Ce travail a été soutenu par une bourse de recherche de l&#39;Institut de Californie pour quantitatives Biosciences (de QB3), Bridging the Award Gap de la Fondation Rogers famille, le / Programme de la Fondation Sandler UCSF pour Breakthrough Biomedical Research, une subvention de BASF, et de la NSF travers Programme Faculté Early Career Development (Carrière) (DBI-1253293).

1. SU8 Fabrication de Maître <ol><li> Concevoir les structures microfluidiques pour deux fabrication de couche en utilisant le logiciel AutoCAD et ont les dessins imprimés par un fournisseur sur le film de la carte de circuit avec 10 um résolution. Les détails de la conception de l&#39;appareil sont données dans une référence attachée 11 et les géométries de canaux sont présentés dans la figure 1. Les couches devraient inclure des repères d&#39;alignement pour aider placez les caractéristiques de chaque couche de fabrication 12. </li><li> Placez une tranche de silicium de 3 pouces de diamètre préalablement nettoyé sur une tournette et tourner sur le vide pour l&#39;apposer sur le mandrin. Appliquer 1 ml de SU8-3035 dans le centre de la plaquette et de spin pendant 20 secondes à 500 tours par minute, puis 30 secondes à 2000 tours par minute, offrant une épaisseur de couche de 50 um. </li><li> Retirer la plaquette et faire cuire sur une plaque de cuisson à 135 ° C pendant 30 min. Laisser refroidir la tranche à la température ambiante avant de passer à l&#39;étape suivante. </li><li> Exposer la tranche revêtue au 1 er masque de calque (figure 2A </strong&gt;) sous un collimaté 190 mW, 365 nm LED pendant 90 secondes. Après l&#39;exposition, placez la galette sur une plaque de cuisson de 135 C ° pendant 1 min, puis refroidir à température ambiante avant de procéder à la prochaine étape. </li><li> Placez la plaquette sur la tournette et tourner sur le vide pour l&#39;apposer sur le mandrin. Appliquer 1 ml de SU8-2050 dans le centre de la plaquette et de spin pendant 20 secondes à 500 tours par minute, puis 30 secondes à 1375 tours par minute, résultant en une couche qui fournit une épaisseur supplémentaire de 135 um. </li><li> Retirer la plaquette et cuire sur une plaque de cuisson à 135 ° C pendant 30 min, puis refroidir à température ambiante avant de passer à l&#39;étape suivante. </li><li> Aligner le 2 ème masque de calque (figure 2B) sur la géométrie à motifs en 1.3 et exposer la plaquette revêtu à une collimaté 190 mW, 365 nm LED pendant 3 min. Après l&#39;exposition, placer sur une plaque de cuisson à 135 ° C pendant 1 min, puis refroidir à température ambiante avant de procéder à la prochaine étape. </li><li> Développer les masques par immersion dans un bain sous agitation de propylèneglycol acétate d&#39;éther monométhylique de 30 min. Laver la plaquettedans de l&#39;isopropanol et de cuisson sur une plaque chauffante à 135 ° C pendant 1 min. Placez le maître développé dans un plat 100 mm Petri pour PDMS moulage. </li></ol> 2. PDMS de fabrication du dispositif <ol><li> Préparation 10: 1 PDMS en combinant 50 g de base de silicone avec 5 g d&#39;agent de durcissement dans un gobelet en plastique. Mélanger le contenu avec un outil rotatif équipé d&#39;un bâtonnet. Dégazer le mélange dans un dessiccateur pendant 30 minutes, ou jusqu&#39;à ce que toutes les bulles d&#39;air sont éliminées. </li><li> Verser les PDMS pour donner une épaisseur de 3 mm sur le maître et remettez-le dans le dessiccateur pour plus de dégazage. Une fois que toutes les bulles sont enlevées, cuire le dispositif à 60 ° C pendant 2 heures. </li><li> Couper l&#39;appareil du moule à l&#39;aide d&#39;un scalpel et le placer sur une surface propre avec le côté à motifs en place. Couper le moule PDMS en deux avec une lame de rasoir pour séparer Master 1 de Master 2 (Figure 3a). Sur la pièce contenant la géométrie de manutention de 50 um de fluide imprimée par Master 1, poinçonner les entrées et les sorties fluidiques avec un 0,75 mm de punch biopsie. </li><li> Plasma traiter les appareils à 1 mbar plasma O 2 pendant 60 secondes dans un nettoyeur à plasma de 300 W. Mouiller la surface de la pièce unpunched de PDMS avec une goutte d&#39;eau DI pour retarder temporairement la liaison PDMS-PDMS et servir comme un lubrifiant. Tout en regardant dans un microscope stéréo, lieu Master 1 Master 2 sur la surface et faites glisser les surfaces relativement jusqu&#39;à un verrouillage mécanique est atteint lorsque les cadres encastrés et cadres en saillie dans la figure 3A compagnon. </li><li> Placer le dispositif dans un four à 60 ° C et cuire le dispositif assemblé (figure 3B) pendant deux jours à 60 ° C pour évaporer l&#39;eau et le collage complet. </li></ol> 3. Préparation des réactifs <ol><li> Remplissez seringue de 1 ml avec de l&#39;eau distillée pour la phase interne. </li><li> Remplissez seringue de 1 ml avec HFE 7500 huile fluorée avec 1 poids. % De tensioactif biocompatible tensioactif 13 pour la phase intermédiaire. </li><li> Remplir la seringue de 10 ml avec 10 poids. % Polyéthylènee glycol (PEG) dans une solution aqueuse contenant 1 en poids. % De Tween 20 et 1 en poids. % De dodécylsulfate de sodium à la phase continue. </li></ol> 4. Preparation System <ol><li> Placer la puce microfluidique sur la platine d&#39;un microscope inversé couplé à une caméra numérique capable de &lt;100 us vitesses d&#39;obturation. </li><li> Montez toutes les seringues sur les pompes à seringues et fixer 27 g aiguilles. Fixez ~ 30 cm de longueur de tube PE-2 sur les aiguilles et insérer les bouts dans le appropriée de poing des trous dans le dispositif. </li><li> Insérer une longueur de 10 cm de PE-2 dans l&#39;orifice de sortie du dispositif et placer l&#39;autre extrémité dans un récipient de collecte des déchets. </li><li> Premier dispositif en exécutant les pompes à seringues à des taux élevés de vitesses (2000 pi / min) jusqu&#39;à ce liquide dans les segments de tube atteint les ports du dispositif d&#39;entrée. </li></ol> 5. Génération Emulsion <ol><li> Concentrer le microscope sur une région qui contient le 50 um x 50 um et l&#39;orificeaval canal de sortie. </li><li> Définissez les pompes de la seringue pour délivrer un fluide à la double générateur d&#39;émulsion à des taux de 250 pi / h pour la phase interne, 100 pl / h pour la phase intermédiaire, et 700 pi / h pour la phase continue débit et attendre 10 minutes pour atteindre l&#39;équilibre. </li><li> Maintenir les taux des phases interne et moyenne écoulement à 250 pi / h et 100 pi / h, respectivement. Fixer le taux de la phase externe de flux à 1050 pi / h. Attendez 3-5 min pour le double génération des émulsions pour stabiliser sous cet ensemble de conditions d&#39;écoulement. </li><li> Acquérir 5 secondes d&#39;images vidéo à 30 Hz pour le traitement hors ligne via une analyse manuelle d&#39;image. </li><li> Répéter 5,3 et 5,4 avec les débits indiqués dans le tableau 1. Les débits de phase interne et intermédiaire sont maintenues constantes et la vitesse d&#39;écoulement de phase de porteuse est variée en ajustant le réglage de la pompe à seringue. </li></ol>

<ol>
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The authors have nothing to disclose.

La géométrie émulsion double générateur décrit ici est conçu pour imiter la physique des dispositifs de capillaires en verre 8. Dans ceux-ci, des capillaires en verre cylindriques alignées sont utilisés pour créer un jet coaxial à trois phases qui est cisaillé en doubles gouttelettes d&#39;émulsion uniforme. La fonction de notre dispositif 3D PDMS dépend de l&#39;alignement central de petits traits formés avec 50 um de hauteur avec les chaînes de fabrication de la phase porteuse qui sont 320 u...

<html> Émulsions doubles se composent de gouttelettes séparées d&#39;une phase de support par une couche de fluide intermédiaire, non miscible, et sont d&#39;un intérêt particulier en raison de leurs utilisations potentielles dans les secteurs industriel, pharmaceutique, et les applications biologiques 1. Dans certains cas, la capacité d&#39;encapsuler des composés à haute valeur dans le noyau permet une émulsion double matière à protéger et libérée de manière contrôlée. Par exemple, les médicaments peuvent être encapsulés dans des conditions de solubilité pas appropriées pour le support fluide externe 2. En outre, la couche huileuse intermédiaire peut être utilisé comme modèle de la capsule pour l&#39;encapsulation et la libération de médicaments, les cosmétiques et les nutriments 3. En biologie, émulsions doubles sont également utiles dans le criblage à haut débit, car ils permettent un nombre massif d&#39;expériences sous-nanolitre à effectuer, alors détecté et triée en utilisant une cellule activé par fluorescence (FACS) 4,5 instrument. ent &quot;&gt; La conception d&#39;émulsions doubles avec des caractéristiques de performance souhaités nécessite un contrôle précis de la double la taille de l&#39;émulsion, la composition et l&#39;uniformité. Bien que les processus d&#39;émulsification en vrac, tels que l&#39;émulsification de la membrane, sont utilisés dans l&#39;industrie, les émulsions résultant sont très polydisperse, présentant une grande variété de propriétés fonctionnelles 1. Le champ des gouttelettes microfluidique est naturellement adapté la génération d&#39;émulsions monodisperses avec la composition soigneusement contrôlé 6. microfluidique doublera la production d&#39;émulsion a été réalisé avec deux stratégies principales, ce qui rend de baisse séquentielle et de flux capillaire en verre de focalisation. émulsions doubles peuvent être générés dans des dispositifs PDMS planes en utilisant une goutte à deux étapes de décision. Tout d&#39;abord, aqueux-dans-huile sont créés en utilisant une émulsion eau-dans-huile région d&#39;un dispositif avec des parois de canal hydrophobes faisant goutte. Ensuite, l&#39;émulsion peut être coulé ou réinjecté dans une région faisant goutte-à parois hydrophiles adaptés à huile dans l&#39;eaudéposez-faire 4. Cependant, le traitement de surface hydrophile du PMDS nécessite une étape de fabrication supplémentaire et il est souvent temporaire 7. La méthode la plus contrôlable et reproductible à former des émulsions doubles est en écoulement co-axial de focalisation, en utilisant une technique pionnier microfluidique capillaire en verre, grâce à quoi un jet concentrique contenant les trois phases est cisaillé à travers un petit orifice pour produire des gouttelettes monodispersées 8. Cette technique permet la production de gouttelettes beaucoup plus petites que les dimensions de canal, avec la taille et la composition précise de la double émulsion étant une fonction des débits de chaque phase de débit. La grande différence entre les gouttelettes et la taille du canal et l&#39;écoulement de la gaine externe de protection empêche les gouttelettes d&#39;entrer en contact les parois des canaux, ce qui rend inutile le traitement de surface. Cependant, de tels dispositifs de verre exigent la fabrication sur mesure de conseils capillaires coniques, avec un montage soigné et étanchéité. Enquêteurs précédents ont utilisé litho douce 3Dgraphie de générer des émulsions doubles en utilisant le flux concentrant physique, mais ces dispositifs produit des émulsions avec des diamètres&gt; 150 um 9,10, soit environ un ordre de grandeur plus grand que les objets généralement triées avec FACS. Une alternative intéressante serait inclure la fonctionnalité robuste et petite génération de gouttelettes d&#39;écoulement coaxial capillaire en verre se concentrant à la facilité de fabrication des PDMS lithographie douce. Dans cet article, nous décrivons un générateur de double émulsion qui utilise les flux de co-axial se concentrant pour produire ≤ 50 um émulsions et est construit en utilisant entièrement 3D lithographie douce 11. Notre dispositif utilise une approche à clapet pour fabriquer des dispositifs qui comprend un petit canal de cisaillement (Figure 1) de rapprocher les processus de formation de l&#39;émulsion dans une buse capillaire de verre tiré. Plus important encore, ces dispositifs nécessitent aucun traitement de surface spécifique, et la construction tout polymère fournit facile et reproductible fabrication scalable à un grand nombre de périphériques en double. Ici, nous décrivons la conception, la fabrication et les tests du générateur double émulsion. Double génération d&#39;émulsion se révèle être robuste et reproductible jusqu&#39;à des diamètres de gouttelettes de 14 um. Le couplage de la fonctionnalité avec la facilité de fabrication rend ce dispositif une option attrayante pour le développement de nouvelles applications doubles d&#39;émulsion.

<table border="0" cellpadding="0" cellspacing="0" width="691">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Photomasks</td>
			<td style="width:135px;">CadArt Servcies</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">3&#34; silicon wafers, P type, virgin test grade</td>
			<td style="width:135px;">University Wafers</td>
			<td style="width:119px;">447</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">SU-8 3035</td>
			<td style="width:135px;">Microchem</td>
			<td style="width:119px;">Y311074</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">SU-8 2050</td>
			<td style="width:135px;">Microchem</td>
			<td style="width:119px;">Y111072</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Sylgard 184 silicone elastomer kit</td>
			<td style="width:135px;">Krayden</td>
			<td style="width:119px;">4019862</td>
			<td></td>
		</tr>
		<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>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">10 ml syringes</td>
			<td style="width:135px;">BD</td>
			<td style="width:119px;">309604</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">27 gaugue needles</td>
			<td style="width:135px;">BD</td>
			<td style="width:119px;">305109</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">PE 2 polyethylene tubing</td>
			<td style="width:135px;">Scientific Commodities, Inc.</td>
			<td style="width:119px;">B31695-PE/2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Novec 7500</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:119px;">98-0212-2928-5</td>
			<td>Commonly knowns as HFE 7500</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">Biocompatable surfactant</td>
			<td style="width:135px;">Ran Biotechnologies</td>
			<td style="width:119px;">008-FluoroSurfactant</td>
			<td></td>
		</tr>
		<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>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Tween 20</td>
			<td style="width:135px;">Sigma Aldrich</td>
			<td style="width:119px;">P1369</td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Le générateur double émulsion se compose d&#39;un dispositif de co-axiale flux concentrant créé en utilisant 3D PDMS fabrication (figure 1A). La géométrie permet que la formation d&#39;une co-axiale jet en trois phases pour être cisaillé dans un carré, 50 um x 50 um orifice, permettant la formation d&#39;eau / huile / eau émulsions doubles (figure 1B, 1C Figure). La phase aqueuse interne et la phase d&#39;huile de milieu sont ...

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.

Double Emulsion génération utilisant un flux focus périphérique (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 vues.  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.

Vidéo: 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.

A Novel tridimensionnelle chambre d&#39;écoulement périphérique pour étudier chimiokine dirigée extravasation des cellules circulantes des conditions d&#39;écoulement physiologiques

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

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Bio-ingénierie

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.

Stretching cellules Micropatterned sur une membrane 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.

Bicouche lipidique des vésicules génération utilisant microfluidique jets

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.

Formation automatisée Lipid bicouche Membrane Utilisation d&#39;un Thin Film Polydiméthylsiloxane

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 microcontrôleur exploité le dispositif pour la génération d’extraits liquides d’aérosol de Cigarette fumée et électronique de Cigarette classique

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.

Immobilisation des individus vivants Caenorhabditis elegans en utilisant une puce de microfluidique de polydiméthylsiloxane ultra-mince avec rétention d’eau

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.

Génération de rats transgéniques utilisant une approche vectorielle lentivirale

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.

Génération de conditions environnementales dynamiques à l’aide d’un dispositif microfluidique à haut débit

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.

Estimation de la pression assistée par sous-harmonique améliorée par contraste (SHAPE) à l’aide de l’imagerie par ultrasons en mettant l’accent sur l’identification de l’hypertension portale

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