Para obtener elconcentración final de células, se multiplica el número de células en un 10 4. NOTA: Se obtienen los mejores resultados del conteo de células cuando el número de células en cada cuadrante hemocitómetro es 20 - 50. </li></ol></li><li> Cultivar las células en el ensayo de rigidez PA en un incubador humidificado a 37 ° C y 5% de CO 2. Cambie medios cada 2-3 días. Recoger las células cuando sea necesario por la exposición a 5% de tripsina / EDTA a 37 ° C durante 5 min. Utilice ~ 80 l de tripsina / EDTA por cm2 de matraz de cultivo de tejidos. Durante todas las etapas de manipulación celular, tener especial cuidado de no perturbar la superficie del hidrogel: aspirado o medio pipeta inclinando ligeramente la placa de pocillos múltiples lados y tocar la punta de la pipeta sobre la pared lateral de cada bien, en lugar de tocar el hidrogel. NOTA: Excepto por teniendo especial cuidado de no dañar la superficie del hidrogel durante la manipulación de cultivo de tejidos estándar, las células sembradas en el ensayo de rigidez puede ser manipulado de la misma manera como si se sembraron ena una placa de múltiples pocillos regular. </li></ol> 5. Obtención de imágenes de células sembradas en Rigidez Ensayo PA <ol><li> Celdas de imagen directamente en el ensayo de rigidez PA. NOTA: Cualquier microscopio - invertida, fluorescente o confocal, se puede utilizar para obtener imágenes de células. NOTA: El soporte de plástico flexible utilizado para construir el ensayo de rigidez es completamente transparente y no autofluoresce o interfiere con imágenes de células. Sin embargo, a pesar de que el propio soporte de plástico flexible es transparente, las capacidades de formación de imágenes estarán limitados por la distancia de trabajo del objetivo. El soporte de plástico flexible tiene un espesor de 0,23 mm y una distancia de trabajo típica de un objetivo de 10X es ~ 4 mm, fuertemente decreciente para ampliaciones superiores. <ol><li> Para imágenes de células vivas, la posición de ensayo de rigidez PA en soporte de la placa de microscopio y la imagen. Mantenga las sesiones de formación de imágenes por debajo de 2 horas, o utilizar un microscopio equipado con una cámara ambiental para tiempos de imagen más largos. </li><li> Cuando im células vivasel envejecimiento no es el objetivo, fijar las células en solución de formaldehído al 4% suplementado con 0,1% de detergente. Remojar las células en fijador a temperatura ambiente durante un mínimo de 2 horas. Enjuague con PBS dos veces. Deseche los residuos fijador en un contenedor de residuos designado. NOTA: Las células se pueden obtener imágenes inmediatamente o almacenada sumergidos en PBS a 4 ° C durante un máximo de 2 semanas. PRECAUCIÓN: El formaldehído es tóxico por inhalación y por contacto. Manipular con guantes en una campana de extracción química. NOTA: protocolo de fijación de la célula tiene que ser elegido en base a los protocolos de tinción celular y la manipulación posterior, debido a que algunos fijadores dañan algunas proteínas de la célula. </li><li> Para imágenes fluorescentes, mancha células con tinción celular deseada directamente en la placa de múltiples pocillos fijo. Imagen de inmediato. </li></ol></li></ol>

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulation+of+adipogenesis+of+adult+adipose-derived+stem+cells+using+substrates+that+mimic+the+stiffness+of+adipose+tissue.">Stimulation of adipogenesis of adult adipose-derived stem cells using substrates that mimic the stiffness of adipose tissue.</a> Biomaterials. 34 (34), 8581-8588 (2013).</li><li>Semler, E. J., Lancin, P. A., Dasgupta, A., Moghe, P. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+hepatocellular+morphogenesis+and+function+via+ligand-presenting+hydrogels+with+graded+mechanical+compliance.">Engineering hepatocellular morphogenesis and function via ligand-presenting hydrogels with graded mechanical compliance.</a> Biotechnology Bioengineering. 89 (3), 296-307 (2005).</li><li>Reinhart-King, C. A., Dembo, M., Hammer, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-cell+mechanical+communication+through+compliant+substrates.">Cell-cell mechanical communication through compliant substrates.</a> Biophysical journal. 95 (12), 6044-6051 (2008).</li><li>Fischer, R. S., Myers, K. A., Gardel, M. L., Waterman, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stiffness-controlled+three-dimensional+extracellular+matrices+for+high-resolution+imaging+of+cell+behavior.">Stiffness-controlled three-dimensional extracellular matrices for high-resolution imaging of cell behavior.</a> Nature protocols. 7 (11), 2056-2066 (2012).</li><li>Quinlan, A. M., Billiar, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigating+the+role+of+substrate+stiffness+in+the+persistence+of+valvular+interstitial+cell+activation.">Investigating the role of substrate stiffness in the persistence of valvular interstitial cell activation.</a> Journal of Biomedical Materials Research Part A. 100 (9), 2474-2482 (2012).</li><li>Zustiak, S. P., Leach, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrolytically+degradable+poly(ethylene+glycol)+hydrogel+scaffolds+with+tunable+degradation+and+mechanical+properties.">Hydrolytically degradable poly(ethylene glycol) hydrogel scaffolds with tunable degradation and mechanical properties.</a> Biomacromolecules. 11 (5), 1348-1357 (2010).</li><li>Chrambach, A., Rodbard, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyacrylamide+gel+electrophoresis.">Polyacrylamide gel electrophoresis.</a> Science. 172 (3982), 440-451 (1971).</li><li>Lin, Y. C., 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=Mechanosensing+of+substrate+thickness.">Mechanosensing of substrate thickness.</a> Physical review. 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The authors have nothing to disclose.

Geles de poliacrilamida, desarrollados originalmente para la electroforesis, 28 ahora se utilizan rutinariamente como sustratos de cultivo celular para estudiar los efectos de la rigidez del sustrato sobre la morfología celular, la motilidad y la comunicación 3,24,29 entre otras características de las células. Poliacrilamida permite la manipulación de la rigidez del sustrato para abarcar la rigidez de los tejidos blandos del cuerpo (0,3 a 300 kPa) 1 con un simple cambio en la concen...

La mayoría de los tejidos en el cuerpo son materiales viscoelásticos suaves con un módulo de Young que varía de 0,1 kPa para el cerebro a 100 kPa para el cartílago suave, sin embargo, la mayoría en la investigación de células in vitro se lleva a cabo en poliestireno de cultivo de tejido (TCP), que tiene un módulo de ~ 1 GPa . 1 Este desajuste rigidez afecta en gran medida la forma en las células responden a su medio ambiente. Un cuerpo creciente de investigación es, pues, dedicada a elucidar el efecto de la rigidez del sustrato sobre el destino de diversos tipos de células, incluidas las células madre 2,3. 4 Como resultado, múltiples hidrogeles han sido desarrollados para ayudar en la comprensión de la célula de rigidez dependiente biología incluyendo poliacrilamida (PA), 5-7 polietilenglicol (PEG), 8,9 polidimetilsiloxano (PDMS), 10 y alginato. 11 Mientras que la evidencia de que la rigidez del sustrato tiene un impacto sustancial en el destino celular está creciendo, se llevan a cabo en la mayoría de los estudios una pequeña escala con un pequeño número de sejemplos. Los estudios sistemáticos, multidimensionales sobre el efecto de la rigidez del sustrato para una gran variedad de tipos de células o las condiciones ambientales son raros. 12 Varias tecnologías de hidrogel de alto rendimiento prometedores se han desarrollado, incluyendo microarrays basados ​​en PEG, 13 dispositivos de microfluidos para la producción de microperlas de agarosa de hidrogel, 14 o micro y nano-barras, donde la rigidez es modulada por el diámetro y la altura de los los micro. 15 Sin embargo , las tecnologías de la preparación de dichos sustratos son sofisticados y disponible para un número limitado de laboratorios. Mucha investigación que implica rigidez modulada respuestas de células utiliza poliacrilamida (PA) geles que no sólo son de bajo costo y simple de implementar, sino también exhibir una gama fisiológicamente relevante del módulo de Young, es decir, 0,3 -. 300 kPa 16-22 Sin embargo, los métodos existentes para fabricar PA geles para el cultivo celular son mano de obra intensiva y, en consecuencia prepared en pequeños lotes. Algunas de las dificultades asociadas con la preparación de geles PA como sustratos de células madre a partir de la exigencia de que los geles tienen que estar preparados: 1) en ausencia de oxígeno para permitir la polimerización completa, 2) con una superficie plana y lisa para permitir celular uniforme apego y difusión, y 3) fijado de forma permanente a la parte inferior de la placa de cultivo celular para prevenir flotante. Varios grupos han intentado producir geles PA para el cultivo de células en grandes lotes. Semler et al. Láminas gruesas preparados de geles PA que fueron luego &quot;cortada&quot; con un punzón y se colocaron en placas de 96 pocillos. 23 Sin embargo, este método está limitado a geles más rígidos, es decir,&gt; 1 kPa en el módulo de Young, ya que más suave geles son &quot;pegajosos&quot;, difícil de cortar, y se dañan fácilmente. Mih et al. Desarrolló una técnica más sofisticada que permite a los geles a polimerizar directamente en una placa de múltiples pocillos con fondo de cristal. <sarriba&gt; 6 Esto se logró mediante el vertido de las soluciones de gel en placas con fondo de vidrio funcionalizadas y formar geles por &quot;sandwich&quot; con una matriz cubreobjetos personalizado. 6 A pesar de que, los efectos de borde ligeras muy prometedores todavía se observaron con esta técnica. Además, la técnica requiere una matriz de diseño personalizado no inmediatamente accesible a muchos laboratorios, así como costosas placas de múltiples pocillos con fondo de cristal. Este documento describe una manera sencilla y económica de montar geles PA en una placa de múltiples pocillos que podría ser adoptado fácilmente por cualquier laboratorio. Aquí, se utiliza un soporte de plástico flexible, que tiene dos lados - un uno hidrofóbico, que es repelente a los geles PA, y uno hidrofílico, que se une covalentemente el gel de PA sobre la deposición. Una vez láminas de gel de PA se depositan y permanentemente fijada en el soporte de plástico flexible, permite un manejo geles de cualquier grosor o rigidez y cortarlos en cualquier forma deseada. Este aproach no sólo produce personalizados &#39;&#39; cubreobjetos de plástico en tamaños no disponibles comercialmente, sino que también elimina la necesidad de las superficies de vidrio-tratamiento previo, ya sea cubreobjetos de vidrio o los pozos de las costosas placas de múltiples pocillos con fondo de cristal, con una solución PA vinculante, que es un tedioso y un paso de tiempo. Por último, las hojas de geles PA uniformes se pueden preparar en grandes lotes y se almacenaron-de hidratado durante varios meses. En resumen, el ensayo que aquí se presenta es una mejora sobre los métodos existentes en varios aspectos. En primer lugar, el proceso de montaje de placa de pocillos múltiples, es eficiente, y el coste global de los materiales requeridos es baja. En segundo lugar, los hidrogeles se producen en grandes lotes en una única película de gel homogénea. Finalmente, sólo se requieren materiales que están disponibles comercialmente. La utilidad del ensayo se ilustra por explorar el efecto de la rigidez del sustrato sobre la morfología celular y el área de difusión.

<table border="0" cellpadding="0" cellspacing="0" width="898">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Reagents</td>
			<td style="width:200px;"></td>
			<td style="width:187px;"></td>
			<td style="width:208px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:303px;">40% Acrylamide</td>
			<td style="width:200px;">Bio-Rad</td>
			<td style="width:187px;">161-0140</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:303px;">2% Bis-acrylamide</td>
			<td style="width:200px;">Bio-Rad</td>
			<td style="width:187px;">161-0142</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:303px;">Ammonium Persulfate</td>
			<td style="width:200px;">Bio-Rad</td>
			<td style="width:187px;">161-07000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">TEMED</td>
			<td style="width:200px;">Sigma Aldrich</td>
			<td style="width:187px;">T9281</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:303px;">Sulfo-SANPAH</td>
			<td style="width:200px;">Thermo Scientific</td>
			<td style="width:187px;">22589</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:303px;">Collagen Type 1, from Rat tail, 3.68 mg/mL</td>
			<td style="width:200px;">BD Biosciences</td>
			<td style="width:187px;">354236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Dimethyl sulfoxide (DMSO)</td>
			<td style="width:200px;">Fisher Scientific&#160;</td>
			<td style="width:187px;">BP231-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:303px;">Hydrophobic solution - Repel Silane&#160;</td>
			<td style="width:200px;">GE Healthcare Bio-Sciences</td>
			<td style="width:187px;">17-1332-01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:303px;">PBS (1x), pH 7.4</td>
			<td style="width:200px;">HyClone</td>
			<td style="width:187px;">SH30256.01</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:42px;width:303px;">Polydimehylsiloxane (PDMS) [Slygard 182 Elastomer Kit]</td>
			<td style="width:200px;">Elsworth Adhesives</td>
			<td style="width:187px;">3097358-1004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Tyrpsin/EDTA (10x)</td>
			<td style="width:200px;">Sigma Aldrich</td>
			<td style="width:187px;">44174</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">RPMI-1640 Medium (1x)</td>
			<td style="width:200px;">HyClone</td>
			<td style="width:187px;">SH30027-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Fetal Bovine Serum</td>
			<td style="width:200px;">HyClone</td>
			<td style="width:187px;">SH30073-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Penicillin Streptomycin</td>
			<td style="width:200px;">MP Biomedicals</td>
			<td style="width:187px;">1670046</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Detergent - Triton-X</td>
			<td style="width:200px;">Sigma Aldrich</td>
			<td style="width:187px;">T8787</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Formaldehyde 37% Solution</td>
			<td style="width:200px;">Sigma Aldrich</td>
			<td style="width:187px;">F1635</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:200px;">Sigma Aldrich</td>
			<td style="width:187px;">A2153</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">BSA-based cell adhesion blocking kit - ECM Cell Adhesion Array Kit</td>
			<td style="width:200px;">Chemicon International</td>
			<td style="width:187px;">ECM540</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;"></td>
			<td style="width:200px;"></td>
			<td style="width:187px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Disposable lab equipment</td>
			<td style="width:200px;"></td>
			<td style="width:187px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">flexible plastic support - GelBond PAG Film for Polyacrylamide Gels</td>
			<td style="width:200px;">GE Healthcare Bio-Sciences</td>
			<td style="width:187px;">309819</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Glass Plates</td>
			<td style="width:200px;">Slumpys</td>
			<td style="width:187px;">GBS4100SFSL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">50 mL Conicals</td>
			<td style="width:200px;">Fisher Scinetific</td>
			<td style="width:187px;">3181345107</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">15 mL Conicals</td>
			<td style="width:200px;">FALCON</td>
			<td style="width:187px;">352097</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Micro centrifuge tubes</td>
			<td style="width:200px;">Fisher Scinetific</td>
			<td style="width:187px;">2 mL: 02681258</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">96-well plate (flat bottom)</td>
			<td style="width:200px;">Fisher Scinetific</td>
			<td style="width:187px;">12565501</td>
			<td></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:303px;">Disposable Pipettes (1 mL, 2mL, 5mL, 10mL, 25 mL, 50mL)</td>
			<td style="width:200px;">Fisher Scinetific</td>
			<td style="width:187px;">1 mL: 13-678-11B, 2mL: 05214038, 5mL(FALCON): 357529, 10mL: 13-678-11E, 25mL: 13-678-11, 50mL: 13-678-11F</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">Glass Transfer Pipettes</td>
			<td style="width:200px;">Fisher Scinetific</td>
			<td style="width:187px;">5 3/4&#34;: 1367820A, 9&#34;:136786B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Pipette Tips (1-200uL, 101-1000uL)</td>
			<td style="width:200px;">Fisher Scinetific</td>
			<td style="width:187px;">2707509</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">Plastic Standard Disposable Transfer Pipettes</td>
			<td style="width:200px;">Fisher Scientific</td>
			<td style="width:187px;">13-711-9D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Parafilm</td>
			<td style="width:200px;">PARAFILM&#160;</td>
			<td style="width:187px;">PM992</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Powder Free Examination Gloves</td>
			<td style="width:200px;">Quest</td>
			<td style="width:187px;">92897</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">Silicone spacers - Silicone sheet, 0.5 mm thick/13 cm x 18 cm</td>
			<td style="width:200px;">Grace Bio-Labs</td>
			<td style="width:187px;">JTR-S-0.5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;"></td>
			<td style="width:200px;"></td>
			<td style="width:187px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Large/non-disposable lab equipment</td>
			<td style="width:200px;"></td>
			<td style="width:187px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">Light and Flourescent Microscope (Axiovert 200M)</td>
			<td style="width:200px;">Zeiss</td>
			<td style="width:187px;">3820005619</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Microscope Software</td>
			<td style="width:200px;">Zeiss</td>
			<td style="width:187px;">AxioVision Rel. 4.8.2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">UV oven</td>
			<td style="width:200px;">UVITRON</td>
			<td style="width:187px;">UV1080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Vacuum chamber/degasser</td>
			<td style="width:200px;">BelArt</td>
			<td style="width:187px;">999320237</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Vacuum pump for degasser</td>
			<td style="width:200px;">KNF Lab</td>
			<td style="width:187px;">5097482</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Tissue Culture Hood</td>
			<td style="width:200px;">NUAIRE</td>
			<td style="width:187px;">NU-425-600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Chemical Fume Hood</td>
			<td style="width:200px;">KEWAUNEE</td>
			<td style="width:187px;">99151</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Inverted Microscope (Axiovert 25)</td>
			<td style="width:200px;">Zeiss</td>
			<td style="width:187px;">663526</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Incubator</td>
			<td style="width:200px;">NUAIRE</td>
			<td style="width:187px;">NU-8500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Pipette Aid</td>
			<td style="width:200px;">Drummond Scientific Co.</td>
			<td style="width:187px;">P-76864</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Hemacytometer</td>
			<td style="width:200px;">Bright-Line</td>
			<td style="width:187px;">383684</td>
			<td></td>
		</tr>
	</tbody>
</table>

simple polyacrylamide-based multiwell stiffness assay for the study of stiffness-dependent cell responses

Currently, most of the in vitro cell research is performed on rigid tissue culture polystyrene (~1 GPa), while most cells in the body are attached to a matrix that is elastic and much softer (0.1 &#8211; 100 kPa). Since such stiffness mismatch greatly affects cell responses, there is a strong interest in developing hydrogel materials that span a wide range of stiffness to serve as cell substrates. Polyacrylamide gels, which are inexpensive and cover the stiffness range of all soft tissues in the body, are the hydrogel of choice for many research groups. However, polyacrylamide gel preparation is lengthy, tedious, and only suitable for small batches. Here, we describe an assay which by utilizing a permanent flexible plastic film as a structural support for the gels, enables the preparation of polyacrylamide gels in a multiwell plate format. The technique is faster, more efficient, and less costly than current methods and permits the preparation of gels of custom sizes not otherwise available. As it doesn&#8217;t require any specialized equipment, the method could be easily adopted by any research laboratory and would be particularly useful in research focused on understanding stiffness-dependent cell responses.

Poliacrilamida (PA) hidrogeles son ampliamente utilizados para probar las respuestas de células de rigidez dependiente. 17,24 Mediante la mezcla de varias concentraciones de acrilamida (A) y bis-acrilamida (B) se puede hacer geles de PA que abarcan el rango de rigidez de la mayoría de los tejidos blandos en el cuerpo - 0,3 -.. 300 módulo de kPa joven 1 Sin embargo, la preparación de geles de poliacrilamida es tedioso y consume mucho tiempo, a menudo limita su utilidad en aplicaciones de &quot;a...

Watch this Scientific Journal Video about Simple Polyacrylamide-based Multiwell Stiffness Assay for the Study of Stiffness-dependent Cell Responses at JoVE.com

Simple Polyacrylamide-based Multiwell Stiffness Assay for the Study of Stiffness-dependent Cell Responses

Here, a method that enables quick, efficient, and inexpensive preparation of polyacrylamide gels in a multiwell plate format is described. The method does not require any specialized equipment and could be easily adopted by any research laboratory. It would be particularly useful in research focused on understanding stiffness-dependent cell responses.

Multiwell Rigidez Ensayo para el Estudio de las respuestas de las células Rigidez dependientes simple basado poliacrilamida-

Currently, most of the in vitro cell research is performed on rigid tissue culture polystyrene (~1 GPa), while most cells in the body are attached to a ...

simple-polyacrylamide-based-multiwell-stiffness-assay-for-study

Research

JoVE Journal

Bioengineering

19.4K vistas.  Saint Louis University. Here, a method that enables quick, efficient, and inexpensive preparation of polyacrylamide gels in a multiwell plate format is described. The method does not require any specialized equipment and could be easily adopted by any research laboratory. It would be particularly useful in research focused on understanding stiffness-dependent cell responses.

Video: Simple Polyacrylamide-based Multiwell Stiffness Assay for the Study of Stiffness-dependent Cell Responses

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue (EHT)

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or bioartificial scaffold materials, entrapment of cardiac myocytes in hydrogels such as fibrin or collagen and stacking of myocyte monolayers 1. These concepts aim at restoration of compromised cardiac function (e.g. after myocardial infarction) or as experimental models (e.g. predictive toxicology and substance screening or disease modelling). Precise monitoring of cell survival after implantation of engineered heart tissue (EHT) has now become possible using in-vivo bioluminescence imaging (BLI) techniques 2. Here we describe the generation of fibrin-based EHT from a transgenic rat strain with ubiquitous expression of firefly luciferase (ROSA/luciferase-LEW Tg; 3). Implantation is performed into the greater omentum of different rat strains to assess immune responses of the recipient organism following EHT implantation. Comparison of results generated by BLI and the Enzyme Linked Immuno Spot Technique (ELISPOT) confirm the usability of BLI for the assessment of immune responses.

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue EHT

This video demonstrates the use of in vivo bioluminescence imaging to study immune responses after implantation of Engineered Heart Tissue (EHT) in rats.

Bioluminiscencia de imágenes para la evaluación de la respuesta inmune después de la implantación de tejido cardiaco de Ingeniería (EHT)

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or ...

Investigación

Bioingeniería

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells within a tissue, however, are typically encased within a closely packed tissue mass in which cells establish intimate connections with many near-neighbors and with extracellular matrix components. Accordingly, the chemical milieu and physical forces experienced by cells within a 3D tissue are fundamentally different than those experienced by cells grown in monolayer culture. This has been shown to markedly impact cellular morphology and signaling. Several methods have been devised to generate 3D cell cultures including encapsulation of cells in collagen gels1or in biomaterial scaffolds2. Such methods, while useful, do not recapitulate the intimate direct cell-cell adhesion architecture found in normal tissues. Rather, they more closely approximate culture systems in which single cells are loosely dispersed within a 3D meshwork of ECM products. Here, we describe a simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which cells are in direct contact with each other and with extracellular matrix components. The method requires no specialized equipment and can be adapted to include addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell-cell or cell-ECM interaction. The method can also be used to co-culture two (or more) different cell populations so as to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between cells. Cell-cell cohesion and cell-ECM adhesion are the cornerstones of studies of embryonic development, tumor-stromal cell interaction in malignant invasion, wound healing, and for applications to tissue engineering. This simple method will provide a means of generating tissue-like cellular aggregates for measurement of biomechanical properties or for molecular and biochemical analysis in a physiologically relevant model.

We describe a simple, rapid method of generating 3D tissue-like spheroids and their potential application to quantify differences in cell-cell interactions.

Una simple gota colgante celular Cultura Protocolo para la generación de esferoides 3D

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells ...

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and metastasis of tumor cells. Migration has been studied on glass coverslips in order to make cellular dynamics amenable to investigation by light microscopy. However, it has become clear that many aspects of cell migration depend on features of the local environment including its elasticity, protein composition, and pore size, which are not faithfully represented by rigid two dimensional substrates such as glass and plastic 1. Furthermore, interaction with other cell types, including stromal fibroblasts 2 and immune cells 3, has been shown to play a critical role in promoting the invasion of cancer cells. Investigation at the molecular level has increasingly shown that molecular dynamics, including response to drug treatment, of identical cells are significantly different when compared in vitro and in vivo 4. 
Ideally, it would be best to study cell migration in its naturally occurring context in living organisms, however this is not always possible. Intermediate tissue culture systems, such as cell derived matrix, matrigel, organotypic culture (described here) tissue explants, organoids, and xenografts, are therefore important experimental intermediates. These systems approximate certain aspects of an in vivo environment but are more amenable to experimental manipulation such as use of stably transfected cell lines, drug treatment regimes, long term and high-resolution imaging. Such intermediate systems are especially useful as proving grounds to validate probes and establish parameters required to image the dynamic response of cells and fluorescent reporters prior to undertaking imaging in vivo 5. As such, they can serve an important role in reducing the need for experiments on living animals.

A method is described for the preparation of a 3-dimensional matrix consisting of collagen type I and primary human fibroblasts. This organotypic gel serves as a useful substrate to assess invasive cell migration because it mimics basic features of tissue stroma and is amenable to many forms of microscopy.

El colágeno organotípicos I del ensayo: Una plataforma maleable para evaluar el comportamiento celular en un contexto de 3 dimensiones

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

Cell-based Calcium Assay for Medium to High Throughput Screening of TRP Channel Functions using FlexStation 3

The Molecular Devices' FlexStation 3 is a benchtop multi-mode microplate reader capable of automated fluorescence measurement in multi-well plates. It is ideal for medium- to high-throughput screens in academic settings. It has an integrated fluid transfer module equipped with a multi-channel pipetter and the machine reads one column at a time to monitor fluorescence changes of a variety of fluorescent reagents. For example, FlexStation 3 has been used to study the function of Ca2+-permeable ion channels and G-protein coupled receptors by measuring the changes of intracellular free Ca2+ levels. Transient receptor potential (TRP) channels are a large family of nonselective cation channels that play important roles in many physiological and pathophysiological functions. Most of the TRP channels are calcium permeable and induce calcium influx upon activation. In this video, we demonstrate the application of FlexStation 3 to study the pharmacological profile of the TRPA1 channel, a molecular sensor for numerous noxious stimuli. HEK293 cells transiently or stably expressing human TRPA1 channels, grown in 96-well plates, are loaded with a Ca2+-sensitive fluorescent dye, Fluo-4, and real-time fluorescence changes in these cells are measured before and during the application of a TRPA1 agonist using the FLEX mode of the FlexStation 3. The effect of a putative TRPA1 antagonist was also examined. Data are transferred from the SoftMax Pro software to construct concentration-response relationships of TRPA1 activators and inhibitors.

This video provides a detailed protocol for studying the pharmacological profile of human TRPA1 channels using FlexStation 3. The protocol covers details of cell preparation, dye loading and operation of the microplate reader, FlexStation 3.

Basados ​​en células de calcio de ensayo para el medio a cribado de alto rendimiento de las funciones del PRT canal utilizando FlexStation 3

The Molecular Devices' FlexStation 3 is a benchtop multi-mode microplate reader capable of automated fluorescence measurement in multi-well plates.  It is ...

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood cell (RBC) as adhesion sensor and presenting cell for one of the interacting molecules. It employs micromanipulation to bring the RBC into contact with another cell that expresses the other interacting molecule with precisely controlled area and time to enable bond formation. The adhesion event is detected as RBC elongation upon pulling the two cells apart. By controlling the density of the ligands immobilized on the RBC surface, the probability of adhesion is kept in mid-range between 0 and 1. The adhesion probability is estimated from the frequency of adhesion events in a sequence of repeated contact cycles between the two cells for a given contact time. Varying the contact time generates a binding curve. Fitting a probabilistic model for receptor-ligand reaction kinetics1 to the binding curve returns the 2D affinity and off-rate.
The assay has been validated using interactions of Fc&gamma; receptors with IgG Fc1-6, selectins with glycoconjugate ligands6-9, integrins with ligands10-13, homotypical cadherin binding14, T cell receptor and coreceptor with peptide-major histocompatibility complexes15-19.
The method has been used to quantify regulations of 2D kinetics by biophysical factors, such as the membrane microtopology5, membrane anchor2, molecular orientation and length6, carrier stiffness9, curvature20, and impingement force20, as well as biochemical factors, such as modulators of the cytoskeleton and membrane microenvironment where the interacting molecules reside and the surface organization of these molecules15,17,19.
The method has also been used to study the concurrent binding of dual receptor-ligand species3,4, and trimolecular interactions19 using a modified model21.
The major advantage of the method is that it allows study of receptors in their native membrane environment. The results could be very different from those obtained using purified receptors17. It also allows study of the receptor-ligand interactions in a sub-second timescale with temporal resolution well beyond the typical biochemical methods.
To illustrate the micropipette adhesion frequency method, we show kinetics measurement of intercellular adhesion molecule 1 (ICAM-1) functionalized on RBCs binding to integrin &alpha;L&beta;2 on neutrophils with dimeric E-selectin in the solution to activate &alpha;L&beta;2.

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

An adhesion frequency assay for measuring receptor-ligand interaction kinetics when both molecules are anchored on the surfaces of the interacting cells is described. This mechanically-based assay is exemplified using a micropipette-pressurized human red blood cell as adhesion sensor and integrin &alpha;L&beta;2 and intercellular adhesion molecule-1 as interacting receptors and ligands.

Ensayo de adherencia de frecuencia para In Situ El análisis de la cinética de unión de la Cruz-Las interacciones moleculares en la interfaz célula-célula

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood ...

Micropipette Aspiration of Substrate-attached Cells to Estimate Cell Stiffness

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell proliferation, differentiation, migration and cell-cell interactions. The two key parameters of cellular biomechanics are cellular deformability or stiffness and the ability of the cells to contract and generate force. Here we describe a quick and simple method to estimate cell stiffness by measuring the degree of membrane deformation in response to negative pressure applied by a glass micropipette to the cell surface, a technique that is called Micropipette Aspiration or Microaspiration.
Microaspiration is performed by pulling a glass capillary to create a micropipette with a very small tip (2-50 &mu;m diameter depending on the size of a cell or a tissue sample), which is then connected to a pneumatic pressure transducer and brought to a close vicinity of a cell under a microscope. When the tip of the pipette touches a cell, a step of negative pressure is applied to the pipette by the pneumatic pressure transducer generating well-defined pressure on the cell membrane. In response to pressure, the membrane is aspirated into the pipette and progressive membrane deformation or "membrane projection" into the pipette is measured as a function of time. The basic principle of this experimental approach is that the degree of membrane deformation in response to a defined mechanical force is a function of membrane stiffness. The stiffer the membrane is, the slower the rate of membrane deformation and the shorter the steady-state aspiration length.The technique can be performed on isolated cells, both in suspension and substrate-attached, large organelles, and liposomes.
Analysis is performed by comparing maximal membrane deformations achieved under a given pressure for different cell populations or experimental conditions. A "stiffness coefficient" is estimated by plotting the aspirated length of membrane deformation as a function of the applied pressure. Furthermore, the data can be further analyzed to estimate the Young's modulus of the cells (E), the most common parameter to characterize stiffness of materials. It is important to note that plasma membranes of eukaryotic cells can be viewed as a bi-component system where membrane lipid bilayer is underlied by the sub-membrane cytoskeleton and that it is the cytoskeleton that constitutes the mechanical scaffold of the membrane and dominates the deformability of the cellular envelope. This approach, therefore, allows probing the biomechanical properties of the sub-membrane cytoskeleton.

Here we describe a quick and simple method to measure cell stiffness. The general principle of this approach is to measure membrane deformation in response to well-defined negative pressure applied through a micropipette to the cell surface. This method provides a powerful tool to study biomechanical properties of substrate-attached cells.

Aspiración Micropipeta de células adheridas a sustratos para estimar la rigidez Celular

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell ...

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 ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

Preparación de geles de colágeno en 3D y microcanales para el estudio de las interacciones 3D<em&gt; En Vivo</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

A Simple and Scalable Fabrication Method for Organic Electronic Devices on Textiles

Today, wearable electronics devices combine a large variety of functional, stretchable, and flexible technologies. However, in many cases, these devices cannot be worn under everyday conditions. Therefore, textiles are commonly considered the best substrate to accommodate electronic devices in wearable use. In this paper, we describe how to selectively pattern organic electroactive materials on textiles from a solution in an easy and scalable manner. This versatile deposition technique enables the fabrication of wearable organic electronic devices on clothes.

In this paper, we present a protocol to selectively deposit organic materials on textiles, which allows for the direct integration of organic electronic devices with wearables. The fabricated devices can be fully integrated in textiles, respecting their mechanical appearance and enabling sensing capabilities.

Un método de fabricación simple y escalable para dispositivos electrónicos orgánicos sobre los Textiles

Today, wearable electronics devices combine a large variety of functional, stretchable, and flexible technologies. However, in many cases, these devices ...

3D Analysis of Multi-cellular Responses to Chemoattractant Gradients

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and chemical complexity of living tissues and mimic in vivo tissue functions. Recent advances in microfabrication technologies have facilitated the development of 3D in vitro environments in which cells can be integrated into a well-defined extracellular matrix (ECM) and a defined set of soluble or matrix associated biomolecules. However, technological barriers have limited their widespread use in research laboratories. Here, we describe a method to construct simple devices for 3D culture and experimentation with cells and multicellular organoids in 3D microenvironments with a defined chemoattractant gradient. We illustrate the use of this platform for analysis of the response of epithelial cells and organoids to gradients of growth factors, such as epidermal growth factor (EGF). EGF gradients were stable in the devices for several days leading to directed branch formation in breast organoids. This analysis allowed us to conclude that collective gradient sensing by groups of cells is more sensitive vs. single cells. We also describe the fabrication method, which does not require photolithography facilities nor advanced soft lithography techniques. This method will be helpful to study 3D cellular behaviors in the context of the analysis of development and pathological states, including cancer.

We describe a method to construct devices for 3D culture and experimentation with cells and multicellular organoids. This device allows analysis of cellular responses to soluble signals in 3D microenvironments with defined chemoattractant gradients. Organoids are better than single cells at detection of weak noisy inputs.

Análisis 3D de las respuestas multicelulares a los degradados Quimioattractant

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and ...