The authors would like to thank Professor Howard Ceri from the Biological Sciences Department of the University of Calgary for providing bacterial strains. A. Kumar acknowledges support from NSERC. T. Thundat acknowledges financial support from the Canada Excellence Research Chair (CERC) program. The authors would also like to acknowledge help from Ms. Zahra Nikakhtari for help with videography.

Eseguire i protocolli sperimentali qui nell&#39;ordine descritto di seguito. Protocolli di microfabbricazione per la creazione della piattaforma microfluidica sono discussi nel Passo 1 Passo 2 descrive il protocollo di coltura batterica (Figura 2), e punto 3 riguarda il montaggio del setup sperimentale (Figura 3). Infine, la fase sperimentale attuale è descritta al punto 4. 1 Chip Fabrication Procedura NOTA: La corretta procedure di sicurezza devono essere seguite per i processi descritti di seguito. Consultare il responsabile della sicurezza istituzionale per i dettagli. <ol><li> Progettare la maschera con un software appropriato (ad esempio, L-Edit). La progettazione canale è composto da un micro-canale di larghezza di 625 micron. La regione centrale del canale contiene una serie di micro-messaggi di 50 micron di diametro, distanziati di 25 micron a parte (vedi Supplemental Files). </li><li> Stampa questo disegno su vetro (5 &quot;x 5&quot; vetro soda lime), Che ha uno spessore di 0.09 &quot;ed è rivestito da circa 70 nm spesso strato di cromo (Cr), utilizzando mascheratura per preparare una maschera fotografico. Autore Utilizzo AZ400K per 1 min. Poi, etch strato Cr utilizzando Cr mordenzante per circa 1 min Usare acetone per togliere il resistere e pulirlo con soluzione piranha freddo. (H 2 SO 4 e H 2 O 2 in un rapporto di 3: 1). </li><li> Fotolitografia <ol><li> Pulire un wafer da 4 &quot;silicio standard chimicamente con una soluzione piranha per 20 min. </li><li> Sciacquare la cialda con acqua deionizzata e asciugare. </li><li> Riscaldare il wafer su una piastra calda (200 ° C per 15 min). </li><li> Rivestire il wafer di silicio con photoresist. Qui, il fotoresist AZ4620 positivo è stato spin-rivestito su un wafer di silicio a 2.000 rpm per 25 secondi per ottenere uno strato di spessore di 12,5 micron. </li><li> Rimuovere tutto il solvente dalla cottura morbida della fetta su una piastra calda per galleggiamento della fetta per 90 secondi in flusso di azoto a 100 ° C. Poi, tenerlo in vuoto a tegli stessa temperatura per 60 sec. </li><li> Posizionare la cialda in una scatola scura per 24 ore per disidratazione. </li><li> Esporre il wafer alla luce UV per trasferire il pattern progettato per il fotoresist. </li><li> Immergere il wafer in soluzione di sviluppo di fotoresist (AZ400K) per 240 sec. Poi, risciacquare il wafer con alcool isopropilico e asciugare mettendo in corrente di azoto. </li></ol></li><li> ICP-DRIE (plasma accoppiato induttivamente - Deep Reactive Ion Etching) Processo <ol><li> Applicare DRIE incisione. Scegliere la profondità etch appropriata in base alla profondità finale richiesta per il dispositivo (50 micron in questa indagine). Photoresist agisce come uno strato di mascheratura durante questo processo. </li><li> Rimuovere il photoresist rimanente con acetone e pulire la cialda. </li></ol></li><li> PDMS (polidimetilsilossano) Casting <ol><li> Utilizzare trichloromethylsilane (TCMS) per silanizzanti stampo maestro di silicio. Versare 2 o 3 gocce di trichloromethylsilane in un flaconcino e metterlo in un essiccatore accanto alstampo maestro silicio. Consenti 2-3 ore per il processo silanizzante per completare. </li><li> In un contenitore separato, mescolare la base di silicone Sylgard 184 con induritore dal rapporto in peso di 10: 1 per preparare PDMS. Degassarla PDMS sottoponendolo a vuoto condizioni (circa 2 ore). </li><li> Mettete lo stampo maestro di silicio in un supporto. Poi, versare il PDMS sullo stampo maestro di silicio per formare il timbro PDMS. Assicurarsi che le bolle non si formano nei PDMS durante questo processo. </li><li> Curare le PDMS per 2 ore a 80 ° C. </li><li> Staccare il timbro PDMS dallo stampo master. Quindi, tagliare le PDMS bollo in microchip separati. Infine, utilizzare un nucleo di taglio per perforare fori per l&#39;ingresso (s) e di uscita (s). </li></ol></li><li> Incollaggio di PDMS al vetro <ol><li> Esporre il vetrino e PDMS bollo di plasma di ossigeno per 30 sec. Legame PDMS bollo per la polizza di copertura. </li><li> Per ottenere una corretta tenuta tra il timbro PDMS e il vetrino, temprare il dispositivo mettendolo in forno a 70 ° C per 10 min. </li&gt; </ol></li></ol> 2 batterica Cultura NOTA: i protocolli di corretta biosicurezza deve essere seguita per i passaggi 2-4. Consultare il responsabile della sicurezza istituzionale per i dettagli. <ol><li> Preparare piastre LB Agar <ol><li> Aggiungere 20 g di Luria-Bertani (LB) agar (Miller) polveri e 500 ml di acqua ultrapura per un pallone da 1 L. Mescolare per sciogliere la polvere. </li><li> Sterilizzare in autoclave a 15 psi, 121 ° C per 15 min. </li><li> Lasciare raffreddare il pallone fino a 50-55 ° C su una panchina o in un bagno d&#39;acqua nella cappa di biosicurezza. </li><li> Aggiungere l&#39;antibiotico tetraciclina per ottenere una concentrazione finale di 50 mg / ml. Mescolare bene agitando. </li><li> Versare il composto in lastre. Riempire ogni piatto fino a 1/2 - 2/3 pieno. </li><li> Aria Fiamma bolle brevemente per farli scoppiare se fanno. Bolle d&#39;aria solidificati sono difficili da diffondere la cultura batterica sopra. </li><li> Lasciare le piastre a raffreddare a temperatura ambiente per una notte. </li><li> Quando sono freschi,mettere lastre di nuovo nella loro manica, sigillare il sacchetto, etichetta (antibiotico e data), e conservare a 4 ° C. NOTA: Coprire lo stock di piastre con carta stagnola, come la luce disattiva molti antibiotici. </li></ol></li><li> LB Broth Preparazione <ol><li> Aggiungere 20 g Luria-Bertani (LB) brodo (Miller) polveri e 1 L di acqua ultrapura in un pallone. Mescolare per sciogliere la polvere. </li><li> Sterilizzare in autoclave a 15 psi, 121 ° C per 15 min. </li><li> Lasciare pallone raffreddare a 50-55 ° C su una panchina o in un bagno d&#39;acqua nella cappa di biosicurezza. </li><li> Aggiungere l&#39;antibiotico tetraciclina per ottenere una concentrazione finale di 50 mg / ml. Mescolare bene agitando. </li><li> Lasciar raffreddare, posizionare la bottiglia etichettata a 4 ° C. NOTA: Coprire la bottiglia con carta stagnola, come la luce disattiva molti antibiotici. </li></ol></li><li> Coltura dei batteri su una piastra di LB Agar (Questo protocollo utilizza Pseudomonas fluorescens) <ol><li> Prendete il magazzino batterica dal congelatore (-80 °, C) e posizionarlo sul ghiaccio. </li><li> Posizionare l&#39;° C magazzino batterica -80 e un piatto di agar LB all&#39;interno di una cappa di biosicurezza. </li><li> Streak il ceppo batterico su una piastra di agar LB a zig-zag. Coprire la piastra di agar e incubare a 30 ° C durante la notte. Infine, riporre il piatto in frigorifero a 4 ° C. </li></ol></li><li> Preparare la soluzione batterica (S1) <ol><li> Versare 50 ml di brodo LB supporto a un pallone autoclavato. Eseguire questa operazione all&#39;interno di una cappa di biosicurezza. </li><li> Trasferire una singola colonia batterica dalla piastra di agar LB al pallone. Questa operazione deve essere eseguita all&#39;interno di una cappa di biosicurezza. </li><li> Mettere il pallone in un incubatore shaker a 30 ° C e 150 rpm per un tempo adeguato (4 ore). </li></ol></li><li> Preparare la soluzione batterica Diluire (S2) <ol><li> Versare 5 ml di LB supporti brodo in un tubo di plastica sterilizzata. </li><li> Diluire S1 mescolando con mezzi brodo LB. Quindi, agitare la soluzione. Diluire la soluzione raggiungere l&#39;ottica desiderataAl densità (OD misurata a 600 nm = 0,1). NOTA: esperimenti Biofilm tipicamente impiegano valori di OD in questa zona. </li></ol></li></ol> 3 Preparare il setup sperimentale <ol><li> Utilizzando le pinzette collegano tubi di plastica flessibili (0.20 &quot;ID) nella presa (s) e di uscita (s) del microchip. Gli ingressi e le uscite sono state precedentemente perforati nella porzione PDMS del microchip (fase 1.5.5). In questa indagine , il microchip è costituito da due ingressi ed una uscita. </li><li> Riempire la siringa (s) con la soluzione batterica (soluzione S2) e rimuovere tutte le bolle nella siringa (s). </li><li> Collegare punta della siringa (s) (30 G 0.5 &quot;ago smussato) in tubo di ingresso (s). Quindi, collegare il tubo di uscita (s) per i rifiuti del contenitore. </li></ol> 4 Esecuzione dell&#39;esperimento <ol><li> Collegare la siringa (s) per la punta della siringa (s). </li><li> Luogo e siringa fix (s) sulla pompa a siringa. Poi mettere il microchip sotto un microscopio ottico con lenti dell&#39;obiettivo di Desired ingrandimento (ad esempio, 40X). Coprire il microchip con un dispositivo di camera di cellule vive per mantenere un ambiente a temperatura costante per la crescita batterica (30 ° C per P. fluorescens). </li><li> Impostare la pompa al livello di portata desiderata (per esempio 10 ml / ora) ed avviare il pompaggio del fluido. </li><li> Una volta che i batteri vengono introdotti nella camera, è anche avviata la formazione di biofilm. Formazione di biofilm e maturazione si verificano in genere in un periodo di diverse ore o addirittura giorni. Osservare e prendere le immagini della crescita di biofilm attraverso il microscopio. </li></ol>

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The authors have nothing to disclose.

Abbiamo dimostrato un semplice dispositivo di microfluidica che imita mezzi porosi per lo studio dello sviluppo del biofilm in ambienti complessi. Ci sono diversi passaggi critici che determinano l&#39;esito degli esperimenti. Essi comprendono la geometria del dispositivo. Mentre la geometria del palo può variare, adeguato poro-spazio per bandierine per formare è necessario. Inoltre, Valiei et al. 1 hanno dimostrato che la formazione streamer si verifica solo in un determinato campo di portata. A p...

Recentemente, abbiamo dimostrato la dinamica di formazione di biofilm batterici in un dispositivo di microfluidica che imita mezzi porosi 1. Biofilm batterici sono essenzialmente colonie di batteri di superficie aggregati che sono racchiusi da sostanze polimeriche extracellulari (EPS) 2-4. Questi film sottili di batteri possono formare in quasi ogni nicchia immaginabile che vanno da superfici lisce all&#39;habitat molto più complesso di mezzi porosi. Valiei et al. 1 usato un dispositivo microfluidico con una matrice di micro-pilastro per simulare una struttura di supporto porosa e studiato la formazione di biofilm in questo dispositivo in funzione della portata del fluido. Essi hanno scoperto che in un certo regime di flusso, biofilm filamentosi noti come stelle filanti hanno cominciato ad emergere tra i diversi pilastri. Streamers possono essere legati ad una o entrambe le estremità per superfici solide, ma il resto della struttura è sospesa in un liquido. Formazione Streamer inizia in genere dopo un primo strato di biofilm ha formato e il suo formatoione può dettare l&#39;evoluzione a lungo termine di biofilm in tali habitat complessi. Recentemente, alcuni ricercatori hanno studiato le dinamiche di formazione streamer. Yazdi et al. 5 ha mostrato che le fiamme possono formare nei flussi vorticosi provenienti da una bolla oscillante. In un altro esperimento, Rusconi et al. 6 studiato l&#39;effetto di curvatura del canale e della geometria del canale sulla formazione di stelle filanti. Essi hanno scoperto che le fiamme possono formare nelle sezioni curve di microcanali, e streamer morfologia è correlata alla motilità. Recenti ricerche hanno dimostrato che le fiamme possono avere ampie ripercussioni in vari scenari naturali e artificiali in quanto possono fungere da precursori della formazione di strutture maturi in interfacce porosi, portare a una rapida e catastrofica proliferazione di biofilm in sistemi biomedici, e anche causare notevoli flow interazioni struttura, ecc 1,7-9. Filanti Biofilm spesso formano ihabitat n complesse come mezzi porosi. Crescita biofilm intesa in ambiente mezzi porosi è rilevante per diversi processi ambientali e industriali come il trattamento biologico delle acque reflue 10, mantenendo ben-bore integrità in situazioni come la cattura di CO 2 11 e collegare dei pori nel terreno 12. Osservando la formazione di biofilm in tali habitat complessi spesso può essere difficile a causa della opacità dei mezzi porosi. In tali situazioni, le piattaforme multimediali porosi microfluidica base possono rivelarsi estremamente vantaggiosa in quanto consentono in tempo reale e monitoraggio in situ. Un altro vantaggio della microfluidica è la possibilità di costruire più bioreattori su un&#39;unica piattaforma bio-microfluidica e contemporaneamente consentire il monitoraggio e / o integrazione di sensori on-line. La flessibilità per implementare più esperimenti di laboratorio in un unico dispositivo e la capacità di raccogliere importanti dati pertinenti per l&#39;analisi statistica accurata è un adv importanteantage di sistemi microfluidici 13,14. Nel contesto della discussione di cui sopra, le dinamiche di formazione streamer comprensione in un ambiente mezzi porosi sarebbe utile per diverse applicazioni. In questo studio, sviluppiamo il protocollo per indagare la formazione streamer in un dispositivo che imita mezzi porosi. Realizzazione della piattaforma microfluidica, sono descritti i passi necessari per la cultura e la sperimentazione di cellule. Nei nostri esperimenti, il ceppo batterico tipo selvaggio di Pseudomonas fluorescens è stato impiegato. P. fluorescens, che si trova naturalmente nel suolo, svolge un ruolo chiave nel mantenimento ecologia del suolo 15. Il ceppo batterico impiegato era stato geneticamente per esprimere la proteina fluorescente verde (GFP) costitutivamente.

<table border="0" cellpadding="0" cellspacing="0" width="1038">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">Name of Material/ Equipment</td>
			<td style="width:255px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:339px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">Flourescent Microscope</td>
			<td style="width:255px;">Nikon</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">LB agar</td>
			<td style="width:255px;">Fisher</td>
			<td style="width:119px;">BP1425-500</td>
			<td>suspend 40 g in 1 L of purified water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">LB broth</td>
			<td style="width:255px;">Fisher</td>
			<td style="width:119px;">BP1427-500</td>
			<td>suspend 20 g in 1 L of purified water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">Biosafety hood</td>
			<td style="width:255px;">Microzone corporation</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">Petri-dish</td>
			<td style="width:255px;">Fisher</td>
			<td style="width:119px;">875712</td>
			<td>sterile 100mmx15mm polystyrene petri dish</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:325px;">Incubator shaker</td>
			<td>New Brunswick Scientific</td>
			<td style="width:119px;"></td>
			<td style="width:339px;">Excella E24incubator shaker series</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 mL sterilized centrifuge tube</td>
			<td style="width:255px;">Corning</td>
			<td style="width:119px;">430828</td>
			<td>Polypropylene Rnase-/Dnase-free</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">Tetracycline free base</td>
			<td style="width:255px;">MP Biomedicals</td>
			<td style="width:119px;">103012</td>
			<td>50 ug/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">SYLGARD 184 silicone</td>
			<td style="width:255px;">Dow Corning Corporation</td>
			<td style="width:119px;">68037-59-2</td>
			<td>Elastomer Base and curing agent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">Positive photoresist (AZ4620)</td>
			<td style="width:255px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">Plastic tube</td>
			<td style="width:255px;">Cole- Parmer</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

protocol for biofilm streamer formation in a microfluidic device with micro-pillars

Diverse specie batteriche possiedono la capacità di attaccare alle superfici e colonizzare in forma di film sottili chiamati biofilm. I biofilm che crescono in mezzi porosi sono rilevanti per diversi processi industriali e ambientali come il trattamento delle acque reflue e di CO 2 sequestro. Abbiamo usato Pseudomonas fluorescens, un batterio aerobico Gram-negativi, per indagare la formazione di biofilm in un dispositivo di microfluidica che imita mezzi porosi. Il dispositivo di microfluidica è costituito da una serie di micro-posti, che sono stati fabbricati utilizzando soft-litografia. In seguito, la formazione di biofilm in questi dispositivi a flusso è stato studiato e ci dimostrano la formazione di biofilm filamentosi note come stelle filanti nel nostro dispositivo. Qui vengono fornite insieme ai protocolli di coltura batterica I protocolli dettagliati per la fabbricazione e l&#39;assemblaggio del dispositivo di microfluidica. Le procedure dettagliate per la sperimentazione con il dispositivo di microfluidica sono anche presentati insieme con il rappresentanterisultati.

Utilizzando il protocollo microfabbricazione sopra menzionato, un dispositivo microfluidico basato PDMS è stato costruito. Figura 1 mostra il microscopio elettronico a scansione (SEM) immagini del PDMS dispositivo. Figura 1a mostra la sezione di ingresso del dispositivo. Un ingresso a forcella è stato creato per equalizzare la testa di pressione attraverso il dispositivo. Ulteriori immagini SEM ha anche mostrato che le pareti pilastro sono quasi verticale (Figura 1b)....

Watch this Scientific Journal Video about Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars at JoVE.com

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Protocols for the study of biofilm formation in a microfluidic device that mimics porous media are discussed. The microfluidic device consists of an array of micro-pillars and biofilm formation by Pseudomonas fluorescens in this device is investigated.

Protocollo per il Biofilm Streamer formazione in un dispositivo a microfluidi con Micro-pilastri

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in ...

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Research

JoVE Journal

Bioengineering

12.1K Views.  University of Alberta. Protocols for the study of biofilm formation in a microfluidic device that mimics porous media are discussed. The microfluidic device consists of an array of micro-pillars and biofilm formation by Pseudomonas fluorescens in this device is investigated. 

Video: Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in vitro. Nutrients delivered by vasculature fail to reach all parts of tumors, giving rise to heterogeneous microenvironments consisting of viable, quiescent and necrotic cell types. Many cancer drugs fail to effectively penetrate and treat all types of cells because of this heterogeneity. Monolayers of cancer cells do not mimic this heterogeneity, making it difficult to test cancer drugs with a suitable in vitro model. Our microfluidic devices were fabricated out of PDMS using soft lithography. Multicellular tumor spheroids, formed by the hanging drop method, were inserted and constrained into rectangular chambers on the device and maintained with continuous medium perfusion on one side. The rectangular shape of chambers on the device created linear gradients within tissue. Fluorescent stains were used to quantify the variability in apoptosis within tissue. Tumors on the device were treated with the fluorescent chemotherapeutic drug doxorubicin, time-lapse microscopy was used to monitor its diffusion into tissue, and the effective diffusion coefficient was estimated. The hanging drop method allowed quick formation of uniform spheroids from several cancer cell lines. The device enabled growth of spheroids for up to 3 days. Cells in proximity of flowing medium were minimally apoptotic and those far from the channel were more apoptotic, thereby accurately mimicking regions in tumors adjacent to blood vessels. The estimated value of the doxorubicin diffusion coefficient agreed with a previously reported value in human breast cancer. Because the penetration and retention of drugs in solid tumors affects their efficacy, we believe that this device is an important tool in understanding the behavior of drugs, and developing new cancer therapeutics.

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We present the procedure for fabrication and operation of a microfluidic device that recreates heterogeneous tumor microenvironments in vitro. The variability in apoptosis within tumor tissue was quantified using fluorescent stains and the effective diffusion coefficient of the chemotherapeutic drug doxorubicin into tumor tissue was evaluated.

Dispositivo Microfluidic per ricreare un microambiente tumorale<em&gt; In Vitro</em

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in ...

Ricerca

Bioingegneria

A Microfluidic Device for Studying Multiple Distinct Strains

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a comparative manner. Multiwell plates are routinely used to compare many different strains or cell lines, but allow limited control over the environment dynamics. Microfluidic devices, on the other hand, allow exquisite dynamic control over the surrounding conditions, but it is challenging to image and distinguish more than a few strains in them. Here we describe a method to easily and rapidly manufacture a microfluidic device capable of applying dynamically changing conditions to multiple distinct yeast strains in one channel. The device is designed and manufactured by simple means without the need for soft lithography. It is composed of a Y-shaped flow channel attached to a second layer harboring microwells. The strains are placed in separate microwells, and imaged under the exact same dynamic conditions. We demonstrate the use of the device for measuring protein localization responses to pulses of nutrient changes in different yeast strains.

We present a simple method to produce microfluidic devices capable of applying similar dynamic conditions to multiple distinct strains, without the need for a clean room or soft lithography.

Un dispositivo a microfluidi per lo studio più ceppi distinti

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a ...

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and used to dictate the shape as well as the diameter of a core stream.&#160;Grooves in the top and bottom of a microfluidic channel were designed to direct the sheath fluid and shape the core fluid.&#160;By matching the viscosity and hydrophilicity of the sheath and core fluids, the interfacial effects are minimized and complex fluid shapes can be formed.&#160;Controlling the relative flow rates of the sheath and core fluids determines the cross-sectional area of the core fluid.&#160;Fibers have been produced with sizes ranging from 300 nm to ~1 mm, and fiber cross-sections can be round, flat, square, or complex as in the case with double anchor fibers. Polymerization of the core fluid downstream from the shaping region solidifies the fibers.&#160;Photoinitiated click chemistries are well suited for rapid polymerization of the core fluid by irradiation with ultraviolet light.&#160;Fibers with a wide variety of shapes have been produced from a list of polymers including liquid crystals, poly(methylmethacrylate), thiol-ene and thiol-yne resins, polyethylene glycol, and hydrogel derivatives. Minimal shear during the shaping process and mild polymerization conditions also makes the fabrication process well suited for encapsulation of cells and other biological components.

Two adjacent fluids passing through a grooved microfluidic channel can be directed to form a sheath around a prepolymer core; thereby&#160;determining both shape and cross-section. Photoinitiated polymerization, such as thiol click chemistry, is well suited for rapidly solidifying the core fluid into a microfiber with predetermined size and shape.

Fabbricazione microfluidica di fibre polimeriche e bioibride con dimensioni e forma predesignate

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

Analyzing Mixing Inhomogeneity in a Microfluidic Device by Microscale Schlieren Technique

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren system is constructed from a Hoffman modulation contrast microscope, which provides easy access to the rear focal plane of the objective lens, by removing the slit plate and replacing the modulator with a knife-edge. The working principle of microscale schlieren technique relies on detecting light deflection caused by variation of refractive index1-3. The deflected light either escapes or is obstructed by the knife-edge to produce a bright or a dark band, respectively. If the refractive index of the mixture varies linearly with its composition, the local change in light intensity in the image plane is proportional to the concentration gradient normal to the optical axis. The micro-schlieren image gives a two-dimensional projection of the disturbed light produced by three-dimensional inhomogeneity.
To accomplish quantitative analysis, we describe a calibration procedure that mixes two fluids in a T-microchannel. We carry out a numerical simulation to obtain the concentration gradient in the T-microchannel that correlates closely with the corresponding micro-schlieren image. By comparison, a relationship between the grayscale readouts of the micro-schlieren image and the concentration gradients presented in a microfluidic device is established. Using this relationship, we are able to analyze the mixing inhomogeneity from associate micro-schlieren image and demonstrate the capability of microscale schlieren technique with measurements in a microfluidic oscillator4. For optically transparent fluids, microscale schlieren technique is an attractive diagnostic tool to provide instantaneous full-field information that retains the three-dimensional features of the mixing process.

Herein, we describe a procedure that employs microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. Through calibration, distribution of concentration gradient can be derived from the micro-schlieren image.

Analizzando disomogeneità Mescolando in un dispositivo a microfluidi da Tecnica microscala Schlieren

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren ...

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human gut-on-a-chip microphysiological device. We recapitulate the intestinal lumen-capillary tissue interface in a microfluidic device, where physiological mechanical deformations and fluid shear flow are constantly applied to mimic peristalsis. In the lumen microchannel, human intestinal epithelial Caco-2 cells are cultured to form a &#39;germ-free&#39; villus epithelium and regenerate small intestinal villi. Pre-cultured microbial cells are inoculated into the lumen side to establish a host-microbe ecosystem. After microbial cells adhere to the apical surface of the villi, fluid flow and mechanical deformations are resumed to produce a steady-state microenvironment in which fresh culture medium is constantly supplied and unbound bacteria (as well as bacterial wastes) are continuously removed. After extended co-culture from days to weeks, multiple microcolonies are found to be randomly located between the villi, and both microbial and epithelial cells remain viable and functional for at least one week in culture. Our co-culture protocol can be adapted to provide a versatile platform for other host-microbiome ecosystems that can be found in various human organs, which may facilitate in vitro study of the role of human microbiome in orchestrating health and disease.

We describe an in vitro protocol to co-culture gut microbiome and intestinal villi for an extended period using a human gut-on-a-chip microphysiological system.

Co-cultura del vivere Microbiome con Microengineered umana intestinale Villi in un Gut-on-a-chip microfluidico dispositivo

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human ...

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the spheroid cultures, a perfusable vascular network in the spheroids remains a critical challenge for long-term culture required to maintain and develop their functions, such as protein expressions and morphogenesis. The protocol presents a novel method to integrate a perfusable vascular network within the spheroid in a microfluidic device. To induce a perfusable vascular network in the spheroid, angiogenic sprouts connected to microchannels were guided to the spheroid by utilizing angiogenic factors from human lung fibroblasts cultured in the spheroid. The angiogenic sprouts reached the spheroid, merged with the endothelial cells co-cultured in the spheroid, and formed a continuous vascular network. The vascular network could perfuse the interior of the spheroid without any leakage. The constructed vascular network may be further used as a route for supply of nutrients and removal of waste products, mimicking blood circulation in vivo. The method provides a new platform in spheroid culture toward better recapitulation of living tissues.

The protocol describes how to engineer a perfusable vascular network in a spheroid. The spheroid's surrounding microenvironment is devised to induce angiogenesis and connect the spheroid to the microchannels in a microfluidic device. The method allows the perfusion of the spheroid, which is a long-awaited technique in three-dimensional cultures.

Perfusable rete vascolare con un modello di tessuto in un dispositivo microfluidico

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the ...

A Microfluidic Platform for Stimulating Chondrocytes with Dynamic Compression

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth plate cartilage architecture and results in growth modulation of long bones of children. To determine the role of compressive stress in bone growth, we created a microfluidic device actuated by pneumatic pressure, to dynamically (or statically) compress growth plate chondrocytes embedded in alginate hydrogel cylinders. In this article, we describe detailed methods for fabricating and characterizing this device. The advantages of our protocol are: 1) Five different magnitudes of compressive stress can be generated on five technical replicates in a single platform, 2) It is easy to visualize cell morphology via a conventional light microscope, 3) Cells can be rapidly isolated from the device after compression to facilitate downstream assays, and 4) The platform can be applied to study mechanobiology of any cell type that can grow in hydrogels.

This article provides detailed methods for fabricating and characterizing a pneumatically actuating microfluidic device for chondrocyte compression.

Una piattaforma microfluidica per stimolare i condrociti con la compressione dinamica

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth ...

Establishing Single-Cell Based Co-Cultures in a Deterministic Manner with a Microfluidic Chip

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases including cancer. However, it is challenging to clarify the complex mechanism of intercellular interactions in highly heterogeneous cell populations using conventional co-culture systems because the heterogeneity of the cell subpopulation is obscured by the average values; the conventional co-culture systems can only be used to describe the population signal, but are incapable of tracking individual cells behavior. Furthermore, conventional single-cell experimental methods have low efficiency in cell manipulation because of the Poisson distribution. Microfabricated devices are an emerging technology for single-cell studies because they can accurately manipulate single cells at high-throughput and can reduce sample and reagent consumption. Here, we describe the concept and application of a microfluidic chip for multiple single-cell co-cultures. The chip can efficiently capture multiple types of single cells in a culture chamber (~46%) and has a sufficient culture space useful to study the cells&#39; behavior (e.g., migration, proliferation, etc.) under cell-cell interaction at the single-cell level. Lymphatic endothelial cells and oral squamous cell carcinoma were used to perform a single-cell co-culture experiment on the microfluidic platform for live multiple single-cell interaction studies.

This report describes a microfluidic chip-based method to set up a single cell culture experiment in which high-efficiency pairing and microscopic analysis of multiple single cells can be achieved.

Stabilire coculture basate su una singola cellula in modo deterministico con un chip microfluidico

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases ...

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 ...

Configurazione di un dispositivo microfluidico per la produzione di tappi combinatoriali

Bilayer Microfluidic Device for Combinatorial Plug Production

Droplet microfluidics is a versatile tool that allows the execution of a large number of reactions in chemically distinct nanoliter compartments. Such systems have been used to encapsulate a variety of biochemical reactions - from incubation of single cells to implementation of PCR reactions, from genomics to chemical synthesis. Coupling the microfluidic channels with regulatory valves allows control over their opening and closing, thereby enabling the rapid production of large-scale combinatorial libraries consisting of a population of droplets with unique compositions. In this paper, protocols for the fabrication and operation of a pressure-driven, PDMS-based bilayer microfluidic device that can be utilized to generate combinatorial libraries of water-in-oil emulsions called plugs are presented. By incorporating software programs and microfluidic hardware, the flow of desired fluids in the device can be controlled and manipulated to generate combinatorial plug libraries and to control the composition and quantity of constituent plug populations. These protocols will expedite the process of generating combinatorial screens, particularly to study drug response in cells from cancer patient biopsies.

Autore in primo piano: Integrazione di approcci computazionali e sperimentali nell'oncologia di precisione

The fabrication of a polydimethylsiloxane (PDMS)-based bilayer device for the production of combinatorial libraries in water-in-oil emulsions (plugs) is presented here. The necessary hardware and software required to automate plug production are detailed in the protocol, and the production of a quantitative library of fluorescent plugs is also demonstrated.

Dispositivo microfluidico a doppio strato per la produzione di tappi combinatori

Droplet microfluidics is a versatile tool that allows the execution of a large number of reactions in chemically distinct nanoliter compartments. Such ...