El financiamiento fue proporcionado por el NIH MH-64611 y el Premio Joven Investigador NARSAD. Los autores también desean agradecer a Adán Beagley, Dikopf Marcos, y Ben Smith por su asistencia técnica.

SU-8 de fabricación de moldes Maestro de la preparación <ol><li> El maestro de SU-8 sobre un sustrato de obleas de silicio se prepara utilizando una de dos etapas estándar negativo resistir proceso de litografía. </li><li> Las marcas de alineación de la oblea de silicio se eliminan utilizando una cuchilla de afeitar como la altura de estas estructuras (que se encuentra a lo largo de la periferia externa de la oblea) es más que la estructura real del dispositivo. </li><li> La oblea de silicio se limpia con alcohol isopropílico y se secan en una corriente de N 2. Pilares de apoyo hechas de cintas con un espesor de menos de la estructura más alta de dispositivos reemplazan a las marcas de alineación en los cuatro lados de la oblea. Nota: Si la altura del soporte de los pilares es más que la estructura más alta del dispositivo, los agujeros por los que no están formados en el molde de PDMS. </li><li> La oblea de silicio maestro se coloca en un plato caliente a la temperatura ambiente. </li></ol> PDMS preparación de la solución <ol><li> Cuatro gramos de polidimetilsiloxano (PDMS) solución se prepara mediante la mezcla de 10 partes de elastómero de silicona con una parte del agente de curado. Nota: Asegúrese de que las dos soluciones se mezclan de manera uniforme para obtener propiedades físicas similares en todo el molde de PDMS. </li><li> Las burbujas generadas en la solución de PDMS durante el proceso de mezcla se eliminan usando un secador de vacío. </li></ol> PDMS recubrimiento y curado <ol><li> La burbuja de una solución libre de PDMS es lenta dispensada en el patrón de SU-8 de asegurarse de que las burbujas no se generan durante el proceso de dispensación. </li><li> Uno de los extremos de una película de transparencia para escribir en ella se coloca en el plato caliente y poco a poco puestos en la solución de PDMS para repartir uniformemente el PDMS en el SU-8 maestro. Las burbujas generadas durante este proceso deben ser eliminadas mediante una sonda. [Nota: No levante la transparencia para evitar la generación de más burbujas.] </li><li> Una losa Borofloat se coloca en la parte superior de la hoja de transparencia para aplicar una presión uniforme. Ejercer presión suave sobre la superficie superior de las estructuras más altas circular (que se encuentra a medio camino de las longitudes de canal y centrado a lo largo de la anchura de los canales) de tal manera que existe una mínima o PDMS no intercalado entre la transparencia y el SU-8 la superficie de la circular estructuras. </li><li> Otros tres losas Borofloat se colocan en la parte superior de la oblea de silicio-Borofloat sándwich losa para aplicar una presión constante sobre la superficie superior de las estructuras de abertura circular antes y durante el proceso de curado. </li><li> La temperatura de la placa caliente se incrementó a 75 ° C y el PDMS se cura a esta temperatura durante 1 hora. La placa caliente es llevado a una temperatura de 50 ° C. </li><li> Las placas se retiran y la transparencia se quita suavemente dejando atrás el maestro y la delgada hoja de PDMS recubierto en la parte superior de la misma. </li></ol> Construcción del dispositivo de microfluidos La eliminación de la hoja de PDMS de maestro <ol><li> El límite exterior de la cámara de perfusión está tallada en la hoja de PDMS con una cuchilla de afeitar después de alinear los puertos de la cámara de perfusión con los puertos en el SU-8 maestro. </li><li> La hoja de PDMS es suavemente eliminado de la maestra, tirando a lo largo de los canales para evitar la ruptura de PDMS. La hoja de PDMS se coloca sobre una hoja de transparencia con la superficie de la red de microfluidos hacia arriba. </li><li> Los puertos de entrada y salida entonces se hacen mediante una sonda. </li></ol> Unión de la hoja de vidrio cubreobjetos PDMS <ol><li> La superficie de unión de la hoja de PDMS (la superficie que contiene la red de microfluidos) y un cubreobjetos son suavemente limpiar con una cinta adhesiva 3M, colocado sobre una hoja de transparencia, y por último, ponerlas en la cámara de O 2 en plasma. </li><li> Las superficies a unir se tratan con plasma 165 vatios durante 10 s. La superficie de plasma tratado del cubreobjetos está inmediatamente unido a la superficie tratado con plasma de la hoja de PDMS. Nota: Aplique una presión suave sobre la superficie del vidrio para eliminar cualquier burbuja de aire atrapada entre las superficies a unir. </li><li> Deje pasar 5 minutos para obtener una buena unión entre el PDMS y vidrio. Después de 5 minutos retire con cuidado la transparencia en la que se coloca la hoja de PDMS para el proceso de unión. </li><li> El dispositivo de microfluidos se coloca en el sistema de plasma de oxígeno para que los canales hidrofílicos. El tratamiento de plasma se realiza a 165 vatios durante 1 minuto. </li></ol> Integración de dispositivos de microfluídica y cámara de perfusión Preparación de la cámara de perfusión <ol><li> Una cámara de perfusión fuera de la plataforma se le ordenó, y los puertos de entrada y salida fueron perforados para que cuando el dispositivo de microfluidos y la cámara están en condiciones de servidumbre, los puertos de la cámara y el dispositivo estén alineados. La superficie inferior de la cámara de perfusión está recubierta con PDMS para obtener un sello hermético entre la cámara y el dispositivo de microfluidos. </li><li> Una escritura en la hoja de transparencia se coloca en un plato caliente a la temperatura ambiente. </li><li> Un gramo PDMS solución se prepara en un proceso similar al descrito anteriormente. La burbuja de una solución libre de PDMS es lentamente dispensado sobre la generación de hoja de transparencia para evitar las burbujas. </li><li> La cámara de perfusión se coloca en la solución de PDMS. </li><li> Una losa Borofloat se coloca en la parte superior de la cámara para aplicar una presión uniforme y para obtener una fina capa de PDMS sobre la superficie inferior de la cámara. </li><li> La temperatura de la placa caliente se incrementó a 75 ° C y el PDMS se cura a esta temperatura durante 1 hora. </li></ol> Unión de dispositivo de microfluidos y cámara de perfusión <ol><li> La losa está eliminado y la transparencia se quita suavemente dejando tras de sí la cámara de perfusión y la delgada hoja de recubrimiento PDMS en su superficie inferior. No deseados PDMS se elimina mediante una cuchilla de afeitar y una sonda de punta afilada para la eliminación de PDMS de puertos de acceso. </li><li> La superficie de PDMS del dispositivo de microfluidos y la superficie inferior de la cámara de PDMS se limpian suavemente con una cinta 3M, colocado sobre una hoja de transparencia, y por último, ponerlas en la cámara de plasma de oxígeno. </li><li> Las superficies se tratan con plasma 165 vatios durante 10 s. La superficie recubierta PDMS de la cámara inmediatamente se adhiere a la superficie PDMS del dispositivo de microfluidos. Nota: Aplique una presión suave sobre la superficie del vidrio para eliminar cualquier burbuja de aire atrapada entre las superficies a unir. </li><li> Inmediatamente antes del uso, el dispositivo se coloca en el sistema de plasma de oxígeno para que los canales hidrofílicos. El tratamiento de plasma se realiza a 165 vatios durante 1 minuto. </li></ol> La exposición de las secciones de cerebro de microambiente neuroquímicos utilizando el dispositivo de microfluidos <ol><li> Los microcanales hidrofílicos de la (dispositivo de microfluidos) - (cámara de perfusión) combinación se rellenan con el estándar de ACSF (artificial líquido cefalorraquídeo) solución. </li><li> Prescindir de pequeñas gotas de la solución de ACSF en los puertos de entrada y permitir que la solución sea malo hasta en los canales. Eliminar las burbujas de la izquierda con una jeringa en el puerto de salida. Prescindir de una gran gota de solución de ACSF en el puerto de salida para permitir pasiva de bombeo de líquidos desde la entrada a los puertos de salida del dispositivo de microfluidos. Nota: Se debe tener cuidado de eliminar todas las burbujas de los canales para permitir el flujo de fluidos usando el método pasivo de bombeo. </li><li> Fijar el (dispositivo de microfluidos) - (cámara de perfusión) combo en la plataforma simple y fijar la plataforma normal en el adaptador de microscopio. </li><li> Conecte la entrada estándar y la salida (succión) tubo a la cámara de perfusión para la perfusión continua de los cortes de cerebro con el estándar de solución de ACSF. La solución ACSF continuamente aspirado, con un 95% de O2, CO2 al 5%. Nota: Ajuste la posición del tubo de succión para mantener un nivel constante de ACSF en el baño de la cámara de perfusión. </li><li> Una vez que la cámara de perfusión se llena con una solución de ACSF, coloque una rebanada del cerebro en la cámara de perfusión con un gotero. Mediante una sonda, la posición de la parte del cerebro por encima de las aberturas circulares en el dispositivo de microfluidos. Una vez que la parte del cerebro está en la posición deseada sobre las aberturas circulares, use un trozo de anclaje para inmovilizar la parte del cerebro. </li><li> El dispositivo de microfluidos ahora se puede utilizar para exponer las rodajas de cerebro (que se encuentra en la cámara de perfusión y en la parte superior de la red de microfluidos) de varios neurotransmisores con pasivos de bombeo de líquidos. </li></ol>

<ol>
	<li>Blake, A. J., Pearce, T. M., Rao, N. S., Johnson, S. M., Williams, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multilayer+PDMS+microfluidic+chamber+for+controlling+brain+slice+microenvironment.">Multilayer PDMS microfluidic chamber for controlling brain slice microenvironment.</a> Lab on a Chip. 7, 842-849 (2007).</li><li>Walker, G. M., Beebe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+passive+pumping+method+for+microfluidic+devices.">A passive pumping method for microfluidic devices.</a> Lab on a Chip. 2, 131-134 (2002).</li></ol>

Los autores están abiertos a colaboraciones involucran la tecnología de microfluidos demostrado en diferentes campos de la biología.

Existentes macroescala o micro cámaras de perfusión cerebral rebanada son limitados en términos de la resolución espacial que ofrecen a exponer las secciones de cerebro de neurotransmisores. La tecnología de los dispositivos de microfluidos demostrado aquí supera esta limitación utilizando técnicas bioMEMS simple. Se prevé que la simplicidad en la fabricación del dispositivo de microfluidos y la facilidad de integración con las configuraciones existentes de electrofisiología permite la aplicación generalizada de la tecnología ...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>RC-26GPL</td>
<td>Tool</td>
<td>Warner Instruments</td>
<td>W2-64-0236</td>
<td>Low Profile Large Bath RC-26GLP Recording Chamber</td>
</tr>
<tr>
<td>SHD-26GH/10</td>
<td>Tool</td>
<td>Warner Instruments</td>
<td>W2-64-0253</td>
<td>Stainless steel slice hold-down for RC-26G, 1.0 mm thread spacing </td>
</tr>
<tr>
<td>PDMS (polydimethylsiloxane)</td>
<td>Reagent</td>
<td>Dow Corning</td>
<td>Sylgard 184</td>
<td>Silicone Elastomer Kit</td>
</tr>
<tr>
<td>Plasma Preen-II 862</td>
<td>Tool</td>
<td>Plasmatic Systems, Inc.</td>
<td>&nbsp;</td>
<td>Microwave plasma system</td>
</tr>
<tr>
<td>Model P-1</td>
<td>Tool</td>
<td>Warner Instruments</td>
<td>W2-64-0277</td>
<td>Series 20 Plain Platform, Model P-1</td>
</tr>
<tr>
<td>SA-NIK</td>
<td>Tool</td>
<td>Warner Instruments</td>
<td>W2-64-0291</td>
<td>Adapter for Nikon Diaphot/TE200/TE2000, SA-NIK</td>
</tr>
<tr>
<td>Oxygenated, heated ACSF (Artificial cerebro-spinal fluid)</td>
<td>Reagent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Exact composition will vary with application</td>
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brain slice stimulation using a microfluidic network and standard perfusion chamber

Hemos demostrado la fabricación de un dispositivo de microfluidos de dos niveles que se pueden integrar fácilmente con las configuraciones existentes de electrofisiología. El dispositivo de microfluidos de dos niveles se fabrica utilizando un estándar de dos etapas negativas resistir un proceso de litografía. El primer nivel contiene microcanales con entrada y salida en cada extremo. El segundo nivel contiene micro orificios circulares situado a medio camino de la longitud del canal y centrado a lo largo con un ancho de canal. Método pasivo de bombeo se utiliza para bombear los líquidos desde el puerto de entrada al puerto de salida 2. El dispositivo de microfluidos está integrado en off-the-shelf cámaras de perfusión y permite una integración perfecta con la configuración de electrofisiología. Los líquidos introducidos en el flujo de entrada de los puertos a través de los microcanales hacia los puertos de salida y escapar a través de las aberturas circulares situados en la parte superior de los microcanales en el baño de la perfusión. Así, la superficie inferior de la parte del cerebro situado en el baño y la cámara de perfusión por encima del dispositivo de microfluidos pueden estar expuestos a diferentes neurotransmisores. El espesor de la microescala dispositivo de microfluidos y el carácter transparente de los materiales [de vidrio cubreobjetos y PDMS (polidimetilsiloxano)] Se utiliza para hacer que el dispositivo de microfluidos permiten microscopía de la rebanada del cerebro. El dispositivo de microfluidos permite la modulación (espacial y temporal) de los estímulos químicos introducidos en el microambiente cerebral rebanada.

Watch this Scientific Journal Video about Brain Slice Stimulation Using a Microfluidic Network and Standard Perfusion Chamber at JoVE.com

Brain Slice Stimulation Using a Microfluidic Network and Standard Perfusion Chamber

Se demuestra la fabricación de un dispositivo de microfluidos sencillos que pueden ser integrados con el estándar de configuraciones de electrofisiología para exponer las superficies a microescala de un trozo del cerebro de una manera bien controlada para los diferentes neurotransmisores.

Estimulación Cerebral sector mediante una red de microfluidos y de la Cámara de perfusión estándar

We have demonstrated the fabrication of a two-level microfluidic device that can be easily integrated with existing electrophysiology setups. The ...

brain-slice-stimulation-using-microfluidic-network-standard-perfusion

Research

JoVE Journal

Biology

10.8K vistas.  University of Illinois, Chicago. Se demuestra la fabricación de un dispositivo de microfluidos sencillos que pueden ser integrados con el estándar de configuraciones de electrofisiología para exponer las superficies a microescala de un trozo del cerebro de una manera bien controlada para los diferentes neurotransmisores.

Video: Brain Slice Stimulation Using a Microfluidic Network and Standard Perfusion Chamber

Fabrication of a Microfluidic Device for the Compartmentalization of Neuron Soma and Axons

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for culturing neurons. Previously, a silicon wafer was patterned with the design for the neuron microfluidic device using SU-8 and photolithography to create a master mold, or what we simply refer to as a "master". Next, we pour the silicon polymer PDMS on top of the master which is then cured by heating the PDMS to 80&deg;C for 1 hour. The PDMS forms a negative mold of the device. The PDMS is then carefully cut and lifted away from the master. Holes are punched where the reservoirs will be and the excess PDMS trimmed away from the device. Nitrogen is used to blow away any excess debris from the device. At this point the devices are now ready for use and can either bonded to corning No. 1 cover glass with a plasma sterilizer/cleaner or can be reversibly bound to the cover glass by simply placing the device on top of the cover glass. The reversible bonding of the device to glass is covered in a separate video and requires first that the device be sterilized either with 70% ethanol or by autoclaving. Plasma treating sterilizes the devices so no further treatment is necessary. It is, however, important, when plasma-treating the devices, to add liquid to the devices within 10 minutes of the plasma treatment while the surfaces are still hydrophilic. Waiting longer than 10 minutes to add liquid to the device makes it difficult for the liquid to enter the device. The neuron devices are typically plasma-bound to cover glass and 0.5 mg/ml poly-L-lysine (PLL) in pH 8.5 borate buffer is immediately added to the device. After a minimum of 3 hours incubating with PLL, the devices are washed with dH2O water a minimum of 3 times with at least 15 minutes between each wash. Next, the water is removed and fresh media is added to the device. At this point the device is ready for use. It is important to remember at this point to never remove all the media from the device. Always leave media in the main channel.

In this video we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to farbricate a microfluidic device for culturing neurons.

Fabricación de un dispositivo de microfluidos para la compartimentación de las neuronas y los axones Soma

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for ...

Investigación

Biología

Preparing E18 Cortical Rat Neurons for Compartmentalization in a Microfluidic Device

In this video, we demonstrate the preparation of E18 cortical rat neurons. E18 cortical rat neurons are obtained from E18 fetal rat cortex previously dissected and prepared. The E18 cortex is, upon dissection, immediately dissociated into individual neurons. It is possible to store E18 cortex in Hibernate E buffer containing B27 at 4&deg;C for up to a week before the dissociation is performed. However, there will be a drop in cell viability. Typically we obtain our E18 Cortex fresh. It is transported to the lab in ice cold Calcium free Magnesium free dissection buffer (CMFM). Upon arrival, trypsin is added to the cortex to a final concentration of 0.125%. The cortex is then incubated at 37&deg;C for 8 minutes. DMEM containing 10% FBS is added to the cortex to stop the reaction. The cortex is then centrifuged at 2500 rpm for 2 minutes. The supernatant is removed and 2 ml of Neural Basal Media (NBM) containing 2% B27 (vol/vol) and 0.25% Glutamax (vol/vol) is added to the cortex which is then re-suspended by pipetting up and down. Next, the cortex is triturated with previously fire polished glass pipettes, each with a successive smaller opening. After triturating, the cortex is once again centrifuged at 2500 rpm for 2 minutes. The supernatant is then removed and the cortex pellet re-suspended with 2 ml of NBM containing B27 and Glutamax. The cell suspension is then passed through a 40 um nylon cell strainer. Next the cells are counted. The neurons are now ready for loading into the neuron microfluidic device.

In this video we demonstrate the preparation of E18 Cortical Rat Neurons.

Preparación de neuronas de rata E18 cortical de compartimentación en un dispositivo de microfluidos

In this video, we demonstrate the preparation of E18 cortical rat neurons. E18 cortical rat neurons are obtained from E18 fetal rat cortex previously ...

CD4+ T-Lymphocyte Capture Using a Disposable Microfluidic Chip for HIV

CD4 + T-linfocitos capturar usando un chip de microfluidos desechables para el VIH

Cell Capture Using a Microfluidic Device

Captura de células utilizando un dispositivo de microfluidos

Assessing Neural Stem Cell Motility Using an Agarose Gel-based Microfluidic Device

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is largely due to the difficulty with which one can create a cell-compatible and steady microenvironment using conventional microfabrication techniques and materials. We address this problem via the introduction of a novel microfabrication material, agarose gel, as the base material for the microfluidic device. Agarose gel is highly malleable, and permeable to gas and nutrients necessary for cell survival, and thus an ideal material for cell-based assays. We have shown previously that agarose gel based devices have been successful in studying bacterial and neutrophil cell migration2. In this report, three parallel microfluidic channels are patterned in an agarose gel membrane of about 1mm thickness. Constant flows with media/buffer are maintained in the two side channels using a peristaltic pump. Cells are maintained in the center channel for observation. Since the nutrients and chemicals in the side channels are constantly diffusing from the side to center channel, the chemical environment of the center channel is easily controlled via the flow along the side channels. Using this device, we demonstrate that the movement of neural stem cells can be monitored optically with ease under various chemical conditions, and the experimental results show that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells. Motility of neural stem cells is an important biomarker for assessing cells aggressiveness, thus tumorigenic factor3. Deciphering the mechanism underlying NSC motility will yield insight into both disorders of neural development and into brain cancer stem cell invasion.

We demonstrate that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells(NSCs) using a novel agarose gel based microfluidic device. This technology can be readily adaptable to other mammalian cell systems where cell sources are scarce, such as human neural stem cells, and the turn around time is critical.

La evaluación de la motilidad de células madre neurales uso de un gel de agarosa al dispositivo basado en microfluídica

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is ...

Trypsinizing and Subculturing Mammalian Cells

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in cell death. The first step in subculturing is to detach cells from the surface of the primary culture vessel by trypsinization or mechanical means. The resultant cell suspension is then subdivided, or reseeded, into fresh cultures. Secondary cultures are checked for growth and fed periodically, and may be subsequently subcultured to produce tertiary cultures. The time between passaging of cells varies with the cell line and depends on the growth rate.

As cells reach confluency, they must be subcultured or passaged. This video will demonstrate a procedure for subculturing both adherent and suspension cells.

Trypsinizing Siembra y células de mamífero

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in ...

The Microfluidic Probe: Operation and Use for Localized Surface Processing

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. Microfluidics is commonly associated with closed microchannels which limit their use to samples that can be introduced, and cultured in the case of cells, within a confined volume. On the other hand, micropipetting system have been used to locally perfuse cells and surfaces, notably using push-pull setups where one pipette acts as source and the other one as sink, but the confinement of the flow is difficult in three dimensions. Furthermore, pipettes are fragile and difficult to position and hence are used in static configuration only. 
The microfluidic probe (MFP) circumvents the constraints imposed by the construction of closed microfluidic channels and instead of enclosing the sample into the microfluidic system, the microfluidic flow can be directly delivered onto the sample, and scanned across the sample, using the MFP. . The injection and aspiration openings are located within a few tens of micrometers of one another so that a microjet injected into the gap is confined by the hydrodynamic forces of the surrounding liquid and entirely aspirated back into the other opening. The microjet can be flushed across the substrate surface and provides a precise tool for localized deposition/delivery of reagents which can be used over large areas by scanning the probe across the surface. 
In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer.

In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer solution.

La sonda de microfluidos: Operación y Uso de tratamiento de superficies localizadas

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. ...

Platelet Adhesion and Aggregation Under Flow using Microfluidic Flow Cells

Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium has been exposed. The platelet adhesion cascade takes place in the presence of shear flow, a factor not accounted for in conventional (static) well-plate assays. This article reports on a platelet-aggregation assay utilizing a microfluidic well-plate format to emulate physiological shear flow conditions. Extracellular proteins, collagen I or von Willebrand factor are deposited within the microfluidic channel using active perfusion with a pneumatic pump. The matrix proteins are then washed with buffer and blocked to prepare the microfluidic channel for platelet interactions. Whole blood labeled with fluorescent dye is perfused through the channel at various flow rates in order to achieve platelet activation and aggregation. Inhibitors of platelet aggregation can be added prior to the flow cell experiment to generate IC50 dose response data.

The platelet adhesion cascade takes place in the presence of shear flow, a factor not accounted for in conventional (static) well-plate assays. This article reports on a platelet-aggregation assay utilizing a microfluidic well-plate format to emulate physiological shear flow conditions.

Adhesión y agregación plaquetaria bajo flujo con células de microfluidos de flujo

Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium has been exposed. The platelet adhesion ...

Mapping the After-effects of Theta Burst Stimulation on the Human Auditory Cortex with Functional Imaging

Auditory cortex pertains to the processing of sound, which is at the basis of speech or music-related processing1. However, despite considerable recent progress, the functional properties and lateralization of the human auditory cortex are far from being fully understood. Transcranial Magnetic Stimulation (TMS) is a non-invasive technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses, and represents a unique method of exploring plasticity and connectivity. It has only recently begun to be applied to understand auditory cortical function 2. 
An important issue in using TMS is that the physiological consequences of the stimulation are difficult to establish. Although many TMS studies make the implicit assumption that the area targeted by the coil is the area affected, this need not be the case, particularly for complex cognitive functions which depend on interactions across many brain regions 3. One solution to this problem is to combine TMS with functional Magnetic resonance imaging (fMRI). The idea here is that fMRI will provide an index of changes in brain activity associated with TMS. Thus, fMRI would give an independent means of assessing which areas are affected by TMS and how they are modulated 4. In addition, fMRI allows the assessment of functional connectivity, which represents a measure of the temporal coupling between distant regions. It can thus be useful not only to measure the net activity modulation induced by TMS in given locations, but also the degree to which the network properties are affected by TMS, via any observed changes in functional connectivity.
Different approaches exist to combine TMS and functional imaging according to the temporal order of the methods. Functional MRI can be applied before, during, after, or both before and after TMS. Recently, some studies interleaved TMS and fMRI in order to provide online mapping of the functional changes induced by TMS 5-7. However, this online combination has many technical problems, including the static artifacts resulting from the presence of the TMS coil in the scanner room, or the effects of TMS pulses on the process of MR image formation. But more importantly, the loud acoustic noise induced by TMS (increased compared with standard use because of the resonance of the scanner bore) and the increased TMS coil vibrations (caused by the strong mechanical forces due to the static magnetic field of the MR scanner) constitute a crucial problem when studying auditory processing. 
This is one reason why fMRI was carried out before and after TMS in the present study. Similar approaches have been used to target the motor cortex 8,9, premotor cortex 10, primary somatosensory cortex 11,12 and language-related areas 13, but so far no combined TMS-fMRI study has investigated the auditory cortex. The purpose of this article is to provide details concerning the protocol and considerations necessary to successfully combine these two neuroscientific tools to investigate auditory processing. 
Previously we showed that repetitive TMS (rTMS) at high and low frequencies (resp. 10 Hz and 1 Hz) applied over the auditory cortex modulated response time (RT) in a melody discrimination task 2. We also showed that RT modulation was correlated with functional connectivity in the auditory network assessed using fMRI: the higher the functional connectivity between left and right auditory cortices during task performance, the higher the facilitatory effect (i.e. decreased RT) observed with rTMS. However those findings were mainly correlational, as fMRI was performed before rTMS. Here, fMRI was carried out before and immediately after TMS to provide direct measures of the functional organization of the auditory cortex, and more specifically of the plastic reorganization of the auditory neural network occurring after the neural intervention provided by TMS. 
Combined fMRI and TMS applied over the auditory cortex should enable a better understanding of brain mechanisms of auditory processing, providing physiological information about functional effects of TMS. This knowledge could be useful for many cognitive neuroscience applications, as well as for optimizing therapeutic applications of TMS, particularly in auditory-related disorders.

Auditory processing is the basis of speech and music-related processing. Transcranial Magnetic Stimulation (TMS) has been used successfully to study cognitive, sensory and motor systems but has rarely been applied to audition. Here we investigated TMS combined with functional Magnetic Resonance Imaging to understand the functional organization of auditory cortex.

Mapeo de las secuelas de la theta ráfaga de estimulación sobre la corteza auditiva humana con imágenes funcionales

Auditory cortex pertains to the processing of sound, which is at the basis of speech or music-related processing1. However, despite considerable recent ...

Mechanical Stimulation of Chondrocyte-agarose Hydrogels

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy this deficiency, several medical interventions have been developed. One such method is to resurface the damaged area with tissue-engineered cartilage; however, the engineered tissue typically lacks the biochemical properties and durability of native cartilage, questioning its long-term survivability. This limits the application of cartilage tissue engineering to the repair of small focal defects, relying on the surrounding tissue to protect the implanted material. To improve the properties of the developed tissue, mechanical stimulation is a popular method utilized to enhance the synthesis of cartilaginous extracellular matrix as well as the resultant mechanical properties of the engineered tissue. Mechanical stimulation applies forces to the tissue constructs analogous to those experienced in vivo. This is based on the premise that the mechanical environment, in part, regulates the development and maintenance of native tissue1,2. The most commonly applied form of mechanical stimulation in cartilage tissue engineering is dynamic compression at physiologic strains of approximately 5-20% at a frequency of 1 Hz1,3. Several studies have investigated the effects of dynamic compression and have shown it to have a positive effect on chondrocyte metabolism and biosynthesis, ultimately affecting the functional properties of the developed tissue4-8. In this paper, we illustrate the method to mechanically stimulate chondrocyte-agarose hydrogel constructs under dynamic compression and analyze changes in biosynthesis through biochemical and radioisotope assays. This method can also be readily modified to assess any potentially induced changes in cellular response as a result of mechanical stimuli.

The biosynthesis of cartilaginous extracellular matrix by chondrocytes can be affected by application of mechanical stimuli. This method describes the technique of applying dynamic compressive strains to chondrocytes encapsulated in 3D constructs and the evaluation of induced changes in chondrocyte metabolism.

La estimulación mecánica de condrocitos-agarosa hidrogeles

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy ...