This work is financially supported by the Ministry of Science and Technology, Taiwan (Contract no. MOST 103-2113-M-001 -003 -MY2) and the Research Program on Nanoscience and Nanotechnology, Academia Sinica, Taiwan.

1. Diseño y Fabricación de la viruta de MDF <ol><li> Dibuja un patrón de capa acrílica individual utilizando software comercial, tales como AutoCAD, y guardar el patrón. <ol><li> Revisar el diseño del patrón de lámina acrílica de cuatro capas en la Figura 1A y confirmar las conexiones entre capas. </li></ol></li><li> Fabrique todas las hojas de acrílico y la cinta de doble cara mediante ablación láser usando un láser scriber CO 2 (Figuras 1B, 2A y 2B). 16 <ol><li> Encienda el Scriber láser y conectar el equipo a la computadora portátil de control. </li><li> Ejecute el software comercial y abrir el patrón diseñado en el paso 1.1, haga clic en &quot;archivo de patrones diseñados.&quot; </li><li> Coloque un pedazo de hoja de acrílico blanco o cinta de doble cara en el escenario XYZ del scriber láser. </li><li> Establecer el foco del haz de láser en la superficie de la lámina de acrílico o cinta de doble cara con un auto-alignment stick proporcionado por el fabricante de la punta de trazar láser. </li><li> Enviar el patrón diseñado para la punta de trazar de láser para el mecanizado directo de la lámina de acrílico o cinta de doble cara. </li></ol></li><li> Retire el papel protector de las hojas de acrílico utilizando pinzas y soplar la superficie limpia con gas nitrógeno. </li><li> Pila de las hojas de acrílico y unir juntos bajo una presión de 2 kg / cm 2 en un dispositivo de unión térmica durante 45 minutos a 110 ° C para formar el / conjunto de canal estimulación eléctrica de flujo. </li><li> Preparar la cubierta de vidrio de microscopio como el sustrato de cultivo celular en el chip. <ol><li> Ponga la cubierta de vidrio a una cubeta y llenar el frasco con una dilución de diez veces del detergente que figuran en la lista de materiales. </li><li> Ponga la cubierta de vidrio y jarra de tinción en una Steri-limpiador ultrasónico y limpiar la cubierta de vidrio durante 15 minutos. </li><li> Vierta el detergente diluido fuera del tarro tinción, vuelva a llenar el recipiente con agua destilada y repetir el paso 1.5.2 3 veces. </li><li> Se seca la cubierta de vidrio limpiado mediante soplado con gas nitrógeno antes de adherir a la cinta de doble cara. </li></ol></li><li> Pegue la cubierta de vidrio limpiado a la / conjunto de canal estimulación eléctrica de flujo con la cinta de doble cara con dibujos en el paso 1.2. </li><li> Adherirse 13 piezas de adaptadores de acrílico a las aberturas individuales en la capa 1 del conjunto de chips MDF con pegamento. El conjunto de chips MDF es entonces completa (Figura 2D). </li><li> Esterilizar el conjunto completo de chip MDF con 30 min de irradiación UV antes de su uso. </li></ol> 2. Configuración de la Red Puente La sal de la viruta de MDF <ol><li> Antes de su uso, esterilizar todos los tubos de plástico, las tuercas con los dedos, y tubos de microcentrífuga que se muestran en la Figura 3B mediante el establecimiento de un tiempo de retención de 15 min a 121 ° C en el autoclave. </li><li> Conecte los tubos fluoroplástico (Figura 3B-i) a la asamblea chip de MDF a través de la entrada del medioy los adaptadores de salida muestran en la Figura 1A. </li><li> Conecte el cono Luer de la entrada del medio y la salida de los tubos de plástico en la figura 3B-i a las llaves de paso de 3 vías. </li><li> Tomar 2,5 ml de CO 2 salina tamponada con fosfato -equilibrated (PBS) usando una jeringa de 3 ml y conectar la jeringa a la llave de paso de 3 vías del tubo de plástico de entrada. Conectar una jeringa vacía de 3 ml a la llave de paso de 3 vías del tubo de plástico de salida. Las dos jeringas son así interconectados a través del chip MDF. NOTA: Incubar la PBS en el 37 ° C 5% -CO 2 incubadora de células de cultivo O / N para obtener CO 2 -equilibrated PBS. </li><li> Selle las aberturas del azul y los adaptadores verdes (Figura 1A) en la hoja de PMMA Capa 1 con las tuercas con los dedos sólidos blancos (Figura 3B-iii). </li><li> Llene los canales del puente de sal y cámaras de cultivo con el CO 2 -equilibrated PBS en la jeringa se describe en el paso de instalación 2.4. Evitarla formación de burbujas. </li><li> A continuación, poner el CO 2 -equilibrated PBS que contiene chips de MDF en el 37 ° C 5% CO 2 incubadora de cultivo celular para O / N de incubación. Esto permite que el aire disuelto en la cinta de doble cara para formar burbujas en las cámaras. </li><li> Enjuague de distancia las burbujas en los canales por un flujo rápido de PBS en el canal utilizando las dos jeringas. Bombear el PBS de ida y vuelta si es necesario. </li><li> Escurrir el PBS en los canales a través de la llave de paso de 3 vías conectada al tubo de salida medio. </li><li> Utilizando una nueva jeringa de 3 ml, tomar 2,5 ml de CO 2 -equilibrated Medio Eagle Modificado de Dulbecco (DMEM) (véase el paso 3.5), y reemplazar la jeringa conectada a la entrada con el nuevo. Vuelva a llenar los canales con el medio. </li><li> Preparación de la red de puente de sal. <ol><li> Eliminar temporalmente las tuercas sólidos (Figura 3B-iii) en los adaptadores verdes (Figura 1A) e inyectar 3% caliente (&gt; 70 ° C) de agarosa al bri salcanal dge a través de la abertura en la adaptadores verdes (Figura 1). NOTA: Preparación de agarosa caliente: Disolver 1,5 g de polvo de agarosa en 50 ml de PBS. Esterilizar estableciendo un tiempo de retención de 20 min a 121 ° C en el autoclave. </li><li> Detener la inyección de la agarosa cuando el líquido llena tres cuartas partes de la longitud del canal de puente salino. </li><li> Sellar los poros en los adaptadores verdes (Figura 1A) atornillando las tuercas de sólidos (Figura 3B-iii) después de la inyección de agarosa. </li><li> Vuelva a colocar las tuercas sólidos sobre los adaptadores azules (Figura 1A) con las tuercas con los dedos tubulares translúcidos (Figura 3B). </li><li> Cargue el 3% precalentado agarosa en las tuercas tubulares (Figura 3B-IV). </li><li> Insertar los electrodos de Ag / AgCl (Figura 3B-V) en las tuercas tubulares (Figura 3B-iv) antes de lasolidificación de la agarosa. </li></ol></li><li> Después de completar la configuración de la red de puente de sal, inyectar las células de cáncer de pulmón CL1-5 en las cámaras de cultivo, una de las cámaras a la vez. </li></ol> 3. Preparación de las células cancerosas y puesta en marcha para el Experimento Electrotactic <ol><li> Cultura regular de línea celular de cáncer de pulmón CL1-5. <ol><li> Cultura de las células de cáncer de pulmón, CL1-5 obtenidos del Prof. Pan-Chyr Yang, 17 en el medio completo en un frasco de cultivo celular 75T a 37 ° C en una atmósfera de 5% de CO 2. El medio completo se compone de DMEM y suero bovino fetal al 10% (FBS). Sub-cultivar las células cada 3 ó 4 días. Las células utilizadas para la realización de experimentos electrotaxis están a menos de 25 pasajes de la fuente original. </li></ol></li><li> Añadir 2 ml de tampón de tripsina 0,25% a las células en crecimiento exponencial CL1-5 y se incuba durante 2 min en el 37 ° C 5% -CO 2 incubadora de cultivo celular para detachmen celularest. </li><li> Terminar el proceso de desprendimiento con 6 ml de DMEM 10% de FBS y centrifugar las células a 300 g durante 5 min a TA. Entonces desechar el medio y suspender el sedimento celular con 5 ml de PBS. </li><li> Contar el número de células en el PBS. Luego tomar 1 x 10 6 células y centrifugar a 300 g durante 5 minutos a temperatura ambiente. </li><li> Deseche la PBS. Suspender las células con pre-calentado CO 2 -equilibrated DMEM y ajustar la densidad de las células a ser de 1 × 10 6 células / ml. NOTA: Incubar el medio completo en el 37 ° C 5% CO 2 cultivo celular incubadora O / N para obtener el CO 2 -equilibrated DMEM. </li></ol> 4. Configuración para las Experimento Electrotactic <ol><li> Desde la salida del chip MDF, inyectar 0,3 ml, 1 × 10 6 / ml, de las células en el chip. </li><li> Incubar el chip MDF sembrado de células en la incubadora de cultivo celular (fijado en 37 ° C y 5% de CO 2) durante 2-4 horas. </li><li> Configuración de MDF microsistema fluídico. <ol><li> Instale el sistema de microfluidos MDF en un calentador de cristal de óxido de indio y estaño transparente (ITO). Medir la temperatura del chip de MDF con un termopar de tipo K recortado entre el chip MDF y vidrio ITO. Controle el calentador de ITO con un controlador proporcional-integral-derivativo (PID). Ajuste la temperatura de incubación de microfluidos sistema de MDF de 37 ° C con el controlador PID. NOTA: La fabricación del calentador de ITO y la configuración del sistema de calentamiento de células de cultivo se describen en los informes anteriores 18,19. </li><li> Montar el conjunto de chip de MDF de temperatura controlada en la etapa motorizada XYZ controlada por ordenador en un microscopio invertido. El uso de la platina motorizada se describe en el paso 5.1. Bombear el medio completo en las cámaras de cultivo a través de las entradas con una bomba de jeringa de cuatro canales. </li></ol></li><li> Bombear el medio completo en el chip MDF con un caudal de 20 l / hr. Los residuos cultura del cabolets se recoge en los tubos de microcentrífuga (que se muestran como &quot;residuos&quot; en la figura 3A). </li><li> Se incuban las células para otro 16-18 horas a 37 ° C en el chip de MDF. </li><li> Para reemplazar el medio, bombear el medio que contiene 50, 25, 5 y 0 inhibidor de quinasa mu M, respectivamente, en cada cámara de cultivo a una velocidad de flujo de 20 l / min durante 10 min. NOTA: Preparar una solución madre 10 mM del inhibidor de la proteína quinasa asociada a Rho Y27632 mediante la adición de 3 ml de agua estéril para el inhibidor de 10,6 mg quinasa. Diluir la solución madre 10 mM a 50, 25, y 5 mM, respectivamente, utilizando 10% de medio FBS DMEM. </li><li> Se incuban las células en el inhibidor de la quinasa durante otros 60 min a 37 ° C. Ajuste el caudal medio de 20 l / h. Utilice la misma temperatura cultura y la tasa se ha descrito anteriormente en los pasos posteriores fluir. </li><li> Utilice cables eléctricos para conectar una fuente de alimentación DC para el chip MDF a través de los electrodos de Ag / AgCl en el chip. </li><li> Conectar en serie un i amperímetron el circuito eléctrico. Utilice el amperímetro para vigilar la corriente eléctrica en el chip de MDF. </li><li> Encienda la fuente de alimentación de CC y ajuste el amperímetro actual a 86.94 mu mediante el ajuste de la tensión de alimentación de entre 15 y 19 V. NOTA: Teniendo en cuenta la ley de Ohm, E = I / (s A eff), donde I es la corriente eléctrica que fluye a través del material a granel, es decir, el medio de cultivo en la cámara de electrotactic, σ (= 1,38 Ω -1 m - 1) es el conductividad del medio de cultivo, A eff (= 0,21 mm 2 para una anchura de 3 mm x 0,07 mm de altura) es el área de sección transversal efectiva de la cámara de electrotactic, y la corriente eléctrica (I) necesaria para generar un 300 mV / mm EF en el chip de MDF es 86,94 mu. </li></ol> 5. Adquisición y Análisis de Imágenes celulares <ol><li> Controlar la platina motorizada mediante un programa MATLAB GUI. Con el sistema de microfluidos MDF montado en el microscope, mueva el chip a las regiones de observación mediante la platina motorizada. </li><li> Imágenes de células registro utilizando una cámara digital réflex de lente única (SLR) montadas en el microscopio. </li><li> Tomar imágenes microscópicas utilizando una lente objetivo 4X a intervalos de 15 min durante 2 horas. </li><li> Análisis de la migración celular. <ol><li> Ejecute NIH ImageJ 1,47 V. </li><li> Analizar la distancia desde la inicial a las posiciones finales de centroide de la célula. </li><li> Ir a &quot;Archivo&quot; → &quot;Importar&quot; → &quot;Secuencia de imágenes,&quot; e importar nueve imágenes para el análisis de la migración celular. La primera imagen es el resultado del tiempo cero, y la última imagen es el resultado de 120 min. </li><li> Ir a &quot;Analizar&quot; → &quot;Establecer Medidas&quot; y marque la casilla junto a &quot;centroide&quot; </li><li> Haga clic en el icono &quot;Freehand Selecciones&quot;. </li><li> Representar el borde de las celdas seleccionadas en la primera imagen. </li><li> Ir a &quot;Editar&quot; → </em&gt; &quot;Selección&quot; → &quot;Añadir a Manager&quot; </li><li> Ir a la novena imagen y representar el borde de las mismas células seleccionadas en la primera imagen. Las posiciones de células pueden ser rastreados utilizando el segundo a las imágenes octavo. </li><li> Ir a &quot;Editar&quot; → &quot;Selección&quot; → &quot;Añadir a Manager&quot; </li><li> Repita los pasos 5.4.5 al 5.4.9 para recopilar datos de 90 a 100 células. </li><li> Ir a &quot;Analizar&quot; → &quot;Medida&quot; para obtener el resultado de las posiciones inicial y final de las celdas seleccionadas. NOTA: La velocidad de migración se define como la longitud media de movimiento de la célula por hora. La direccionalidad se define como coseno, donde es el ángulo entre el vector de la dcEF (cátodo del ánodo al) y el vector desde el punto de partida de una célula a su posición final. La direccionalidad es -1 para las células que migran hacia el ánodo y 1 para las células que migran hacia el cátodo. Para un grupo de azar mirejilla de celdas, la direccionalidad es 0. 2 </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

Nos pareció que el proceso de adherirse adaptadores de acrílico en la capa 1 del chip MDF a ser complicado. La aplicación de sólo 1 a 2 l de pegamento es suficiente para adherirse firmemente el adaptador en el chip de MDF. Grandes cantidades de cola como resultado una polimerización incompleta del pegamento y la falta de adhesión. Una vez que los adaptadores de acrílico estaban firmemente adherido sobre el chip MDF, fuga de líquido en el sistema de microfluidos rara vez se produjo. Además, O / N de incubación ...

El comportamiento de las células adherentes que se mueven hacia un ánodo o cátodo bajo una corriente de campo eléctrico directo (dcEF) se conoce como electrotaxis. El comportamiento electrotactic de las células juega un papel importante en la embriogénesis, la regeneración del nervio, y la curación de la herida. 1 Las células tumorales tales como células de cáncer de próstata de rata, células de cáncer de mama 2, 3 y pulmonares células de adenocarcinoma 4-8 han demostrado movimiento electrotactic bajo una dcEF aplicado . El EF fisiológica se ha medido en tejidos de la glándula. 9,10 electrotaxis También se ha informado en las células tumor de la glándula asociada a 2,3. Tomados en conjunto, los electrotaxis de células de cáncer se considera que es un factor de metástasis. 11 El control de la orientación eléctrica de las células cancerosas en virtud de dcEF puede ser un enfoque potencial para el tratamiento futuro de cáncer. Sin embargo, hoy en día, el mecanismo molecular detallado de electrotaxis sigue siendo controvertido. Por lo tanto, una investigación de la influence de la estimulación eléctrica sobre la migración de células de cáncer puede facilitar el desarrollo de estrategias para el tratamiento del cáncer. Recientemente, los dispositivos bio-microfluidos se han fabricado para el estudio de las respuestas celulares a fluir la fuerza de corte, 12 gradientes químicos, 13 y eléctrica estímulos 4 in vitro. La fabricación de dispositivos de microfluidos utilizando bio-polidimetilsiloxano (PDMS) o polimetilmetacrilato (PMMA, también conocido como acrílico) ha reducido con éxito la tasa de fracaso de tales experimentos. Por otra parte, el uso de dispositivos de microfluidos de base acrílica como un prototipo para la investigación de temas biológicos es más simple que el uso de chips de PDMS. Varias funciones en los dispositivos de base acrílica se han desarrollado para electrotaxis estudio. Sin embargo, ninguno de los diseños anteriores son capaces de probar simultáneamente los efectos de varias condiciones químicas y el campo eléctrico en las células para electrotaxis estudio. Por lo tanto, hemos desarrollado un dispositivo de microfluidos-multichannel doble de campo eléctrico (MDF) cuatro canales de cultivo independientes y ocho condiciones experimentales diferentes en un chip que contiene chips. El chip de MDF de base acrílica, primero reportada por Hou et al., 8 integra la estimulación eléctrica y varios canales aislados químicamente. Estos canales aislados químicamente se pueden utilizar para la cultura diferentes tipos de células en un experimento. El dcEF en los canales es producida por una fuente de alimentación eléctrica. Dos campos eléctricos independientes, uno con la fuerza aplicada de campo eléctrico (EFS) y otro con 0 EFS, se llevan a cabo en cada canal aislado químicamente. De esta manera, el chip proporciona EF coexistente mejor controlado y la estimulación química. Además, los resultados de la simulación numérica de la difusión química dentro del chip MDF indican que no se produjo la contaminación cruzada entre los canales después de un periodo experimental de 24 hr. 8 En comparación con el Device reportado por Li et al., 14 el chip MDF proporciona un área de cultivo a mayor, lo que permite para su posterior análisis bioquímico de las células estimuladas eléctricamente. Además, con área de observación más grande del chip MDF, más células pueden ser observados en la prueba, por lo que el análisis de la velocidad de migración o direccionalidad de las células estimuladas eléctricamente es más preciso. Los diseños de chips de un solo canal de estudios previos reportados por Huang et al. 4 y Tsai et al. 15 sólo permiten un tipo de célula o químico a ensayar. Sin embargo, el chip MDF puede utilizarse para investigar los efectos de diversos productos químicos en electrotaxis, así como los efectos de la estimulación eléctrica en diferentes tipos de células. En otras palabras, el chip MDF permite el estudio de dosis eficaz de dependencias químicas.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Reagent</td>
			<td ></td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >DMEM medium</td>
			<td >Gibco,Invitrogen, USA</td>
			<td >12800-017</td>
			<td></td>
		</tr>
		<tr >
			<td >Fetal Bovine Serum</td>
			<td >Gibco,Invitrogen, USA</td>
			<td >16000-044</td>
			<td></td>
		</tr>
		<tr >
			<td >Trypsin</td>
			<td >Gibco,Invitrogen, USA</td>
			<td >25200-072</td>
			<td></td>
		</tr>
		<tr >
			<td >PBS</td>
			<td >Basic Life</td>
			<td >BL2651</td>
			<td></td>
		</tr>
		<tr >
			<td >Y-27632 (hydrochloride)</td>
			<td >Cayman Chemical Co</td>
			<td >10005583</td>
			<td></td>
		</tr>
		<tr >
			<td >agarose</td>
			<td >LONZO, USA</td>
			<td >SeaKem LE AGAROSE</td>
			<td></td>
		</tr>
		<tr >
			<td >syringe</td>
			<td>Terumo</td>
			<td>3 ml with Luer taper</td>
			<td></td>
		</tr>
		<tr >
			<td >3-way stopcock</td>
			<td>Nipro</td>
			<td >with Luer taper</td>
			<td></td>
		</tr>
		<tr >
			<td >PMMA (acrylic)</td>
			<td >HiShiRon Industries CO., Ltd, Taiwan</td>
			<td ></td>
			<td >thickness 1mm, 2mm</td>
		</tr>
		<tr >
			<td >acrylic adaptor</td>
			<td >KuanMin Technology Co., Ltd, Taichung, Taiwan</td>
			<td >1/4-28 port, 10x10x6 mm</td>
			<td>customized</td>
		</tr>
		<tr >
			<td >nut</td>
			<td >Thermo Fisher Scientific Inc.</td>
			<td >UPCHURCH:P-206x, P-200x, F120x, P-659, P-315x</td>
			<td></td>
		</tr>
		<tr >
			<td >Microscope cover glass</td>
			<td >Deckgl&#228;ser, Germany</td>
			<td >24x60 mm</td>
			<td></td>
		</tr>
		<tr >
			<td >double-sided tape</td>
			<td >3M</td>
			<td >PET 8018</td>
			<td></td>
		</tr>
		<tr >
			<td >super glue</td>
			<td >3M</td>
			<td >Scotch Liquid Plus Super Glue</td>
			<td></td>
		</tr>
		<tr >
			<td >Teflon tube</td>
			<td >HENG YI ENTERPRISE CO., LTD., Taiwan</td>
			<td >UPTB_06, DUPONT TEFLON BRAND RESIN FEP TUBING</td>
			<td>outer diameter 1/16 in., inner diameter 0.03 in.; Upchurch Scienti&#64257;c</td>
		</tr>
		<tr >
			<td >TFD4 detergent</td>
			<td >Franklab, France</td>
			<td >TFD4</td>
			<td></td>
		</tr>
		<tr >
			<td >ultrasonic steri cleaner</td>
			<td >LEO ULTRASONIC CO., LTD., Taiwan</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Thermo bonder</td>
			<td >KuanMin Technology Co., Ltd, Taichung, Taiwan</td>
			<td>customized</td>
			<td></td>
		</tr>
		<tr >
			<td >CO2 laser scriber</td>
			<td >LTT group, Taiwan</td>
			<td >ISL-II</td>
			<td></td>
		</tr>
		<tr >
			<td >indium tin oxide glass (ITO glass)</td>
			<td >AimCore Technology Co., Ltd</td>
			<td >TN/STN, &#8806;10&#937;</td>
			<td></td>
		</tr>
		<tr >
			<td >proportional-integral-derivative (PID) controller</td>
			<td>JETEC Electronics Co., Japen</td>
			<td >TTM-J40-R-AB,</td>
			<td></td>
		</tr>
		<tr >
			<td >K-type thermocouple</td>
			<td >TECPEL</td>
			<td >TPK-02A</td>
			<td></td>
		</tr>
		<tr >
			<td >4-channel syringe pump</td>
			<td >KdScientific, USA</td>
			<td >250P</td>
			<td></td>
		</tr>
		<tr >
			<td >DC power supply</td>
			<td >GWInstek, Taiwan</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >X-Y-Z motor stage</td>
			<td >TanLian, E-O Co. Ltd., Taiwan</td>
			<td>customized</td>
			<td></td>
		</tr>
		<tr >
			<td >inverted microscope</td>
			<td >Olympus, Japan</td>
			<td >CKX41</td>
			<td></td>
		</tr>
		<tr >
			<td >digital SLR camera</td>
			<td >Canon, Japan</td>
			<td >60D</td>
			<td></td>
		</tr>
	</tbody>
</table>

electrotaxis studies of lung cancer cells using a multichannel dual-electric-field microfluidic chip

El comportamiento de la migración celular direccional bajo una corriente de campo eléctrico directo (dcEF) se conoce como electrotaxis. El papel importante de dcEF fisiológica en guiar el movimiento celular durante el desarrollo embrionario, la diferenciación celular, la curación de heridas y se ha demostrado en muchos estudios. Mediante la aplicación de chips de microfluidos a un ensayo electrotaxis, el proceso de investigación se acorta y los errores experimentales se reducen al mínimo. En los últimos años, los dispositivos de microfluidos hechas de sustancias poliméricas (por ejemplo, polimetilmetacrilato, PMMA, o acrílico) o polidimetilsiloxano (PDMS) han sido ampliamente utilizados en el estudio de las respuestas de las células a la estimulación eléctrica. Sin embargo, a diferencia de los numerosos pasos requeridos para fabricar un dispositivo de PDMS, la construcción simple y rápida de los micro acrílicos fl chip de uidic hace que sea adecuado tanto para la creación de prototipos dispositivo y la producción. Sin embargo, ninguno de los dispositivos reportados facilitar el estudio eficiente de la sustancia química simultánea y DCEF efectos sobre las células. En este informe, describimos nuestro diseño y fabricación de un multicanal basado en acrílico de campo eléctrico dual (MDF) de chips para investigar el efecto simultáneo de química y la estimulación eléctrica de las células del cáncer de pulmón. El chip MDF proporciona ocho combinaciones de estímulos eléctricos / químicos en una sola prueba. El chip no sólo acorta considerablemente el tiempo experimental requerido sino también aumenta la precisión en electrotaxis estudios.

Fabricación y montaje del dispositivo de MDF Un diagrama esquemático del chip MDF de base acrílica se muestra en la Figura 1A. Cuatro hojas de acrílico, una cubierta de vidrio, 13 adaptadores de acrílico, y un trozo de cinta de doble cara se utilizan en el montaje del chip MDF completado (Figura 2D). Sólo hay cuatro canales independientes de cultivo en el dispositivo de ...

Watch this Scientific Journal Video about Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip at JoVE.com

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

Many microfluidic devices have been developed for use in the study of electrotaxis. Yet, none of these chips allows the efficient study of the simultaneous chemical and electric-field (EF) effects on cells. We developed a polymethylmethacrylate-based device that offers better-controlled coexisting EF and chemical stimulation for use in electrotaxis research.

Estudios electrotaxis de pulmón células de cáncer utilizando un Multicanal Dual-campo eléctrico chip de microfluidos

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of ...

electrotaxis-studies-lung-cancer-cells-using-multichannel-dual

Research

JoVE Journal

Bioengineering

8.6K vistas.  Academia Sinica. Many microfluidic devices have been developed for use in the study of electrotaxis. Yet, none of these chips allows the efficient study of the simultaneous chemical and electric-field (EF) effects on cells. We developed a polymethylmethacrylate-based device that offers better-controlled coexisting EF and chemical stimulation for use in electrotaxis research.

Video: Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

Métodos de detección de fluorescencia para las plataformas de las gotas de microfluidos

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

Investigación

Bioingeniería

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and pathways as well as its ease of cultivation. Several C. elegans disease models have been reported, including neurodegenerative disorders such as Parkinson's disease (PD), which involves the degeneration of dopaminergic (DA) neurons 1. Both transgenes and neurotoxic chemicals have been used to induce DA neurodegeneration and consequent movement defects in worms, allowing for investigations into the basis of neurodegeneration and screens for neuroprotective genes and compounds 2,3.
Screens in lower eukaryotes like C. elegans provide an efficient and economical means to identify compounds and genes affecting neuronal signaling. Conventional screens are typically performed manually and scored by visual inspection; consequently, they are time-consuming and prone to human errors. Additionally, most focus on cellular level analysis while ignoring locomotion, which is an especially important parameter for movement disorders.
We have developed a novel microfluidic screening system (Figure 1) that controls and quantifies C. elegans' locomotion using electric field stimuli inside microchannels. We have shown that a Direct Current (DC) field can robustly induce on-demand locomotion towards the cathode ("electrotaxis") 4. Reversing the field's polarity causes the worm to quickly reverse its direction as well. We have also shown that defects in dopaminergic and other sensory neurons alter the swimming response 5. Therefore, abnormalities in neuronal signaling can be determined using locomotion as a read-out. The movement response can be accurately quantified using a range of parameters such as swimming speed, body bending frequency and reversal time.
Our work has revealed that the electrotactic response varies with age. Specifically, young adults respond to a lower range of electric fields and move faster compared to larvae 4. These findings led us to design a new microfluidic device to passively sort worms by age and phenotype 6.
We have also tested the response of worms to pulsed DC and Alternating Current (AC) electric fields. Pulsed DC fields of various duty cycles effectively generated electrotaxis in both C. elegans and its cousin C. briggsae 7. In another experiment, symmetrical AC fields with frequencies ranging from 1 Hz to 3 KHz immobilized worms inside the channel 8.
Implementation of the electric field in a microfluidic environment enables rapid and automated execution of the electrotaxis assay. This approach promises to facilitate high-throughput genetic and chemical screens for factors affecting neuronal function and viability.

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans&#039; Locomotion

A semi-automated micro-electro-fluidic method to induce on-demand locomotion in Caenorhabditis elegans is described. This method is based on the neurophysiologic phenomenon of worms responding to mild electric fields (&#x201C;electrotaxis&#x201D;) inside microfluidic channels. Microfluidic electrotaxis serves as a rapid, sensitive, low-cost, and scalable technique to screen for factors affecting neuronal health.

Electrotaxis microfluidos basados ​​en On-Demand Análisis cuantitativo de<em&gt; Caenorhabditis elegans</em&gt; &#39;Locomotion

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and ...

Microfluidic Chip Fabrication and Method to Detect Influenza

Fast and effective diagnostics play an important role in controlling infectious disease by enabling effective patient management and treatment. Here, we present an integrated microfluidic thermoplastic chip with the ability to amplify influenza A virus in patient nasopharyngeal (NP) swabs and aspirates. Upon loading the patient sample, the microfluidic device sequentially carries out on-chip cell lysis, RNA purification and concentration steps within the solid phase extraction (SPE), reverse transcription (RT) and polymerase chain reaction (PCR) in RT-PCR chambers, respectively. End-point detection is performed using an off-chip Bioanalyzer (Agilent Technologies, Santa Clara, CA). For peripherals, we used a single syringe pump to drive reagent and samples, while two thin film heaters were used as the heat sources for the RT and PCR chambers. The chip is designed to be single layer and suitable for high throughput manufacturing to reduce the fabrication time and cost. The microfluidic chip provides a platform to analyze a wide variety of virus and bacteria, limited only by changes in reagent design needed to detect new pathogens of interest.

An integrated microfluidic thermoplastic chip has been developed for use as a molecular diagnostic. The chip performs nucleic acid extraction, reverse transcriptase, and PCR. Methods for fabricating and running the chip are described.

Fabricación de chips de microfluidos y método para detectar la influenza

Fast  and effective diagnostics play an important role in controlling infectious  disease by enabling effective patient management and treatment. Here, we ...

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this purpose, the cells are passively trapped in the middle of a microchamber. Upon activation of the control layer, the cell is isolated from the surrounding volume in a small chamber. The surrounding volume can then be exchanged without affecting the isolated cell. However, upon short opening and closing of the chamber, the solution in the chamber can be replaced within a few hundred milliseconds. Due to the reversibility of the chambers, the cells can be exposed to different solutions sequentially in a highly controllable fashion, e.g. for incubation, washing, and finally, cell lysis. The tightly sealed microchambers enable the retention of the lysate, minimize and control the dilution after cell lysis. Since lysis and analysis occur at the same location, high sensitivity is retained because no further dilution or loss of the analytes occurs during transport. The microchamber design therefore enables the reliable and reproducible analysis of very small copy numbers of intracellular molecules (attomoles, zeptomoles) released from individual cells. Furthermore, many microchambers can be arranged in an array format, allowing the analysis of many cells at once, given that suitable optical instruments are used for monitoring. We have already used the platform for proof-of-concept studies to analyze intracellular proteins, enzymes, cofactors and second messengers in either relative or absolute quantifiable manner.

In this article we present a microfluidic chip for single cell analysis. It allows the quantification of intracellular proteins, enzymes, cofactors, and second messengers by means of fluorescent assays or immunoassays.&#160;

Un chip de microfluidos para el análisis químico versátil de células individuales

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this ...

A Microfluidic Chip for ICPMS Sample Introduction

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). The chip produces monodisperse aqueous sample droplets in perfluorohexane (PFH). Size and frequency of the aqueous droplets can be varied in the range of 40 to 60 &#181;m and from 90 to 7,000&#160;Hz, respectively. The droplets are ejected from the chip with a second flow of PFH and remain intact during the ejection. A custom-built desolvation system removes the PFH and transports the droplets into the ICPMS. Here, very stable signals with a narrow intensity distribution can be measured, showing the monodispersity of the droplets. We show that the introduction system can be used to quantitatively determine iron in single bovine red blood cells. In the future, the capabilities of the introduction device can easily be extended by the integration of additional microfluidic modules.

We present a discrete droplet sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). It is based on a cheap and disposable microfluidic chip that generates highly monodisperse droplets in a size range of 40&#8722;60 &#181;m at frequencies from 90 to 7,000 Hz.

Un chip de microfluidos para ICPMS Introducción Muestra

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma ...

The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need for new solutions for substance testing. Many drugs that have been removed from the market due to drug-induced liver injury released their toxic potential only after several doses of chronic testing in humans. However, a controlled microenvironment is pivotal for long-term multiple dosing experiments,&#160;as even minor alterations in extracellular conditions may greatly influence the cell physiology. We focused within our research program on the generation of a microengineered bioreactor, which can be dynamically perfused by an on-chip pump and combines at least two culture spaces for multi-organ applications. This circulatory system mimics the in vivo conditions of primary cell cultures better and assures a steadier,&#160;more quantifiable extracellular relay of signals to the cells. 
For demonstration purposes, human liver equivalents, generated by aggregating differentiated HepaRG cells with human hepatic stellate cells in hanging drop plates, were cocultured with human skin punch biopsies for up to 28 days inside the microbioreactor. The use of cell culture inserts enables the skin to be cultured at an air-liquid interface, allowing topical substance exposure. The microbioreactor system is capable of supporting these cocultures at near physiologic fluid flow and volume-to-liquid ratios, ensuring stable and organotypic culture conditions. The possibility of long-term cultures enables the repeated exposure to substances. Furthermore, a vascularization of the microfluidic channel circuit using human dermal microvascular endothelial cells yields a physiologically more relevant vascular model.

Here, we present a protocol to coculture primary cells, tissue models and punch biopsies in a microfluidic multi-organ chip for up to 28 days. Human dermal microvascular endothelial cells, liver aggregates and skin biopsies were successfully combined in a common media circulation.

El chip de varios órganos - Una plataforma de microfluidos para el largo plazo co-cultivo de múltiples tejidos

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need ...

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human gut-on-a-chip microphysiological device. We recapitulate the intestinal lumen-capillary tissue interface in a microfluidic device, where physiological mechanical deformations and fluid shear flow are constantly applied to mimic peristalsis. In the lumen microchannel, human intestinal epithelial Caco-2 cells are cultured to form a &#39;germ-free&#39; villus epithelium and regenerate small intestinal villi. Pre-cultured microbial cells are inoculated into the lumen side to establish a host-microbe ecosystem. After microbial cells adhere to the apical surface of the villi, fluid flow and mechanical deformations are resumed to produce a steady-state microenvironment in which fresh culture medium is constantly supplied and unbound bacteria (as well as bacterial wastes) are continuously removed. After extended co-culture from days to weeks, multiple microcolonies are found to be randomly located between the villi, and both microbial and epithelial cells remain viable and functional for at least one week in culture. Our co-culture protocol can be adapted to provide a versatile platform for other host-microbiome ecosystems that can be found in various human organs, which may facilitate in vitro study of the role of human microbiome in orchestrating health and disease.

We describe an in vitro protocol to co-culture gut microbiome and intestinal villi for an extended period using a human gut-on-a-chip microphysiological system.

Co-cultivo de estar con microbioma intestinal humana microconstruidos Villi en una Gut-on-a-Chip dispositivo de microfluidos

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human ...

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An easy-to-use and low-cost sensor platform is highly desired to measure various types of analytes (e.g., biomarkers, hormones, and drugs) quantitatively and specifically. For this reason, dry film photoresist technology - enabling cheap, facile, and high-throughput fabrication - was used to manufacture the microfluidic biosensor presented here. Depending on the bioassay used afterwards, the versatile platform is capable of detecting various types of biomolecules. For the fabrication of the device, platinum electrodes are structured on a flexible polyimide (PI) foil in the only clean-room process step. The PI foil serves as a substrate for the electrodes, which are insulated with an epoxy-based photoresist. The microfluidic channel is subsequently generated by the development and lamination of dry film photoresist (DFR) foils onto the PI wafer. By using a hydrophobic stopping barrier in the channel, the channel is separated into two specific areas: an immobilization section for the enzyme-linked assay and an electrochemical measurement cell for the amperometric signal readout.
The on-chip bioassay immobilization is performed by the adsorption of the biomolecules to the channel surface. The glucose oxidase enzyme is used as a transducer for electrochemical signal generation. In the presence of the substrate, glucose, hydrogen peroxide is produced, which is detected at the platinum working electrode. The stop-flow technique is applied to obtain signal amplification along with rapid detection. Different biomolecules can quantitatively be measured by means of the introduced microfluidic system, giving an indication of different types of diseases, or, in regard to therapeutic drug monitoring, facilitating a personalized therapy.

A microfluidic biosensor platform was designed and fabricated using low-cost dry film photoresist technology for the rapid and sensitive quantification of various analytes. This single-use system allows for the electrochemical readout of on-chip-immobilized enzyme-linked assays by means of the stop-flow technique.

Seca de la película basada en la fotoresistencia el Biosensor electroquímico de microfluidos plataforma: Fabricación de dispositivo, preparación del análisis de la en-viruta y el funcionamiento del sistema

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An ...

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means of identifying radiosensitive sites in living organisms. This paper describes a series of on-chip immobilization methods developed for the targeted microbeam irradiation of live individuals of Caenorhabditis elegans. Notably, the treatment of the polydimethylsiloxane (PDMS) microfluidic chips that we previously developed to immobilize C. elegans individuals without the need for anesthesia is explained in detail. This chip, referred to as a worm sheet, is resilient to allow the microfluidic channels to be expanded, and the elasticity allows animals to be enveloped gently. Also, owing to the self-adsorption capacity of the PDMS, animals can be sealed in the channels by covering the surface of the worm sheet with a thin cover film, in which animals are not pushed into the channels for enclosure. By turning the cover film over, we can easily collect the animals. Furthermore, the worm sheet shows water retention and allows C. elegans individuals to be subjected to microscopic observation for long periods under live conditions. In addition, the sheet is only 300 &#181;m thick, allowing heavy ions such as carbon ions to pass through the sheet enclosing the animals, thus allowing the ion particles to be detected and the applied radiation dose to be measured accurately. Because selection of the cover films used for enclosing the animals is very important for successful long-term immobilization, we conducted the selection of the suitable cover films and showed a recommended one among some films. As an application example of the chip, we introduced imaging observation of muscular activities of animals enclosing the microfluidic channel of the worm sheet, as well as the microbeam irradiation. These examples indicate that the worm sheets have greatly expanded the possibilities for biological experiments.

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

A series of immobilization methods has been established to allow the targeted irradiation of live Caenorhabditis elegans individuals using a recently developed ultra-thin polydimethylsiloxane microfluidic chip with water retention. This novel on-chip immobilization is also adequate for imaging observations. The detailed treatment and application examples of the chip are explained.

Inmovilización de personas en Caenorhabditis elegans con un Chip de microfluidos polidimetilsiloxano ultra-delgada de retención de agua

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means ...

Establishing Single-Cell Based Co-Cultures in a Deterministic Manner with a Microfluidic Chip

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases including cancer. However, it is challenging to clarify the complex mechanism of intercellular interactions in highly heterogeneous cell populations using conventional co-culture systems because the heterogeneity of the cell subpopulation is obscured by the average values; the conventional co-culture systems can only be used to describe the population signal, but are incapable of tracking individual cells behavior. Furthermore, conventional single-cell experimental methods have low efficiency in cell manipulation because of the Poisson distribution. Microfabricated devices are an emerging technology for single-cell studies because they can accurately manipulate single cells at high-throughput and can reduce sample and reagent consumption. Here, we describe the concept and application of a microfluidic chip for multiple single-cell co-cultures. The chip can efficiently capture multiple types of single cells in a culture chamber (~46%) and has a sufficient culture space useful to study the cells&#39; behavior (e.g., migration, proliferation, etc.) under cell-cell interaction at the single-cell level. Lymphatic endothelial cells and oral squamous cell carcinoma were used to perform a single-cell co-culture experiment on the microfluidic platform for live multiple single-cell interaction studies.

This report describes a microfluidic chip-based method to set up a single cell culture experiment in which high-efficiency pairing and microscopic analysis of multiple single cells can be achieved.

Establecimiento de coculturas basadas en células únicas de manera determinista con un chip microfluídico

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases ...