The authors would like to thank Professor Howard Ceri from the Biological Sciences Department of the University of Calgary for providing bacterial strains. A. Kumar acknowledges support from NSERC. T. Thundat acknowledges financial support from the Canada Excellence Research Chair (CERC) program. The authors would also like to acknowledge help from Ms. Zahra Nikakhtari for help with videography.

Realice los protocolos experimentales aquí en el orden descrito a continuación. Protocolos de microfabricación para la creación de la plataforma de microfluidos se discuten en el Paso 1 Paso 2 describe el protocolo de cultivo bacteriano (Figura 2), y el Paso 3 se refiere al montaje de la configuración experimental (Figura 3). Finalmente, la etapa experimental real se describe en el paso 4. 1. viruta Procedimiento Fabrication NOTA: los procedimientos de seguridad deben ser seguidas por los procedimientos descritos a continuación. Consulte al encargado de seguridad institucional para más detalles. <ol><li> Diseñar la máscara con un software apropiado (por ejemplo, L-Edit). El diseño del canal consiste en un principal micro-canal de ancho de 625 m. La región central del canal contiene una matriz de micro-mensajes 50 m de diámetro, espaciados 25 m aparte (Ver Archivos Suplementarios). </li><li> Imprimir este diseño en vidrio (5 &quot;x 5&quot; vidrio sódico-cálcico), Que tiene un espesor de 0,09 &quot;y está recubierto en aproximadamente 70 nm de espesor de capa de cromo (Cr), el uso de enmascaramiento con el fin de preparar una máscara foto. Desarrollador Uso AZ400K durante 1 min. Entonces, grabar la capa de Cr Cr utilizando reactivo de ataque durante 1 min con acetona para quitar la capa protectora y limpiarlo con una solución de pirañas frío. (H 2 SO 4 y H 2 O 2 en una proporción de 3: 1). </li><li> Fotolitografia <ol><li> Limpie una oblea de 4 &quot;de silicio estándar químicamente con una solución de pirañas durante 20 min. </li><li> Enjuague la oblea con agua desionizada y séquelo. </li><li> Calentar la oblea en una placa caliente (200 ° C durante 15 min). </li><li> Escudo de la oblea de silicio con fotoprotector. Aquí, el AZ4620 fotoprotector positivo se revistió spin-sobre una oblea de silicio a 2000 rpm durante 25 s para obtener una capa de espesor 12,5 micras. </li><li> Eliminar todo el disolvente por hornear suave de la oblea en una placa caliente por la flotación de la oblea durante 90 segundos en el flujo de nitrógeno a 100 ° C. A continuación, guárdelo en vacío a tél misma temperatura durante 60 seg. </li><li> Coloque las tabletas en una caja oscura durante 24 horas por deshidratación. </li><li> Exponer la oblea a la luz UV con el fin de transferir el patrón diseñado para la fotoprotección. </li><li> Sumergir la oblea en solución de revelador fotorresistente (AZ400K) para 240 seg. Luego, enjuague la oblea con alcohol isopropílico y séquelo colocando en una corriente de gas nitrógeno. </li></ol></li><li> ICP-DRIE (plasma acoplado inductivamente - Deep Reactive Ion Aguafuerte) Proceso <ol><li> Aplicar DRIE grabado. Seleccione la profundidad de ataque apropiado de acuerdo a la profundidad final requerido para el dispositivo (50 micras en esta investigación). Fotoprotector actúa como una capa de enmascaramiento durante este proceso. </li><li> Retire el fotoprotector restante con acetona y limpiar la oblea. </li></ol></li><li> PDMS (polidimetilsiloxano) Casting <ol><li> Utilice triclorometilsilano (TCMS) para silanizar el molde maestro de silicio. Vierta 2 o 3 gotas de triclorometilsilano en un vial y colocarlo en un secador al lado delmolde maestro de silicio. Permita 2-3 horas para el proceso de silanización para completar. </li><li> En un recipiente separado, mezclar la base de silicona Sylgard 184 con agente de curado en relación en peso de 10: 1 para preparar PDMS. Desgasificar la PDMS sometiéndolo a condiciones de vacío (alrededor de 2 h). </li><li> Ponga el molde maestro de silicio en un soporte. Luego, vierta el PDMS en el molde maestro de silicio para formar el sello PDMS. Asegúrese de que las burbujas no se forman en los PDMS durante este proceso. </li><li> Curar el PDMS durante 2 horas a 80 ° C. </li><li> Despegue el sello PDMS del molde maestro. A continuación, cortar el PDMS sello en microchips separados. Por último, utilizar un núcleo de corte para perforar agujeros para la entrada (s) y salida (s). </li></ol></li><li> La unión de PDMS a Glass <ol><li> Exponer la hoja de la cubierta y el sello PDMS de plasma de oxígeno durante 30 segundos. Sello Bond PDMS a la hoja de la cubierta. </li><li> Para lograr un sellado adecuado entre el sello de PDMS y la hoja de la cubierta, recocer el dispositivo al ponerlo en el horno a 70 ° C durante 10 min. </li&gt; </ol></li></ol> 2. bacteriana Cultura NOTA: los protocolos adecuados de bioseguridad debe ser seguido por los pasos 2-4. Consulte al encargado de seguridad institucional para más detalles. <ol><li> Preparar placas de agar LB <ol><li> Añadir 20 g de Luria-Bertani (LB) agar (Miller) polvos y 500 ml de agua ultrapura a un matraz de 1 L. Revuelva para disolver el polvo. </li><li> Esterilizar en autoclave a 15 psi, 121 ° C durante 15 min. </li><li> Dejar el frasco enfriar a 50-55 ° C en un banco o en un baño de agua en la campana de seguridad biológica. </li><li> Añadir el antibiótico tetraciclina para lograr una concentración final de 50 mg / ml. Mezclar bien por agitación. </li><li> Vierta la mezcla en placas. Llene cada plato hasta 1.2 a 2.3 completa. </li><li> Llama burbujas de aire brevemente para hacerlas explotar si forman. Burbujas de aire solidificadas son difíciles de difundir la cultura bacteriana más. </li><li> Deje que los platos se enfríen a temperatura ambiente durante la noche. </li><li> Cuando estén fríos,poner placas de nuevo en su manga, selle la bolsa, la etiqueta (antibiótico y fecha), y se almacenan a 4 ° C. NOTA: Cubra el balance de las placas con papel de aluminio, como la luz se desactiva muchos antibióticos. </li></ol></li><li> LB Preparación Caldo <ol><li> Añadir 20 g de Luria-Bertani (LB) caldo (Miller) polvos y 1 L de agua ultrapura a un matraz. Revuelva para disolver el polvo. </li><li> Esterilizar en autoclave a 15 psi, 121 ° C durante 15 min. </li><li> Permita matraz se enfríe a 50-55 ° C en un banco o en un baño de agua en la campana de seguridad biológica. </li><li> Añadir el antibiótico tetraciclina para lograr una concentración final de 50 mg / ml. Mezclar bien por agitación. </li><li> Cuando esté frío, coloque la botella etiquetada a 4 ° C. NOTA: Cubra la botella con papel de aluminio, como la luz se desactiva muchos antibióticos. </li></ol></li><li> Cultura de las bacterias en una placa de LB Agar (utiliza este protocolo Pseudomonas fluorescens) <ol><li> Tomar la acción bacteriana del congelador (-80 °; C) y colocarlo en hielo. </li><li> Coloque el ° C de stock bacteriana -80 y una placa de agar LB dentro de una campana de bioseguridad. </li><li> Racha de la cepa bacteriana sobre una placa de agar LB en un patrón de zigzag. Cubrir la placa de agar y se incuba a 30 º C durante la noche. Por último, guarde la plancha en el refrigerador a 4 ° C. </li></ol></li><li> Preparar la solución bacteriana (S1) <ol><li> Verter 50 ml de medio de caldo LB a un matraz autoclave. Realice esta operación dentro de una campana de bioseguridad. </li><li> Transferir una sola colonia bacteriana de la placa de agar LB al matraz. Esta operación también se debe realizar dentro de una campana de bioseguridad. </li><li> Ponga el frasco en un incubador con agitación a 30 ° C y 150 rpm durante el tiempo necesario (4 horas). </li></ol></li><li> Preparar la bacteriana solución diluida (S2) <ol><li> Vierta 5 ml de medio LB caldo en un tubo de plástico esterilizado. </li><li> Diluir S1 mezclando con los medios de caldo LB. Entonces, agite la solución. Diluir la solución alcanzar la óptica deseadaal densidad (OD medida a 600 nm = 0,1). NOTA: los experimentos Biofilm suelen emplear valores de DO en esta vecindad. </li></ol></li></ol> 3. Preparar el montaje experimental <ol><li> Con ayuda de pinzas se conectan tubos de plástico flexibles (0.20 &quot;ID) en la entrada (s) y salida (s) del microchip. Las entradas y salidas se perforaron previamente en la porción de PDMS del microchip (etapa 1.5.5). En esta investigación , el microchip consta de dos entradas y una salida. </li><li> Llene la jeringa (s) con la solución bacteriana (solución S2) y eliminar todas las burbujas en la jeringa (s). </li><li> Conecte la punta de la jeringa (s) (30 G 0.5 &quot;aguja roma) en el tubo (s) de entrada. Luego, conecte el tubo (s) de salida al contenedor de residuos. </li></ol> 4. Ejecute el Experimento <ol><li> Conectar la jeringa (s) a la punta (s) jeringa. </li><li> Lugar y jeringa (s) revisión en la bomba de jeringa. A continuación, coloque el microchip bajo un microscopio óptico con lentes objetivas de desirmagnificación ed (por ejemplo, 40X). Cubrir el microchip con un dispositivo de cámara de la celda en vivo para mantener un ambiente de temperatura constante para el crecimiento bacteriano (30 ° C para P. fluorescens). </li><li> Ajuste la bomba al nivel del caudal deseado (por ejemplo, 10 l / h) e iniciar el bombeo de líquidos. </li><li> Una vez que las bacterias se introducen en la cámara, también se inicia la formación de biopelículas. La formación de biopelículas y la maduración ocurren típicamente durante un período de varias horas o incluso días. Observar y tomar imágenes de la formación de biopelículas a través del microscopio. </li></ol>

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The authors have nothing to disclose.

Hemos demostrado un dispositivo de microfluidos sencillo que imita medios porosos para estudiar el desarrollo del biofilm en hábitats complejos. Hay varios pasos críticos que determinan el resultado de los experimentos. Ellos incluyen geometría del dispositivo. Mientras que la geometría mensaje puede variar, adecuada poro-espacio para serpentinas para formar es necesario. Además, Valiei et al. 1 han demostrado que la formación de streamer se produce sólo en un cierto rango de caudal. A caudale...

Recientemente, hemos demostrado la dinámica de la formación de biopelículas bacterianas en un dispositivo de microfluidos que imita medios porosos 1. Los biofilms bacterianos son esencialmente colonias de bacterias de la superficie agregada que están cubiertos de sustancias poliméricas extracelulares (EPS) 2-4. Estas películas delgadas de bacterias pueden formar en casi todos los nichos concebible que van desde superficies lisas para el hábitat mucho más complejo de los medios porosos. Valiei et al. 1 utiliza un dispositivo de microfluidos con una matriz de micro-pilares para simular una estructura de medios porosos y estudió la formación de biopelículas en este dispositivo como una función de la tasa de flujo de fluido. Encontraron que en un régimen de flujo determinado, biofilms filamentosos conocidos como serpentinas comenzaron a surgir entre los diferentes pilares. Serpentinas pueden ser atados en uno o ambos extremos para superficies sólidas, pero el resto de la estructura se suspende en un líquido. Formación Streamer comienza típicamente después de una capa inicial de la biopelícula se ha formado y su formatoion puede dictar la evolución a largo plazo de la biopelícula en tales hábitats complejos. Recientemente, varios investigadores han estudiado la dinámica de formación de serpentina. Yazdi et al. 5 mostró que las serpentinas pueden formar en los flujos de torbellino se originan en una burbuja oscilante. En otro experimento, Rusconi et al. 6 investigó el efecto de la curvatura del canal y la geometría del canal en la formación de serpentinas. Ellos encontraron que las serpentinas se pueden formar en secciones curvas de microcanales, y la morfología streamer está relacionada con la motilidad. La investigación reciente ha demostrado que las cuerdas pueden tener una amplia repercusión en diversos escenarios naturales y artificiales, ya que pueden actuar como precursores para la formación de estructuras porosas maduros en las interfaces, conducir a la proliferación de biopelículas rápido y catastrófico en unos sistemas biomédicos, y también puede causar Flow sustancial interacciones estructura, etc 1,7-9. Serpentinas Biofilm menudo forman in hábitats complejos como medios porosos. El crecimiento de biopelículas Entendimiento en el entorno de medios porosos es relevante para varios procesos industriales y medioambientales como el tratamiento biológico de aguas residuales 10, manteniendo así la integridad de ánima en situaciones como la captura de CO2 11 y el taponamiento de los poros en el suelo 12. Observar la formación de biopelículas en tales hábitats complejos, con frecuencia puede ser un reto debido a la opacidad de medios porosos. En tales situaciones, las plataformas de medios porosos microfluidos basada pueden resultar muy ventajosa, ya que permiten en tiempo real y el seguimiento in situ. Otra ventaja de la microfluídica es la capacidad de construir múltiples biorreactores en una sola plataforma de microfluidos bio-y simultáneamente permitir la supervisión y / o incorporación de sensores en línea. La flexibilidad para implementar múltiples experimentos de laboratorio en un solo dispositivo y la capacidad de recopilar datos pertinentes importantes para el análisis estadístico exacto es un adv importanteantage de los sistemas de microfluidos 13,14. En el contexto de la discusión anterior, la comprensión de la dinámica de formación de serpentina en un entorno de medios porosos sería beneficioso para varias aplicaciones. En este estudio, desarrollamos el protocolo para la investigación de la formación de serpentina en un dispositivo que imita los medios porosos. La fabricación de la plataforma de microfluidos, se describen los pasos necesarios para el cultivo celular y la experimentación. En nuestros experimentos, se empleó la cepa bacteriana de tipo salvaje de Pseudomonas fluorescens. P. fluorescens, que se encuentra naturalmente en el suelo, juega un papel clave en el mantenimiento de la ecología del suelo 15. La cepa bacteriana empleada había sido manipulada genéticamente para expresar la proteína fluorescente verde (GFP) constitutivamente.

<table border="0" cellpadding="0" cellspacing="0" width="1038">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">Name of Material/ Equipment</td>
			<td style="width:255px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:339px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">Flourescent Microscope</td>
			<td style="width:255px;">Nikon</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">LB agar</td>
			<td style="width:255px;">Fisher</td>
			<td style="width:119px;">BP1425-500</td>
			<td>suspend 40 g in 1 L of purified water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">LB broth</td>
			<td style="width:255px;">Fisher</td>
			<td style="width:119px;">BP1427-500</td>
			<td>suspend 20 g in 1 L of purified water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">Biosafety hood</td>
			<td style="width:255px;">Microzone corporation</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">Petri-dish</td>
			<td style="width:255px;">Fisher</td>
			<td style="width:119px;">875712</td>
			<td>sterile 100mmx15mm polystyrene petri dish</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:325px;">Incubator shaker</td>
			<td>New Brunswick Scientific</td>
			<td style="width:119px;"></td>
			<td style="width:339px;">Excella E24incubator shaker series</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 mL sterilized centrifuge tube</td>
			<td style="width:255px;">Corning</td>
			<td style="width:119px;">430828</td>
			<td>Polypropylene Rnase-/Dnase-free</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">Tetracycline free base</td>
			<td style="width:255px;">MP Biomedicals</td>
			<td style="width:119px;">103012</td>
			<td>50 ug/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">SYLGARD 184 silicone</td>
			<td style="width:255px;">Dow Corning Corporation</td>
			<td style="width:119px;">68037-59-2</td>
			<td>Elastomer Base and curing agent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">Positive photoresist (AZ4620)</td>
			<td style="width:255px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">Plastic tube</td>
			<td style="width:255px;">Cole- Parmer</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

protocol for biofilm streamer formation in a microfluidic device with micro-pillars

Varias especies bacterianas poseen la capacidad de adherirse a las superficies y colonizar ellos en la forma de películas delgadas llamadas biopelículas. Las biopelículas que crecen en medios porosos son relevantes para varios procesos industriales y ambientales, tales como el tratamiento de aguas residuales y secuestro de CO 2. Utilizamos Pseudomonas fluorescens, una bacteria aerobia gram-negativos, para investigar la formación de biopelículas en un dispositivo de microfluidos que imita medios porosos. El dispositivo microfluídico consiste de una matriz de micro-mensajes, que fueron fabricados utilizando litografía suave. Posteriormente, la formación de biopelículas en estos dispositivos con flujo fue investigado y se demuestra la formación de biofilms filamentosos conocidos como serpentinas en nuestro dispositivo. Los protocolos detallados para la fabricación y montaje de dispositivo de microfluidos se proporcionan aquí junto con los protocolos de cultivo bacteriano. Los procedimientos detallados para la experimentación con el dispositivo de microfluidos también se presentan junto con el representanteresultados.

Utilizando el protocolo de microfabricación mencionados arriba, se construyó un dispositivo de microfluidos basada PDMS. Figura 1 muestra el microscopio electrónico de barrido (SEM) imágenes de la PDMS dispositivo. Figura 1a muestra la sección de entrada del dispositivo. Se crea una entrada de tenedor para igualar la carga de presión a través del dispositivo. Además de imágenes SEM mostró también que las paredes pilar son casi vertical (Figura 1b). La soluci?...

Watch this Scientific Journal Video about Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars at JoVE.com

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Protocols for the study of biofilm formation in a microfluidic device that mimics porous media are discussed. The microfluidic device consists of an array of micro-pillars and biofilm formation by Pseudomonas fluorescens in this device is investigated.

Protocolo para Biofilm Streamer Formación en un dispositivo de microfluidos con Micro-pilares

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in ...

protocol-for-biofilm-streamer-formation-microfluidic-device-with

Research

JoVE Journal

Bioengineering

12.1K vistas.  University of Alberta. Protocols for the study of biofilm formation in a microfluidic device that mimics porous media are discussed. The microfluidic device consists of an array of micro-pillars and biofilm formation by Pseudomonas fluorescens in this device is investigated. 

Video: Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in vitro. Nutrients delivered by vasculature fail to reach all parts of tumors, giving rise to heterogeneous microenvironments consisting of viable, quiescent and necrotic cell types. Many cancer drugs fail to effectively penetrate and treat all types of cells because of this heterogeneity. Monolayers of cancer cells do not mimic this heterogeneity, making it difficult to test cancer drugs with a suitable in vitro model. Our microfluidic devices were fabricated out of PDMS using soft lithography. Multicellular tumor spheroids, formed by the hanging drop method, were inserted and constrained into rectangular chambers on the device and maintained with continuous medium perfusion on one side. The rectangular shape of chambers on the device created linear gradients within tissue. Fluorescent stains were used to quantify the variability in apoptosis within tissue. Tumors on the device were treated with the fluorescent chemotherapeutic drug doxorubicin, time-lapse microscopy was used to monitor its diffusion into tissue, and the effective diffusion coefficient was estimated. The hanging drop method allowed quick formation of uniform spheroids from several cancer cell lines. The device enabled growth of spheroids for up to 3 days. Cells in proximity of flowing medium were minimally apoptotic and those far from the channel were more apoptotic, thereby accurately mimicking regions in tumors adjacent to blood vessels. The estimated value of the doxorubicin diffusion coefficient agreed with a previously reported value in human breast cancer. Because the penetration and retention of drugs in solid tumors affects their efficacy, we believe that this device is an important tool in understanding the behavior of drugs, and developing new cancer therapeutics.

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We present the procedure for fabrication and operation of a microfluidic device that recreates heterogeneous tumor microenvironments in vitro. The variability in apoptosis within tumor tissue was quantified using fluorescent stains and the effective diffusion coefficient of the chemotherapeutic drug doxorubicin into tumor tissue was evaluated.

Dispositivo de microfluidos para volver a crear un microambiente tumoral<em&gt; In Vitro</em

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in ...

Investigación

Bioingeniería

A Microfluidic Device for Studying Multiple Distinct Strains

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a comparative manner. Multiwell plates are routinely used to compare many different strains or cell lines, but allow limited control over the environment dynamics. Microfluidic devices, on the other hand, allow exquisite dynamic control over the surrounding conditions, but it is challenging to image and distinguish more than a few strains in them. Here we describe a method to easily and rapidly manufacture a microfluidic device capable of applying dynamically changing conditions to multiple distinct yeast strains in one channel. The device is designed and manufactured by simple means without the need for soft lithography. It is composed of a Y-shaped flow channel attached to a second layer harboring microwells. The strains are placed in separate microwells, and imaged under the exact same dynamic conditions. We demonstrate the use of the device for measuring protein localization responses to pulses of nutrient changes in different yeast strains.

We present a simple method to produce microfluidic devices capable of applying similar dynamic conditions to multiple distinct strains, without the need for a clean room or soft lithography.

Un dispositivo de microfluidos para el estudio de varias cepas distintas

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a ...

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and used to dictate the shape as well as the diameter of a core stream.&#160;Grooves in the top and bottom of a microfluidic channel were designed to direct the sheath fluid and shape the core fluid.&#160;By matching the viscosity and hydrophilicity of the sheath and core fluids, the interfacial effects are minimized and complex fluid shapes can be formed.&#160;Controlling the relative flow rates of the sheath and core fluids determines the cross-sectional area of the core fluid.&#160;Fibers have been produced with sizes ranging from 300 nm to ~1 mm, and fiber cross-sections can be round, flat, square, or complex as in the case with double anchor fibers. Polymerization of the core fluid downstream from the shaping region solidifies the fibers.&#160;Photoinitiated click chemistries are well suited for rapid polymerization of the core fluid by irradiation with ultraviolet light.&#160;Fibers with a wide variety of shapes have been produced from a list of polymers including liquid crystals, poly(methylmethacrylate), thiol-ene and thiol-yne resins, polyethylene glycol, and hydrogel derivatives. Minimal shear during the shaping process and mild polymerization conditions also makes the fabrication process well suited for encapsulation of cells and other biological components.

Two adjacent fluids passing through a grooved microfluidic channel can be directed to form a sheath around a prepolymer core; thereby&#160;determining both shape and cross-section. Photoinitiated polymerization, such as thiol click chemistry, is well suited for rapidly solidifying the core fluid into a microfiber with predetermined size and shape.

Fabricación Microfluídica De Fibras Poliméricas Y Biohíbridas Con Tamaño y Forma Prediseñados

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

Analyzing Mixing Inhomogeneity in a Microfluidic Device by Microscale Schlieren Technique

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

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

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

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

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 ...

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the spheroid cultures, a perfusable vascular network in the spheroids remains a critical challenge for long-term culture required to maintain and develop their functions, such as protein expressions and morphogenesis. The protocol presents a novel method to integrate a perfusable vascular network within the spheroid in a microfluidic device. To induce a perfusable vascular network in the spheroid, angiogenic sprouts connected to microchannels were guided to the spheroid by utilizing angiogenic factors from human lung fibroblasts cultured in the spheroid. The angiogenic sprouts reached the spheroid, merged with the endothelial cells co-cultured in the spheroid, and formed a continuous vascular network. The vascular network could perfuse the interior of the spheroid without any leakage. The constructed vascular network may be further used as a route for supply of nutrients and removal of waste products, mimicking blood circulation in vivo. The method provides a new platform in spheroid culture toward better recapitulation of living tissues.

The protocol describes how to engineer a perfusable vascular network in a spheroid. The spheroid's surrounding microenvironment is devised to induce angiogenesis and connect the spheroid to the microchannels in a microfluidic device. The method allows the perfusion of the spheroid, which is a long-awaited technique in three-dimensional cultures.

Perfusable Red Vascular con un modelo de tejido en un dispositivo de microfluidos

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the ...

A Microfluidic Platform for Stimulating Chondrocytes with Dynamic Compression

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth plate cartilage architecture and results in growth modulation of long bones of children. To determine the role of compressive stress in bone growth, we created a microfluidic device actuated by pneumatic pressure, to dynamically (or statically) compress growth plate chondrocytes embedded in alginate hydrogel cylinders. In this article, we describe detailed methods for fabricating and characterizing this device. The advantages of our protocol are: 1) Five different magnitudes of compressive stress can be generated on five technical replicates in a single platform, 2) It is easy to visualize cell morphology via a conventional light microscope, 3) Cells can be rapidly isolated from the device after compression to facilitate downstream assays, and 4) The platform can be applied to study mechanobiology of any cell type that can grow in hydrogels.

This article provides detailed methods for fabricating and characterizing a pneumatically actuating microfluidic device for chondrocyte compression.

Una plataforma microfluídica para estimular los condrocitos con compresión dinámica

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth ...

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 ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

Generación de condiciones ambientales dinámicas utilizando un dispositivo microfluídico de alto rendimiento

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...

Configuración de un dispositivo microfluídico para la producción de tapones combinatorios

Bilayer Microfluidic Device for Combinatorial Plug Production

Droplet microfluidics is a versatile tool that allows the execution of a large number of reactions in chemically distinct nanoliter compartments. Such systems have been used to encapsulate a variety of biochemical reactions - from incubation of single cells to implementation of PCR reactions, from genomics to chemical synthesis. Coupling the microfluidic channels with regulatory valves allows control over their opening and closing, thereby enabling the rapid production of large-scale combinatorial libraries consisting of a population of droplets with unique compositions. In this paper, protocols for the fabrication and operation of a pressure-driven, PDMS-based bilayer microfluidic device that can be utilized to generate combinatorial libraries of water-in-oil emulsions called plugs are presented. By incorporating software programs and microfluidic hardware, the flow of desired fluids in the device can be controlled and manipulated to generate combinatorial plug libraries and to control the composition and quantity of constituent plug populations. These protocols will expedite the process of generating combinatorial screens, particularly to study drug response in cells from cancer patient biopsies.

Autor destacado: Integración de enfoques computacionales y experimentales en oncología de precisión

The fabrication of a polydimethylsiloxane (PDMS)-based bilayer device for the production of combinatorial libraries in water-in-oil emulsions (plugs) is presented here. The necessary hardware and software required to automate plug production are detailed in the protocol, and the production of a quantitative library of fluorescent plugs is also demonstrated.

Dispositivo microfluídico bicapa para la producción de tapones combinatorios

Droplet microfluidics is a versatile tool that allows the execution of a large number of reactions in chemically distinct nanoliter compartments. Such ...