Le financement a été fourni par le NIH et le MH-64611 NARSAD Young Investigator Award. Les auteurs tiennent également à remercier Adam Beagley, Mark Dikopf, et Ben Smith pour leur assistance technique.

SU-8 de fabrication du moule Préparation de Maître <ol><li> Le maître SU-8 sur le substrat de plaquette de silicium est préparé en utilisant une norme en deux étapes de lithographie processus négatif résister. </li><li> Les marques d&#39;alignement sur la plaquette de silicium sont enlevés à l&#39;aide d&#39;une lame de rasoir que la hauteur de ces structures (situé le long de la périphérie extérieure de la plaquette) est plus que les structures périphérique réel. </li><li> La plaquette de silicium est ensuite nettoyé à l&#39;alcool isopropylique et séché dans un courant de N2. Piliers de soutien faite de bandes d&#39;une épaisseur inférieure à la plus haute structure dispositif de remplacer les marques d&#39;alignement sur les quatre côtés de la plaquette. Remarque: Si la hauteur de l&#39;appui des piliers est plus que les structures plus haut appareil, les trous à travers ne sont pas formées dans le moule PDMS. </li><li> La plaquette de silicium master est placé sur une plaque chauffante à la température ambiante. </li></ol> Préparation de la solution PDMS <ol><li> Quatre grammes de polydiméthylsiloxane (PDMS) solution est préparée en mélangeant soigneusement 10 parties d&#39;élastomère de silicone avec 1 partie d&#39;agent de durcissement. Remarque: Assurez-vous que les deux solutions sont mélangées uniformément pour obtenir des propriétés physiques similaires à travers le moule PDMS. </li><li> Les bulles générées dans la solution PDMS pendant le processus de mixage sont retirés en utilisant un dessiccateur à vide. </li></ol> PDMS de revêtement et de séchage <ol><li> La bulle sans solution de PDMS est lentement distribué sur le maître SU-8 en s&#39;assurant que les bulles ne sont pas générées pendant le processus de distribution. </li><li> Une extrémité d&#39;un film transparent en écriture est alors placé sur la sur la plaque chauffante et lentement placé sur la solution d&#39;une répartition régulière PDMS PDMS sur le SU-8 maître. Toute bulles générées au cours de ce processus doivent être retirés à l&#39;aide d&#39;une sonde. [Note: Ne soulevez pas la transparence pour éviter la génération de plus de bulles.] </li><li> Une dalle Borofloat est placé en haut de la feuille de transparence pour appliquer une pression uniforme. Une légère pression est appliquée sur la surface supérieure de la plus haute des structures circulaires (situé à mi-chemin de la longueur de canal et centré le long de la largeur des canaux) telle qu&#39;il ya peu ou pas de PDMS en sandwich entre la transparence et le SU-8 de surface de la circulaire structures. </li><li> Trois dalles Borofloat plus sont placés sur des plaquettes de silicium-Borofloat sandwichs dalle à appliquer une pression constante sur la surface supérieure de l&#39;ouverture circulaire structures avant et pendant le processus de durcissement. </li><li> La température de la plaque chaude est ensuite augmentée à 75 ° C et le PDMS est guéri à cette température pendant 1 heure. La plaque chauffante est portée à une température de 50 ° C. </li><li> Les dalles sont enlevés et la transparence est doucement retiré laissant derrière le maître et la mince feuille PDMS enduit sur le dessus. </li></ol> La construction du dispositif microfluidique Retrait de feuille de PDMS de maître <ol><li> La limite extérieure de la chambre de perfusion est taillé sur la feuille de PDMS en utilisant une lame de rasoir après alignement des ports de la chambre de perfusion avec les ports sur le SU-8 maître. </li><li> La feuille de PDMS est ensuite délicatement retiré du maître, tirant le long des canaux pour empêcher la déchirure de PDMS. La feuille de PDMS est placé sur une feuille de transparence avec la surface du réseau microfluidique vers le haut. </li><li> Les orifices d&#39;entrée et de sortie sont alors fait à l&#39;aide d&#39;un perce-bouchon. </li></ol> Collage de feuilles de verre PDMS à lamelle <ol><li> La surface de collage de la feuille de PDMS (la surface contenant le réseau microfluidique) et une lamelle de verre sont ensuite délicatement nettoyé à l&#39;aide d&#39;un ruban de scotch 3M, placé sur une feuille de transparence, et finalement placé dans la chambre à plasma d&#39;O 2. </li><li> Les surfaces de collage sont traités avec 165 Watts de plasma pendant 10 s. La surface traitée par un plasma de la lamelle de verre est immédiatement liée à la surface traitée par un plasma de la feuille de PDMS. Note: Appliquer une légère pression sur la surface du verre pour enlever les bulles d&#39;air emprisonnées entre les surfaces de collage. </li><li> Laisser 5 minutes pour obtenir une bonne liaison entre le PDMS et le verre. Après 5 minutes retirez délicatement la transparence sur lequel la feuille PDMS a été placé pour le processus de collage. </li><li> Le dispositif microfluidique est placé dans le système de plasma d&#39;oxygène pour faire les canaux hydrophiles. Le traitement plasma est fait à 165 Watts pendant 1 minute. </li></ol> Intégration de dispositif microfluidique et de la chambre de perfusion Préparation de la chambre de perfusion <ol><li> Une chambre de perfusion off-the-shelf a été ordonnée, et les ports d&#39;entrée et de sortie ont été forés afin que, lorsque le dispositif microfluidique et la chambre sont liés, les ports de la chambre et l&#39;appareil sont alignés. La surface inférieure de la chambre de perfusion est enduit avec PDMS pour obtenir un joint étanche entre la chambre et le dispositif microfluidique. </li><li> Une écriture sur la feuille de transparence est placé sur une plaque chaude à la température ambiante. </li><li> Un gramme de PDMS solution est préparée dans un processus similaire à celui décrit précédemment. La bulle sans solution de PDMS est alors lentement distribué sur la génération de feuille de la transparence en évitant des bulles. </li><li> La chambre de perfusion est ensuite placé sur la solution de PDMS. </li><li> Une dalle Borofloat est ensuite placé sur le dessus de la chambre d&#39;appliquer une pression uniforme et pour obtenir une fine couche de PDMS sur la surface inférieure de la chambre. </li><li> La température de la plaque chaude est ensuite augmentée à 75 ° C et le PDMS est guéri à cette température pendant 1 heure. </li></ol> Collage de dispositif microfluidique et de la chambre de perfusion <ol><li> La dalle est enlevée et la transparence est délicatement retiré laissant derrière la chambre de perfusion et de l&#39;enduit mince feuille de PDMS sur sa surface inférieure. Indésirables PDMS est enlevé en utilisant une lame de rasoir et d&#39;une sonde pointu pour l&#39;enlèvement de PDMS à partir des ports d&#39;accès. </li><li> La surface de PDMS du dispositif microfluidique et la surface inférieure de la chambre de PDMS sont ensuite délicatement nettoyé à l&#39;aide d&#39;un ruban 3M, placé sur une feuille de transparence, et finalement placé dans la chambre à plasma d&#39;oxygène. </li><li> Les surfaces sont traitées avec 165 Watts de plasma pendant 10 s. Le PDMS surface revêtue de la chambre est immédiatement liée à la surface de PDMS du dispositif microfluidique. Note: Appliquer une légère pression sur la surface du verre pour enlever les bulles d&#39;air emprisonnées entre les surfaces de collage. </li><li> Immédiatement avant l&#39;utilisation, le dispositif est placé dans le système de plasma d&#39;oxygène pour faire les canaux hydrophiles. Le traitement plasma est fait à 165 Watts pendant 1 minute. </li></ol> Exposer des tranches de cerveau à l&#39;aide de micro-environnement neurochimique du dispositif microfluidique <ol><li> Les microcanaux hydrophile de la (dispositif microfluidique) - (chambre de perfusion) sont remplis avec la combinaison standard de l&#39;ACSF (Artificial liquide céphalo-rachidien) solution. </li><li> Distribuer de petites gouttes de solution ACSF dans les ports d&#39;entrée et de permettre à la solution d&#39;être méchant jusqu&#39;à dans les canaux. Retirer les bulles gauche à l&#39;aide d&#39;une seringue par l&#39;orifice de sortie. Verser une grosse goutte de solution de l&#39;ACSF au port de sortie pour permettre aux passifs de pompage de fluides à partir des ports d&#39;entrée à la sortie du dispositif microfluidique. Remarque: Il faut prendre soin d&#39;enlever toutes les bulles à partir des canaux pour permettre l&#39;écoulement des fluides en utilisant la méthode passive de pompage. </li><li> Fixer le (dispositif microfluidique) - (chambre de perfusion) combo dans la plate-forme simple et fixer la plate-forme de plaine dans l&#39;adaptateur de microscope. </li><li> Connectez l&#39;entrée standard et sortie (d&#39;aspiration) tuyau à la chambre de perfusion pour la perfusion continue de la tranche de cerveau à la norme ACSF solution. La solution est continuellement aspiré ACSF avec 95% d&#39;O2-5% de CO 2. Note: Ajuster la position du tuyau d&#39;aspiration pour maintenir un niveau constant de l&#39;ACSF dans le bain de la chambre de perfusion. </li><li> Une fois la chambre de perfusion est rempli avec une solution ACSF, placer une tranche de cerveau dans la chambre de perfusion en utilisant un compte-gouttes. En utilisant une sonde, la position de la tranche au-dessus du cerveau, des ouvertures circulaires dans le dispositif microfluidique. Une fois la tranche de cerveau est à la position souhaitée sur les ouvertures circulaires, utilisez un point d&#39;ancrage pour immobiliser le morceau de tranches de cerveau. </li><li> Le dispositif microfluidique peut maintenant être utilisé pour exposer les tranches de cerveau (placé dans la chambre de perfusion et sur le dessus du réseau microfluidique) de divers neurotransmetteurs à l&#39;aide passive de pompage de fluides. </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>

Les auteurs sont ouverts à des collaborations impliquant la technologie microfluidique démontré aux différents domaines de la biologie.

Existants macroscopique ou microscopique chambres du cerveau tranche de perfusion sont limitées en termes de la résolution spatiale qu&#39;ils fournissent pour exposer des tranches de cerveau avec des neurotransmetteurs. La technologie dispositif microfluidique démontré ici surmonte cette limitation en utilisant des techniques simples de bioMEMS. Il est prévu que la simplicité dans la fabrication du dispositif microfluidique et de la facilité de son intégration avec les configurations existantes électrophysiologie permettra l&#39;a...

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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>
		</thead>
		<tbody>
		
<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>
</tr>		</tbody>
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		</div>

brain slice stimulation using a microfluidic network and standard perfusion chamber

Nous avons démontré la fabrication d&#39;un dispositif à deux niveaux microfluidique qui peuvent être facilement intégrés avec les configurations existantes électrophysiologie. Le dispositif à deux niveaux microfluidique est fabriqué en utilisant une norme en deux étapes négative résister à une processus de lithographie. Le premier niveau contient des microcanaux avec des orifices d&#39;entrée et de sortie à chaque extrémité. Le deuxième niveau contient micro trous circulaires situées à mi-chemin de la longueur de canal et centré le long avec une largeur de canal. Passif méthode de pompage est utilisée pour pomper des liquides à partir du port d&#39;entrée au port de sortie 2. Le dispositif microfluidique est intégré à off-the-shelf chambres de perfusion et permet une intégration transparente avec l&#39;installation d&#39;électrophysiologie. Les fluides introduits à l&#39;entrée des ports le débit à travers les microcanaux vers les ports de sortie et aussi s&#39;échapper à travers les ouvertures circulaires situées sur le dessus des microcanaux dans le bain de la perfusion. Ainsi, la surface inférieure de la tranche de cerveau placé dans le bain et la chambre de perfusion au-dessus du dispositif microfluidique peut être exposé à différents neurotransmetteurs. L&#39;épaisseur microscopique du dispositif microfluidique et la nature des matériaux transparents [verre lamelle et PDMS (polydiméthylsiloxane)] utilisé pour rendre le dispositif microfluidique permet de microscopie de la tranche de cerveau. Le dispositif microfluidique permet une modulation (à la fois spatiale et temporelle) des stimuli chimiques introduits à l&#39;microenvironnements tranche de cerveau.

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

Nous démontrons la fabrication d&#39;un dispositif simple microfluidique qui peuvent être intégrés avec les configurations standard de l&#39;électrophysiologie pour exposer les surfaces micro d&#39;une tranche du cerveau d&#39;une manière bien contrôlée pour différents neurotransmetteurs.

Stimulation de tranches de cerveau utilisant un réseau microfluidique et de chambre de perfusion standard

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 vues.  University of Illinois, Chicago. Nous démontrons la fabrication d'un dispositif simple microfluidique qui peuvent être intégrés avec les configurations standard de l'électrophysiologie pour exposer les surfaces micro d'une tranche du cerveau d'une manière bien contrôlée pour différents neurotransmetteurs.

Vidéo: 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.

Fabrication d&#39;un dispositif microfluidique pour la compartimentation du Neuron Soma et des axones

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

Recherche

Biologie

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.

Préparation E18 neurones de rat corticale compartimentation dans un dispositif microfluidique

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

T CD4 + des lymphocytes capturer en utilisant une puce jetable microfluidiques pour le VIH

Cell Capture Using a Microfluidic Device

Capturez cellule à l&#39;aide d&#39;un dispositif microfluidique

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.

Évaluer la motilité Neural Stem Cell utilisant un gel d&#39;agarose à base dispositif microfluidique

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.

Trypsinisation et Repiquage des cellules de mammifères

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 sonde microfluidique: Fonctionnement et utilisation pour le traitement de surface localisé

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.

L&#39;adhésion des plaquettes et l&#39;agrégation sous flux en utilisant des cellules de débit microfluidique

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.

Cartographie des séquelles de Theta Burst Stimulation sur le cortex auditif humain à l&#39;imagerie fonctionnelle

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 stimulation mécanique des chondrocytes-agarose hydrogels

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