Este trabajo fue apoyado por un Premio de Investigación del Instituto de California para cuantitativos Biosciences (QB3), el Premio Salvando Gap de la Fundación de la familia de Rogers, el / Programa Fundación Sandler UCSF para Breakthrough Investigación Biomédica, una subvención de BASF, y la NSF a través Programa Facultad Early Career Development (CARRERA) (DBI-1253293).

1. SU8 Fabricación Maestro <ol><li> Diseñar las estructuras de microfluidos para dos fabricación capa utilizando el software AutoCAD y tienen los diseños impresos por un proveedor en la película placa de circuito con 10 micras de resolución. Los detalles de diseño del dispositivo se dan en una referencia vinculada 11 y las geometrías de canal se muestran en la Figura 1. Las capas deben incluir marcas de alineación para ayudar collocate características de cada capa de la fabricación 12. </li><li> Coloque una oblea de silicio de diámetro previamente limpiado 3 pulgadas en una recubridora de rotación y encienda el vacío para pegarla en el mandril. Aplicar 1 ml de SU8-3035 en el centro de la oblea y centrifugado durante 20 segundos a 500 rpm, a continuación, 30 segundos a 2000 rpm, proporcionando un espesor de capa de 50 micras. </li><li> Retire la oblea y hornear en una placa de cocción de 135 ° C durante 30 minutos. Permitir que la oblea se enfríe a RT antes de pasar a la siguiente etapa. </li><li> Exponer la oblea revestida de la 1ª máscara de capa (Figura 2A </strong&gt;) bajo un colimado 190 mW, 365 nm LED durante 90 segundos. Después de la exposición, colocar la oblea en una placa calefactora 135 ° C durante 1 min, después se enfría a RT antes de proceder al siguiente paso. </li><li> Coloque la oblea en el revestidor de giro y encienda el vacío para pegarla en el mandril. Aplicar 1 ml de SU8-2050 en el centro de la oblea y centrifugado durante 20 segundos a 500 rpm, a continuación, 30 segundos a 1.375 rpm, lo que resulta en una capa que proporciona un espesor adicional de 135 micras. </li><li> Retire la oblea y hornear en una placa de cocción de 135 ° C durante 30 minutos, dejar enfriar a temperatura ambiente antes de pasar al siguiente paso. </li><li> Alinear la capa de máscara 2 nd (Figura 2B) en la geometría modelada en 1,3 y exponer la oblea recubierta a un colimado 190 mW, 365 nm LED para 3 min. Después de la exposición, colocar en una placa calefactora 135 ° C durante 1 min, después se enfría a RT antes de proceder al siguiente paso. </li><li> Desarrollar las máscaras mediante la inmersión en un baño agitada de acetato de propilenglicol monometil éter durante 30 min. Lave la obleaen isopropanol y hornear en una placa de cocción de 135 ° C durante 1 min. Coloque el maestro desarrollado en una placa de Petri de 100 mm para el moldeo por PDMS. </li></ol> 2. PDMS la fabricación del dispositivo <ol><li> Preparar 10: 1 PDMS mediante la combinación de 50 g de base de silicona con 5 g de agente de curado en un vaso de plástico. Mezcle el contenido con una herramienta rotativa equipada con un palo revuelva. Desgasificar la mezcla dentro de un desecador durante 30 min, o hasta que se eliminen todas las burbujas de aire. </li><li> Vierta el PDMS para dar un espesor de 3 mm en el maestro y colocar de nuevo en el desecador durante más de desgasificación. Una vez que se eliminan todas las burbujas, hornear el dispositivo a 60 ° C durante 2 horas. </li><li> Cortar el dispositivo del molde utilizando un bisturí y colóquelo sobre una superficie limpia con el lado estampado para arriba. Cortar el molde de PDMS por la mitad con una hoja de afeitar para separar Master 1 de Master 2 (Figura 3a). En la pieza que contiene la geometría de manipulación 50 micras fluido impreso por el Maestro 1, perforar las entradas y salidas de fluidos con un 0,75 mm ponche biopsia. </li><li> Plasma tratar los dispositivos a 1 mbar plasma de O2 durante 60 segundos en un limpiador de plasma 300 W. Humedecer la superficie de la pieza unpunched de PDMS con una gota de agua DI para retardar temporalmente PDMS-PDMS unión y servir como un lubricante. Mientras ve a través de un microscopio estereoscópico, lugar principal 1 en Master 2 de superficie y deslice las superficies relativamente hasta que se logre un bloqueo mecánico cuando los marcos empotrados y salientes marcos en compañero de la figura 3A. </li><li> Coloque el dispositivo en un horno de 60 ° C y hornear el dispositivo ensamblado (Figura 3B) durante dos días a 60 ° C para evaporar el agua y la unión completa. </li></ol> 3. Preparación de los reactivos <ol><li> Llenar 1 ml jeringa con agua destilada durante la fase interna. </li><li> Rellene 1 ml jeringa con HFE 7500 aceite fluorado con 1 peso. % De tensioactivo tensioactivo biocompatible 13 para la fase intermedia. </li><li> Rellene 10 ml jeringa con 10 en peso. % Polietilenoe glicol (PEG) en solución en agua que contiene 1 en peso. % De Tween 20 y 1 en peso. % De dodecilsulfato de sodio para la fase continua. </li></ol> 4. Sistema de Preparación <ol><li> Coloque el chip de microfluidos en el escenario de un microscopio invertido, junto con una cámara digital capaz de &lt;100 microsegundos velocidades de obturación. </li><li> Montar todas las jeringas en las bombas de jeringa y colocar 27 g agujas. Adjuntar ~ 30 cm de longitud de tubería de PE-2 en las agujas e insertar los cabos sueltos en el apropiado puñetazos agujeros en el dispositivo. </li><li> Inserte una longitud 10 cm de PE-2 en el puerto de salida del dispositivo y colocar el otro extremo en un recipiente de recogida de residuos. </li><li> Primer el dispositivo mediante la ejecución de las bombas de jeringa a altas tasas de velocidades (2.000 l / min) hasta que el fluido en los segmentos de tubo llega a los puertos de entrada del dispositivo. </li></ol> 5. Emulsión Generación <ol><li> Enfocar el microscopio en una región que contiene el 50 micras x 50 micras y el orificiocanal de salida aguas abajo. </li><li> Establecer las bombas de jeringa para suministrar fluido al generador de doble emulsión a velocidades de flujo de 250 l / h para la fase interna, 100 l / h durante la fase media, y 700 l / h durante la fase continua y esperar 10 min para el equilibrio. </li><li> Mantener los caudales de las fases interna y media a 250 l / hora y 100 l / h, respectivamente. Ajuste la velocidad de flujo de la fase externa en 1050 l / h. Espere 3-5 minutos para el doble de la generación de emulsiones para estabilizar bajo este conjunto de condiciones de flujo. </li><li> Adquirir 5 segundos de imágenes de vídeo a 30 Hz para el procesamiento sin conexión a través de análisis de imagen manual. </li><li> Repita 5.3 y 5.4 con los caudales dados en la Tabla 1. Las velocidades de flujo de fase interna y media se mantienen constantes y la tasa de flujo de fase de la portadora se varía mediante el ajuste de la configuración de la bomba de jeringa. </li></ol>

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	<li>Van Der Graaf, S., Schro&#235;n, C. G. P. H., Boom, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+double+emulsions+by+membrane+emulsification+-+A+review.">Preparation of double emulsions by membrane emulsification - A review.</a> J. Membrane Sci. 251 (1-2), 7-15 (2005).</li><li>Laugel, C., Baillet, A. P., Youenang Piemi, M., Marty, J., Ferrier, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oil-water-oil+multiple+emulsions+for+prolonged+delivery+of+hydrocortisone+after+topical+application:+comparison+with+simple+emulsions.">Oil-water-oil multiple emulsions for prolonged delivery of hydrocortisone after topical application: comparison with simple emulsions.</a> Int. J. Pharm. 160 (1), 109-117 (1998).</li><li>Kim, S. H., Kim, J. W., Cho, J. C., Weitz, D. 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The authors have nothing to disclose.

La geometría de generación de emulsión doble se describe aquí está diseñado para imitar la física de los dispositivos capilares de vidrio 8. En estos, capilares de vidrio cilíndricos alineados se utilizan para crear un chorro coaxial tres fases que se cizalla en gotitas de emulsión dobles uniformes. La función de nuestro dispositivo 3D PDMS es dependiente de la alineación central de pequeñas características formadas con 50 micras de fabricación de altura con canales de fase de portadora que son ...

<html> Emulsiones dobles consisten en gotas separadas de una fase de la portadora por una capa de fluido intermedio, inmiscibles, y son de particular interés debido a sus usos potenciales en industrial, farmacéutica y aplicaciones biológicas 1. En algunos casos, la capacidad de encapsular compuestos de alto valor en el núcleo de una emulsión doble permite el material a ser protegido y se libera de una manera controlada. Por ejemplo, los fármacos pueden encapsularse en condiciones de solubilidad no es apropiado para el fluido portador externo 2. Además, la capa de aceite intermedio se puede utilizar como una plantilla cápsula para la encapsulación y la entrega de medicamentos, cosméticos, nutrientes y 3. En biología, emulsiones dobles son también útiles en el cribado de alto rendimiento debido a que permiten un número masivo de experimentos sub-nanolitros a llevarse a cabo, a continuación, detecta y ordenadas utilizando una célula activada por fluorescencia (FACS) instrumento 4,5. ent &quot;&gt; El diseño de las emulsiones dobles con características de rendimiento deseadas requiere el control preciso del tamaño de emulsión doble, la composición y uniformidad. Aunque los procesos de emulsificación a granel, tales como emulsificación de membrana, se utilizan en la industria, las emulsiones resultantes son altamente polidispersa, que presenta una amplia variedad de propiedades funcionales 1. El campo de la microfluídica gotita se adapta naturalmente la generación de emulsiones monodispersas con la composición cuidadosamente controlada 6. Microfluidic generación de doble emulsión se ha logrado con dos estrategias principales, toma de gota secuencial y de flujo capilar de vidrio de enfoque. emulsiones dobles pueden pueden generar en dispositivos PDMS planas utilizando una gota de dos pasos de decisiones. En primer lugar, acuosa-en-aceite emulsiones se crean utilizando una emulsión agua-en-aceite gota de decisiones región de un dispositivo con paredes de los canales hidrófobas. A continuación, la emulsión puede ser fluido o reinyectado en una región gota de decisiones con paredes hidrófilas adecuadas para aceite en aguacaer de decisiones 4. Sin embargo, el tratamiento de superficie hidrófila de PMDS requiere un paso adicional de fabricación y es a menudo temporal 7. El método más controlable y repetible para formar emulsiones dobles es por el flujo de co-axial de enfoque, una técnica pionera en el uso de la microfluídica capilar de vidrio, mediante el cual un chorro concéntrica que contiene las tres fases se cizalla a través de un pequeño orificio para producir gotas monodispersas 8. Esta técnica permite la producción de gotitas mucho más pequeñas que las dimensiones del canal, con el tamaño preciso y composición de la emulsión doble es una función de los caudales de cada fase. La gran diferencia entre las gotitas y el tamaño del canal y el flujo de vaina externa de protección evita que las gotas entren en contacto con las paredes del canal, lo que hace innecesario el tratamiento de superficies. Sin embargo, este tipo de dispositivos de cristal requieren fabricación personalizada de consejos capilares cónicos, además de cuidado montaje y sellado. Anterior investigadores han utilizado litografía blanda 3Dgrafía para generar emulsiones dobles utilizando flujo centrándose la física, pero estos dispositivos produce emulsiones con diámetros&gt; 150 micras 9,10, aproximadamente un orden de magnitud mayor que los objetos normalmente ordenados con FACS. Una alternativa atractiva incluiría la funcionalidad robusta y pequeña generación de gotitas de capilar de vidrio de flujo coaxial de enfoque con la facilidad de fabricación de PDMS litografía blanda. En este trabajo se describe un generador de emulsión doble que utiliza coaxial flujo de enfoque para producir ≤ 50 micras emulsiones y está construido enteramente usando litografía blanda 3D 11. Nuestro dispositivo utiliza un enfoque de la cubierta para la fabricación de dispositivos que incluye un canal de cizallamiento pequeño (Figura 1) para aproximarse a los procesos de formación de emulsión en una boquilla capilar de vidrio desmenuzado. Más importante aún, estos dispositivos no requieren tratamiento superficial específica, y la construcción de todo polímero proporciona fácil y repetible sc fabricaciónalable a un gran número de dispositivos duplicados. Aquí, se describe el diseño, fabricación y pruebas del generador de doble emulsión. Generación de doble emulsión se demuestra que es robusto y repetible hacia abajo para diámetros de las gotitas de 14 micras. El acoplamiento de la funcionalidad con la facilidad de fabricación hace que este dispositivo una opción atractiva para el desarrollo de nuevas aplicaciones de emulsión doble.

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			<td height="21" style="height:21px;width:271px;">Photomasks</td>
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			<td height="42" style="height:42px;width:271px;">3&#34; silicon wafers, P type, virgin test grade</td>
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			<td height="21" style="height:21px;width:271px;">Sylgard 184 silicone elastomer kit</td>
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			<td style="width:119px;">4019862</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:271px;">1 ml syringes</td>
			<td style="width:135px;">BD</td>
			<td style="width:119px;">309628</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:271px;">10 ml syringes</td>
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			<td height="21" style="height:21px;width:271px;">27 gaugue needles</td>
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			<td height="42" style="height:42px;width:271px;">PE 2 polyethylene tubing</td>
			<td style="width:135px;">Scientific Commodities, Inc.</td>
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			<td height="21" style="height:21px;width:271px;">Novec 7500</td>
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			<td style="width:119px;">98-0212-2928-5</td>
			<td>Commonly knowns as HFE 7500</td>
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			<td height="42" style="height:42px;width:271px;">Biocompatable surfactant</td>
			<td style="width:135px;">Ran Biotechnologies</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:271px;">35,000 MW PEG</td>
			<td style="width:135px;">Sigma Aldrich</td>
			<td style="width:119px;">1546660</td>
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			<td height="21" style="height:21px;width:271px;">Tween 20</td>
			<td style="width:135px;">Sigma Aldrich</td>
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			<td height="21" style="height:21px;width:271px;">Sodium dodecyl sulfate&#160;</td>
			<td style="width:135px;">Sigma Aldrich</td>
			<td style="width:119px;">L3771</td>
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double emulsion generation using a polydimethylsiloxane (pdms) co-axial flow focus device

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper presents a polydimethylsiloxane (PDMS) microfluidic device capable of generating water/oil/water double emulsions using a coaxial flow focusing geometry that can be fabricated entirely using soft lithography. Similar to emulsion devices using glass capillaries, double emulsions can be formed in channels with uniform wettability and with dimensions much smaller than the channel sizes. Three dimensional flow focusing geometry is achieved by casting a pair of PDMS slabs using two layer soft lithography, then mating the slabs together in a clamshell configuration. Complementary locking features molded into the PDMS slabs enable the accurate registration of features on each of the slab surfaces. Device testing demonstrates formation of double emulsions from 14 &#181;m to 50 &#181;m in diameter while using large channels that are robust against fouling and clogging.

El generador de emulsión doble se compone de un dispositivo de enfoque de flujo co-axial creado usando la fabricación 3D PDMS (Figura 1A). La geometría permite que la formación de un chorro de co-axial de tres fases para ser cortado en un cuadrado, 50 micras x 50 micras orificio, permitiendo la formación de agua / aceite / agua emulsiones dobles (Figura 1B, Figura 1C). La fase acuosa interna y la fase aceite intermedio se juntan en ...

Watch this Scientific Journal Video about Double Emulsion Generation Using a Polydimethylsiloxane PDMS Co-axial Flow Focus Device at JoVE.com

Double Emulsion Generation Using a Polydimethylsiloxane PDMS Co-axial Flow Focus Device

Microfluidic double emulsions generation typically involves devices with patterned wettability or custom-fabricated glass components. Here we describe the fabrication and testing of an all polydimethylsiloxane (PDMS) double emulsion generator that does not require surface treatment or complicated fabrication processes, and is capable of producing double emulsions down to 14 &#181;m.

Generación de doble emulsión utilizando un flujo Focus Dispositivo de polidimetilsiloxano (PDMS) Co-axial

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper ...

double-emulsion-generation-using-polydimethylsiloxane-pdms-co-axial

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Bioengineering

Double Emulsion Generation Using a Polydimethylsiloxane (PDMS) Co-axial Flow Focus Device

15.9K vistas.  University of California, San Francisco. Microfluidic double emulsions generation typically involves devices with patterned wettability or custom-fabricated glass components. Here we describe the fabrication and testing of an all polydimethylsiloxane (PDMS) double emulsion generator that does not require surface treatment or complicated fabrication processes, and is capable of producing double emulsions down to 14 µm.

Video: Double Emulsion Generation Using a Polydimethylsiloxane PDMS Co-axial Flow Focus Device

A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell homing and tumor metastasis. The three-dimensional flow chamber device (hereafter the 3D device) is a novel in vitro technology that recreates physiological shear stress and allows each step of the cell extravasation cascade to be quantified. The 3D device consists of an upper compartment in which the cells of interest circulate under shear stress, and a lower compartment of static wells that contain the chemoattractants of interest. The two compartments are separated by porous inserts coated with a monolayer of endothelial cells (EC). An optional second insert with microenvironmental cells of interest can be placed immediately beneath the EC layer. A gas exchange unit allows the optimal CO2 tension to be maintained and provides an access point to add or withdraw cells or compounds during the experiment. The test cells circulate in the upper compartment at the desired shear stress (flow rate) controlled by a peristaltic pump. At the end of the experiment, the circulating and migrated cells are collected for further analyses. The 3D device can be used to examine cell rolling on and adhesion to EC under shear stress, transmigration in response to chemokine gradients, resistance to shear stress, cluster formation, and cell survival. In addition, the optional second insert allows the effects of crosstalk between EC and microenvironmental cells to be examined. The translational applications of the 3D device include testing of drug candidates that target cell migration and predicting the in vivo behavior of cells after intravenous injection. Thus, the novel 3D device is a versatile and inexpensive tool to study the molecular mechanisms that mediate cellular extravasation.

The three-dimensional flow chamber device is a novel in vitro technology for the quantitative and step-wise evaluation of the extravasation cascade of cells circulating under conditions of physiological shear stress. The device therefore fills a critical need for basic, preclinical, and clinical studies of cell migration.

Un dispositivo de cámara de flujo tridimensional Novel para el Estudio de quimioquinas-dirigida extravasación de células circulantes en condiciones de flujo fisiológicas

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell ...

Investigación

Bioingeniería

PLGA Nanoparticles Formed by Single- or Double-emulsion with Vitamin E-TPGS

Poly(lactic-co-glycolic acid) (PLGA) is a biocompatible member of the aliphatic polyester family of biodegradable polymers. PLGA has long been a popular choice for drug delivery applications, particularly since it is already FDA-approved for use in humans in the form of resorbable sutures. Hydrophobic and hydrophilic drugs are encapsulated in PLGA particles via single- or double-emulsion. Briefly, the drug is dissolved with polymer or emulsified with polymer in an organic phase that is then emulsified with the aqueous phase. After the solvent has evaporated, particles are washed and collected via centrifugation for lyophilization and long term storage. PLGA degrades slowly via hydrolysis in aqueous environments, and encapsulated agents are released over a period of weeks to months. Although PLGA is a material that possesses many advantages for drug delivery, reproducible formation of nanoparticles can be challenging; considerable variability is introduced by the use of different equipment, reagents batch, and precise method of emulsification. Here, we describe in great detail the formation and characterization of microparticles and nanoparticles formed by single- or double-emulsion using the emulsifying agent vitamin E-TPGS. Particle morphology and size are determined with scanning electron microscopy (SEM). We provide representative SEM images for nanoparticles produced with varying emulsifier concentration, as well as examples of imaging artifacts and failed emulsifications. This protocol can be readily adapted to use alternative emulsifiers (e.g. poly(vinyl alcohol), PVA) or solvents (e.g.&#160;dichloromethane, DCM).

We describe the production and characterization of nanoparticles and microparticles composed of poly(lactic-co-glycolic acid) using vitamin E-TPGS as an emulsifier. By varying formulation parameters such as the concentration of emulsifier, it is possible to produce nanoparticles with mean diameters ranging from 220 nm to&#160;1.98 &#181;m.

Poly(lactic-co-glycolic acid) (PLGA) is a biocompatible member of the aliphatic polyester family of biodegradable polymers. PLGA has long been a popular ...

Stretching Micropatterned Cells on a PDMS Membrane

Mechanical forces exerted on cells and/or tissues play a major role in numerous processes. We have developed a device to stretch cells plated on a PolyDiMethylSiloxane (PDMS) membrane, compatible with imaging. This technique is reproducible and versatile. The PDMS membrane can be micropatterned in order to confine cells or tissues to a specific geometry. The first step is to print micropatterns onto the PDMS membrane with a deep UV technique. The PDMS membrane is then mounted on a mechanical stretcher. A chamber is bound on top of the membrane with biocompatible grease to allow gliding during the stretch. The cells are seeded and allowed to spread for several hours on the micropatterns. The sample can be stretched and unstretched multiple times with the use of a micrometric screw. It takes less than a minute to apply the stretch to its full extent (around 30%). The technique presented here does not include a motorized device, which is necessary for applying repeated stretch cycles quickly and/or computer controlled stretching, but this can be implemented. Stretching of cells or tissue can be of interest for questions related to cell forces, cell response to mechanical stress or tissue morphogenesis. This video presentation will show how to avoid typical problems that might arise when doing this type of seemingly simple experiment.

This manuscript presents a technique to apply or release forces on adherent cells or tissues using unidirectional stretching.

El estiramiento células micropatterned en una membrana de PDMS

Mechanical forces exerted on cells and/or tissues  play a major role in numerous processes. We have developed a device to stretch  cells plated on a ...

Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This emerging field engages engineers, chemists, biologists, and physicists to design and assemble basic biological components into complex, functioning systems from the bottom up. Such bottom-up systems could lead to the development of artificial cells for fundamental biological inquiries and innovative therapies1,2. Giant unilamellar vesicles (GUVs) can serve as a model platform for synthetic biology due to their cell-like membrane structure and size. Microfluidic jetting, or microjetting, is a technique that allows for the generation of GUVs with controlled size, membrane composition, transmembrane protein incorporation, and encapsulation3. The basic principle of this method is the use of multiple, high-frequency fluid pulses generated by a piezo-actuated inkjet device to deform a suspended lipid bilayer into a GUV. The process is akin to blowing soap bubbles from a soap film. By varying the composition of the jetted solution, the composition of the encompassing solution, and/or the components included in the bilayer, researchers can apply this technique to create customized vesicles. This paper describes the procedure to generate simple vesicles from a droplet interface bilayer by microjetting.

Microfluidic jetting against a droplet interface lipid bilayer provides a reliable way to generate vesicles with control over membrane asymmetry, incorporation of transmembrane proteins, and encapsulation of material. This technique can be applied to study a variety of biological systems where compartmentalized biomolecules are desired.

Lípidos bicapa vesículas Generación Usando Microfluidic Jetting

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This ...

Automated Lipid Bilayer Membrane Formation Using a Polydimethylsiloxane Thin Film

An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for biosensor applications. However, conventional BLM formation techniques have several drawbacks and they often require specific expertise and laborious processes. In particular, conventional BLMs suffer from low formation success rates and inconsistent membrane formation time. Here, we demonstrate a storable and transportable BLM formation system with controlled thinning-out time and enhanced BLM formation rate by replacing conventionally used films (polytetrafluoroethylene, polyoxymethylene, polystyrene) to polydimethylsiloxane (PDMS). In this experiment, a porous-structured polymer such as PDMS thin film is used. In addition, as opposed to conventionally used solvents with low viscosity, the use of squalene permitted a controlled thinning-out time via slow solvent absorption by PDMS, prolonging membrane lifetime. In addition, by using a mixture of squalene and hexadecane, the freezing point of the lipid solution was increased (~16 &#176;C), in addition, membrane precursors were produced that can be indefinitely stored and readily transported. These membrane precursors have reduced BLM formation time of &#60; 1 hr and achieved a BLM formation rate of ~80%. Moreover, ion channel experiments with gramicidin A demonstrated the feasibility of the membrane system.

We demonstrate a storable, transportable lipid bilayer formation system. A lipid bilayer membrane can be formed within 1 hr with over 80% success rate when a frozen membrane precursor is brought to ambient temperature. This system will reduce laborious processes and expertise associated with ion channels.

Formación Automated bicapa lipídica membrana utilizando un polidimetilsiloxano Thin Film

An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for ...

A Microcontroller Operated Device for the Generation of Liquid Extracts from Conventional Cigarette Smoke and Electronic Cigarette Aerosol

Electronic cigarettes are the most popular tobacco product among middle and high schoolers and are the most popular alternative tobacco product among adults. High quality, reproducible research on the consequences of electronic cigarette use is essential for understanding emerging public health concerns and crafting evidence based regulatory policy. While a growing number of papers discuss electronic cigarettes, there is little consistency in methods across groups and very little consensus on results. Here, we describe a programmable laboratory device that can be used to create extracts of conventional cigarette smoke and electronic cigarette aerosol. This protocol details instructions for the assembly and operation of said device, and demonstrates the use of the generated extract in two sample applications: an in vitro cell viability assay and gas-chromatography mass-spectrometry. This method provides a tool for making direct comparisons between conventional cigarettes and electronic cigarettes, and is an accessible entry point into electronic cigarette research.

Here, we describe a programmable laboratory device that can be used to create extracts of conventional cigarette smoke and electronic cigarette aerosol. This method provides a useful tool for making direct comparisons between conventional cigarettes and electronic cigarettes, and is an accessible entry point into electronic cigarette research.

Un microcontrolador funciona dispositivo para la generación de extractos líquidos de Aerosol de humo y electrónicos cigarrillo cigarrillo convencional

Electronic cigarettes are the most popular tobacco product among middle and high schoolers and are the most popular alternative tobacco product among ...

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 ...

Generation of Transgenic Rats using a Lentiviral Vector Approach

Transgenic animal models are fundamentally important for modern biomedical research. The incorporation of foreign genes into early mouse or rat embryos is an invaluable tool for gene function analysis in living organisms. The standard transgenesis method is based on microinjecting foreign DNA fragments into a pronucleus of a fertilized oocyte. This technique is widely used in mice but remains relatively inefficient and technically demanding in other animal species. The transgene can also be introduced into one-cell-stage embryos via lentiviral infection, providing an effective alternative to standard pronuclear injections, especially in species or strains with a more challenging embryo structure. In this approach, a suspension that contains lentiviral vectors is injected into the perivitelline space of a fertilized rat embryo, which is technically less demanding and has a higher success rate. Lentiviral vectors were shown to efficiently incorporate the transgene into the genome to determine the generation of stable transgenic lines. Despite some limitations (e.g., Biosafety Level 2 requirements, DNA fragment size limits), lentiviral transgenesis is a rapid and efficient transgenesis method. Additionally, using female rats that are mated with a fertile male strain with a different dominant fur color is presented as an alternative to generate pseudopregnant foster mothers.

This article aims to provide the methodology for lentiviral transgenesis in rat embryos using multiple injections of a virus suspension into the zygote perivitelline space. Female rats that are mated with a fertile male strain with a different dominant fur color is used to generate pseudopregnant foster mothers.

Generación de ratas transgénicas utilizando un enfoque vectorial lentiviral

Transgenic animal models are fundamentally important for modern biomedical research. The incorporation of foreign genes into early mouse or rat embryos is ...

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 ...

Contrast-Enhanced Subharmonic Aided Pressure Estimation (SHAPE) Using Ultrasound Imaging with a Focus on Identifying Portal Hypertension

Noninvasive, accurate measurement of pressures within the human body has long been an important but elusive clinical goal. Contrast agents for ultrasound imaging are gas-filled, encapsulated microbubbles (diameter &#60; 10 &#956;m) that traverse the entire vasculature and enhance signals by up to 30 dB. These microbubbles also produce nonlinear oscillations at frequencies ranging from the subharmonic (half of the transmit frequency) to higher harmonics. The subharmonic amplitude has an inverse linear relationship with the ambient hydrostatic pressure. Here an ultrasound system capable of performing real-time, subharmonic aided pressure estimation (SHAPE) is presented. During ultrasound contrast agent infusion, an algorithm for optimizing acoustic outputs is activated. Following this calibration, subharmonic microbubble signals (i.e., SHAPE) have the highest sensitivity to pressure changes and can be used to noninvasively quantify pressure. The utility of the SHAPE procedure for identifying portal hypertension in the liver is the emphasis here, but the technique has applicability across many clinical scenarios.

Contrast-Enhanced Subharmonic Aided Pressure Estimation SHAPE Using Ultrasound Imaging with a Focus on Identifying Portal Hypertension

A protocol for noninvasively estimating ambient pressures utilizing subharmonic ultrasound imaging of infused contrast microbubbles (following appropriate calibration) is described with examples from human patients with chronic liver disease.

Estimación de la presión asistida por subarmónicos (SHAPE) con contraste mejorado mediante imágenes de ultrasonido con un enfoque en la identificación de la hipertensión portal

Noninvasive, accurate measurement of pressures within the human body has long been an important but elusive clinical goal. Contrast agents for ultrasound ...