Questo lavoro è stato sostenuto da un premio di ricerca del California Institute for Quantitative Biosciences (QB3), il Bridging the Gap Award dalla Fondazione Famiglia Rogers, il / Sandler Foundation Program UCSF per Breakthrough Ricerca Biomedica, una borsa di studio dalla BASF, e la NSF attraverso il Programma di Facoltà anticipo di sviluppo di carriera (CARRIERA) (DBI-1.253.293).

1. SU8 Maestro Fabrication <ol><li> Progettare le strutture microfluidica per due fabbricazione strato utilizzando il software AutoCAD e avere i disegni stampati da un fornitore su pellicola circuito con risoluzione di 10 micron. I dettagli del progetto del dispositivo sono riportati in un riferimento collegato 11 e le geometrie dei canali nella Figura 1. Gli strati devono includere segni di allineamento per aiutare colloca caratteristiche da ogni strato fabbricazione 12. </li><li> Posizionare un 3 pollici wafer di silicio del diametro di pre-pulizia su un dispositivo a induzione giro e accendere il vuoto di apporre al mandrino. Applicare 1 ml di SU8-3035 al centro della fetta e centrifugare per 20 sec a 500 rpm, quindi 30 secondi a 2000 rpm, fornendo uno spessore di 50 micron. </li><li> Rimuovere il wafer e cuocere su una piastra riscaldante a 135 ° C per 30 min. Lasciare raffreddare il wafer di RT prima di passare alla fase successiva. </li><li> Esporre il wafer rivestito al 1 maschera di livello (Figura 2A </strong&gt;) sotto un collimato 190 mW, 365 nm LED per 90 secondi. Dopo l&#39;esposizione, posizionare il wafer su una piastra 135 ° C per 1 minuto, poi raffreddare a RT prima di procedere alla fase successiva. </li><li> Posizionare la cialda sul dispositivo a induzione di rotazione e accendere il vuoto di apporre al mandrino. Applicare 1 ml di SU8-2050 al centro della fetta e centrifugare per 20 sec a 500 rpm, quindi 30 secondi a 1.375 rpm, risultante in uno strato che fornisce uno spessore di 135 micron aggiuntivo. </li><li> Rimuovere il wafer e cuocere su una piastra 135 ° C per 30 minuti, poi raffreddare per RT prima di passare alla fase successiva. </li><li> Allineare la maschera di livello 2 nd (Figura 2B) sulla geometria modellata in 1.3 ed esporre il wafer rivestito per un collimato 190 mW, 365 nm LED per 3 min. Dopo l&#39;esposizione, posto su una piastra 135 ° C per 1 minuto, poi raffreddare per RT prima di procedere alla fase successiva. </li><li> Sviluppare le maschere immergendo in un bagno agitata di propilenglicole monometiletere acetato per 30 min. Lavare il waferin isopropanolo e cuocere su una piastra 135 ° C per 1 min. Posizionare il maestro sviluppato in una capsula di Petri 100 mm per lo stampaggio PDMS. </li></ol> 2. PDMS di fabbricazione del dispositivo <ol><li> Preparare 10: 1 PDMS combinando 50 g di base siliconica con 5 g di agente indurente in una tazza di plastica. Mescolare il contenuto con un utensile rotante dotato di un bastoncino. Degassare la miscela all&#39;interno di un essiccatore per 30 minuti, o fino a quando tutte le bolle d&#39;aria vengono rimosse. </li><li> Versare il PDMS per dare uno spessore di 3 mm sopra il maestro e il luogo di nuovo nel essiccatore per ulteriore degasaggio. Una volta che tutte le bolle vengono rimossi, cuocere il dispositivo a 60 ° C per 2 ore. </li><li> Tagliare il dispositivo dallo stampo con un bisturi e posto su una superficie pulita con il lato modellato su. Tagliare lo stampo PDMS a metà con una lama di rasoio per separare Master 1 da Master 2 (Figura 3a). Sul pezzo che contiene la gestione geometria 50 um fluido impresso dal Maestro 1, pugno gli ingressi e le uscite fluidici con 0,75 mm biopsia. </li><li> Plasma trattare i dispositivi a 1 mbar O 2 al plasma per 60 secondi in un pulitore di plasma di 300 W. Bagnare la superficie del pezzo unpunched di PDMS con una goccia di acqua deionizzata a ritardare temporaneamente PDMS-PDMS legame e servire come lubrificante. Durante la visualizzazione attraverso un microscopio stereo, posto Master 1 Master 2 sulla superficie e far scorrere le superfici relativamente fino ad ottenere un blocco meccanico quando i telai da incasso e le cornici sporgenti in figura 3A compagno. </li><li> Posizionare il dispositivo in un forno a 60 ° C e cuocere il dispositivo assemblato (Figura 3B) per due giorni a 60 ° C per evaporare l&#39;acqua e completo incollaggio. </li></ol> 3. Preparazione dei reagenti <ol><li> Riempire siringa da 1 ml con acqua distillata per la fase interna. </li><li> Riempire siringa da 1 ml con HFE 7500 olio fluorurato con 1 peso. % Tensioattivo biocompatibile tensioattivo 13 per fase centrale. </li><li> Riempire 10 ml siringa con 10 WT. % Polietilenee glicole (PEG) in soluzione acquosa contenente 1 wt. % Di Tween 20 e 1 in peso. % Sodio dodecil solfato per la fase continua. </li></ol> 4. Sistema Preparazione <ol><li> Posizionare il chip microfluidico sul palcoscenico di un microscopio invertito accoppiato con una fotocamera digitale in grado di &lt;100 msec tempi di posa. </li><li> Montare tutte le siringhe sulle pompe siringa e collegare 27 aghi G. Attaccare ~ 30 cm di lunghezza di PE-2 tubi sugli aghi e inserire le estremità nell&#39;appropriata perforato fori nel dispositivo. </li><li> Inserire una lunghezza 10 cm di PE-2 alla porta di uscita del dispositivo e posizionare l&#39;altra estremità in un contenitore di raccolta dei rifiuti. </li><li> Prime dispositivo eseguendo le pompe a siringa ad alte percentuali di velocità (2,000 ml / min) fino fluido nei segmenti tubi raggiunge le luci di entrata del dispositivo. </li></ol> 5. Emulsione Generation <ol><li> Fuoco il microscopio su una regione che contiene il 50 um x 50 um orifizio e lacanale di uscita a valle. </li><li> Impostare le pompe a siringa per fornire fluido al doppio generatore di emulsione a portate di 250 ml / h per la fase interna, 100 ml / h per la fase intermedia, e 700 ml / h per la fase continua e attendere 10 minuti per il raggiungimento dell&#39;equilibrio. </li><li> Mantenere le portate delle fasi interni e centrali a 250 microlitri / hr e 100 microlitri / h, rispettivamente. Impostare la portata della fase esterna a 1.050 ml / hr. Attendere 3-5 minuti per il doppio generazione emulsioni per stabilizzare sotto questo insieme di condizioni di flusso. </li><li> Acquisire 5 sec di immagini video a 30 Hz per l&#39;elaborazione offline tramite analisi di immagine manuale. </li><li> Ripetere 5.3 e 5.4 con le portate indicate nella tabella 1. Le portate fase interni e centrali rimangono costanti e la portata fase della portante viene variata regolando l&#39;impostazione della pompa a siringa. </li></ol>

<ol>
	<li>Van Der Graaf, S., Schro&#235;n, C. G. P. H., Boom, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+double+emulsions+by+membrane+emulsification+-+A+review.">Preparation of double emulsions by membrane emulsification - A review.</a> J. Membrane Sci. 251 (1-2), 7-15 (2005).</li><li>Laugel, C., Baillet, A. P., Youenang Piemi, M., Marty, J., Ferrier, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oil-water-oil+multiple+emulsions+for+prolonged+delivery+of+hydrocortisone+after+topical+application:+comparison+with+simple+emulsions.">Oil-water-oil multiple emulsions for prolonged delivery of hydrocortisone after topical application: comparison with simple emulsions.</a> Int. J. Pharm. 160 (1), 109-117 (1998).</li><li>Kim, S. H., Kim, J. W., Cho, J. C., Weitz, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double-emulsion+drops+with+ultra-thin+shells+for+capsule+templates.">Double-emulsion drops with ultra-thin shells for capsule templates.</a> Lab Chip. 11 (18), 3162-3166 (2011).</li><li>Lim, S. W., Abate, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrahigh-throughput+sorting+of+microfluidic+drops+with+flow+cytometry.">Ultrahigh-throughput sorting of microfluidic drops with flow cytometry.</a> Lab Chip. 13 (23), 4563-4572 (2013).</li><li>Bernath, K., Hai, M., Mastrobattista, E., Griffiths, A. D., Magdassi, S., Tawfik, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+compartmentalization+by+double+emulsions:+sorting+and+gene+enrichment+by+fluorescence+activated+cell+sorting.">In vitro compartmentalization by double emulsions: sorting and gene enrichment by fluorescence activated cell sorting.</a> Anal. Biochem. 325 (1), 151-157 (2004).</li><li>Seemann, R., Brinkmann, M., Pfohl, T., Herminghaus, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Droplet+based+microfluidics.">Droplet based microfluidics.</a> Rep. Prog. Phys. 75 (1), 016601 (2012).</li><li>Bauer, W. A. C., Fischlechner, M., Abell, C., Huck, W. T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrophilic+PDMS+microchannels+for+high-throughput+formation+of+oil-in-water+microdroplets+and+water-in-oil-in-water+double+emulsions.">Hydrophilic PDMS microchannels for high-throughput formation of oil-in-water microdroplets and water-in-oil-in-water double emulsions.</a> Lab Chip. 10 (14), 1814-1819 (2010).</li><li>Utada, A. S., Lorenceau, E., Link, D. R., Kaplan, P. D., Stone, H. A., Weitz, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monodisperse+double+emulsions+generated+from+a+microcapillary+device.">Monodisperse double emulsions generated from a microcapillary device.</a> Science. 308 (5721), 537-541 (2005).</li><li>Chang, F. C., Su, Y. 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The authors have nothing to disclose.

La doppia geometria di generazione emulsione qui descritto è stato progettato per simulare la fisica dei dispositivi capillare vetro 8. In questi, capillari di vetro cilindriche allineati vengono usati per creare un getto coassiale trifase che viene tranciata in uniformi goccioline doppia emulsione. La funzione del nostro dispositivo 3D PDMS dipende l&#39;allineamento centrale piccole caratteristiche formate con 50 micron fabbricazione alto con canali di fase portante che sono 320 micron di altezza totale. C...

<html> Emulsioni doppie consistono goccioline separate da una fase portante da uno strato liquido immiscibile intermedio, e sono di particolare interesse per le loro potenziali impieghi industriali, farmaceutici e applicazioni biologiche 1. In alcuni casi, la capacità di incapsulare composti ad alto valore del core doppio dell&#39;emulsione permette materiale da proteggere e rilasciato in modo controllato. Ad esempio, i farmaci possono essere incapsulati in condizioni di solubilità non appropriati per il vettore fluido esterno 2. Inoltre, lo strato di olio intermedio può essere utilizzato come modello per la capsula incapsulamento e la consegna di farmaci, cosmetici e nutrienti 3. In biologia, doppie emulsioni sono anche utili in screening ad alto rendimento in quanto consentono un enorme numero di esperimenti sub-nanolitri da effettuare, quindi rilevato e risolto utilizzando una cella a fluorescenza-attivato (FACS) strumento 4,5. ent &quot;&gt; Il disegno di doppi emulsioni con caratteristiche prestazionali desiderate richiede il controllo preciso della dimensione doppia emulsione, composizione e uniformità. I ​​processi di emulsificazione sfuso, come le membrane emulsificazione, sono utilizzati nell&#39;industria, le emulsioni risultanti sono altamente polidisperso, esibendo un Un&#39;ampia varietà di proprietà funzionali 1. Il campo di goccia microfluidica è naturalmente adatto alla generazione di emulsioni monodisperse con composizione attentamente controllata 6. Microfluidic generazione doppia emulsione è stato raggiunto con due strategie principali, la preparazione di goccia sequenziale e flusso capillare di vetro concentrandosi. emulsioni doppie possono essere generati in dispositivi PDMS planari utilizzando una goccia in due fasi processo. Primo, acquosa-in-olio vengono creati utilizzando un acqua-in-olio regione di un dispositivo con pareti di canale idrofobici fabbricazione goccia. Successivamente, l&#39;emulsione può essere scorreva o reiniettato in una regione-making goccia con pareti idrofili adatti per olio in acquadrop-fare 4. Tuttavia, il trattamento di superficie idrofila di PMDS richiede un ulteriore fase di fabbricazione ed è spesso temporanea 7. Il metodo più controllabile e ripetibile per formare emulsioni doppie è di flusso coassiale focalizzazione, una tecnica sperimentato usando microfluidica capillare di vetro, per cui un getto concentrica contenente le tre fasi viene tagliato attraverso un piccolo orifizio per produrre goccioline monodisperse 8. Questa tecnica permette la produzione di goccioline molto più piccole rispetto alle dimensioni di canale, con la dimensione precisa e composizione del doppio dell&#39;emulsione essendo funzione delle portate di ciascuna fase. La grande differenza tra le dimensioni delle gocce e il canale ed il flusso protettivo guaina esterna impedisce goccioline dal contatto con le pareti del canale, rendendo non necessario il trattamento di superficie. Tuttavia, tali dispositivi richiedono vetro montaggio su ordinazione di estremità dei capillari conici, insieme a un&#39;attenta assemblaggio e sigillatura. Ricercatori precedenti hanno utilizzato 3D litografia sofficegrafia di generare doppi emulsioni utilizzando flusso focalizzazione fisica, ma questi dispositivi prodotta emulsioni con diametri&gt; 150 micron 9,10, circa un ordine di grandezza più grande di oggetti tipicamente ordinati con FACS. Un&#39;alternativa interessante includerebbe la robusta funzionalità e piccola generazione goccia di vetro capillare flusso coassiale messa a fuoco con la facilità di fabbricazione di PDMS morbido litografia. In questo articolo, si descrive un doppio generatore di un&#39;emulsione che utilizza il flusso coassiale di messa a fuoco per produrre ≤ 50 micron emulsioni ed è costruito interamente con 3D litografia soft 11. Il nostro dispositivo utilizza un approccio a conchiglia per fabbricare dispositivi che include un piccolo canale di taglio (figura 1) per approssimare i processi di formazione di emulsione in un ugello capillare di vetro tirato. Ancora più importante, questi dispositivi non richiedono alcun trattamento superficiale specifica, e la costruzione tutto polimero fornisce un facile e ripetibile fabbricazione scalable a un gran numero di dispositivi duplicati. Qui, descriviamo la progettazione, fabbricazione e collaudo del doppio generatore di emulsione. Generazione Doppia emulsione è dimostrato di essere affidabile e ripetibile fino a diametri delle gocce di 14 micron. L&#39;accoppiamento di funzionalità, facilità di fabbricazione rende questo dispositivo un&#39;opzione attraente per sviluppo di nuove applicazioni doppia emulsione.

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	<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<tr height="21">
			<td height="21" style="height:21px;width:271px;">35,000 MW PEG</td>
			<td style="width:135px;">Sigma Aldrich</td>
			<td style="width:119px;">1546660</td>
			<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>
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double emulsion generation using a polydimethylsiloxane (pdms) co-axial flow focus device

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

Il generatore doppia emulsione costituito da un dispositivo di messa a fuoco flusso coassiale creato utilizzando 3D PDMS fabbricazione (Figura 1A). La geometria permette che la formazione di un getto coassiale trifase da tagliare in un quadrato, 50 um x 50 um orifizio, consentendo la formazione di acqua / olio / acqua doppio emulsioni (Figura 1B, Figura 1C). La fase acquosa interna e la fase olio medio sono riuniti in una giunzione con d...

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.

Doppia Emulsione Generazione Utilizzando un Polidimetilsilossano (PDMS) coassiale flusso fuoco Dispositivo

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 ...

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Bioengineering

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

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

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

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

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

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

A Novel tridimensionale del flusso Camera dispositivo per studiare Chemokine-diretto stravaso di cellule circolanti in condizioni di flusso fisiologici

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

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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.

PLGA nanoparticelle formate da singola o doppia emulsione con Vitamina 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 ...

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 celle micropatterned su una membrana 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.

Doppio strato lipidico delle vescicole Generation Uso Microfluidic getto

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.

Formazione automatizzato doppio strato lipidico della membrana Utilizzando un Polidimetilsilossano Thin Film

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

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

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

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

Un microcontrollore azionato il dispositivo per la generazione di estratti liquidi da Aerosol elettronico e fumo di sigaretta sigaretta convenzionale

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.

Immobilizzazione degli individui Live Caenorhabditis elegans utilizzando un Chip microfluidico Polydimethylsiloxane di ultra-sottile con ritenzione idrica

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.

Generazione di ratti transgenici con un approccio al vettore 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.

Generazione di condizioni ambientali dinamiche utilizzando un dispositivo microfluidico ad alta produttività

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.

Stima della pressione subarmonica assistita da contrasto (SHAPE) utilizzando l'imaging a ultrasuoni con particolare attenzione all'identificazione dell'ipertensione portale

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