Sostegno da parte dei National Institutes of Health (concede GM092804, DE019938) e Shriners Hospital Burns.

1. Fabbricazione di dispositivi Microfluidic <ol><li> Utilizzando tecniche fotolitografiche standard fabbricare il maestro stampo wafer in una camera pulita di classe 1000. Reticolo i primi 3 micron-sottile strato di fotoresist negativo a base epossidica per definire i canali di migrazione secondo le istruzioni del produttore. Reticolo secondo strato 50 micron di spessore per definire le camere cellulare carico e chemochine. </li><li> Utilizzare il wafer fantasia di lanciare dispositivi (PDMS) polidimetilsilossano. Agitare vigorosamente PDMS (20 g) con iniziatore (2 g) per 5 minuti usando forchetta di plastica in grande vassoio pesatura plastica. </li><li> Versare cautamente PDMS sulla muffa. </li><li> Degassarla PDMS mettendo stampo con PDMS riversato in un essiccatore a vuoto per 1 ora. </li><li> Cuocere e curare dispositivi microfluidici PDMS per almeno 3 ore in forno a 65 ° C. </li><li> Punch i WBLCs centrali con un perforatore con un diametro della punta di 1,5 mm. </li><li> Punch out interi dispositivi a forma di ciambella con un punclei con un diametro della punta di 5,0 mm. </li><li> Rimuovere le particelle da dispositivi a ciambella con del nastro adesivo. </li><li> Risciacquare una piastra 12 pozzetti con acqua deionizzata e poi asciugare con azoto. Posizionare la piastra in 60 ° C forno per 5 min. </li><li> Ossigeno plasma trattare la piastra 12 pozzetti due volte; una volta sola per 35 secondi e poi di nuovo con i dispositivi ciambella per un altro 35 sec. </li><li> Posizionare con cura i dispositivi nei pozzetti della piastra con una pinzetta. </li><li> Piatto Cuocere con i dispositivi aderenti, su una piastra calda impostato a 80 ° C per 10 min. </li></ol> 2. Microfluidic saggio Preparazione <ol><li> Dispositivi microfluidici Prime immediatamente dopo il trattamento al plasma di ossigeno, quando il dispositivo è idrofilo ed effetti capillari può favorire l&#39;innesco dei piccoli canali nel dispositivo. </li><li> Crea soluzione chemoattractant mescolando 5 ml fMLP [soluzione stock 10 mM] con 5 microlitri fibronectina [soluzione stock 1 mg / ml] e 490 microlitri HBSS. </li><li> Lentamente pipetta &amp; #160; soluzione chemoattractant in WBLC, con una punta di gel di carico (Figura 1A). Pipettare altri 20 ml di fattore chemiotattico intorno alla parte esterna del dispositivo. </li><li> Posizionare la piastra in un essiccatore per 15 minuti. Applicando un vuoto al dispositivo, la soluzione viene instillata laterali canali del dispositivo e l&#39;aria spostata diffusa attraverso il PDMS. </li><li> Rimuovere la piastra di essiccatore e confermare la bagnatura del canale dispositivo sotto il microscopio. Guarda come la bolla diventa più piccolo come soluzione chemoattractant entra nella camera. Nessuna bolla dovrebbe essere presente all&#39;interno del dispositivo dopo trattamento sottovuoto. </li><li> Lavare la WBLC e al di fuori del dispositivo a fondo per rimuovere la soluzione in eccesso chemoattractant. Questo passaggio genera un gradiente di chemiotattico da ciascuna delle camere chemiotattici focali (FCC) al centro del dispositivo. <ol><li> Riempire una siringa da 1 ml con PBS, aggiungere una punta di ago smussato 30 G per siringa. Delicatamente, inserire la punta dell&#39;ago della siringa inal centro del foro ciambella. Spingere delicatamente 100 ml di PBS nel foro in modo che una goccia di forme PBS sulla parte superiore del dispositivo. </li><li> Piastra Tilt e Pipettare 1 ml di PBS intorno dispositivo in modo che il liquido si raccoglie sul fondo del pozzo. Aspirare il liquido di processo e ripetere per un totale di 3x con soluzione tampone fresco (uso dei media al posto del tampone se verranno caricati i neutrofili separate in media) 9. </li></ol></li><li> Riempire ogni pozzetto con i media fino alle cime dei dispositivi sono sommerse sotto liquido. Lasciare che i dispositivi riposare per 15 min per permettere gradiente di stabilizzarsi. Risultati sperimentali e dati teorici di simulazioni agli elementi finiti mostrano che i gradienti di questi dispositivi sono stabili fino a 24 ore per le piccole molecole (ad esempio, fMLP) e fino a diversi giorni per peso molecolare più grande (ad esempio, IL8). </li><li> Usando le punte gel di carico, lentamente pipetta 2 ml di sangue (o neutrofili isolati) in ciascun tutta loa sangueding camera (WBLC) (Figura 1A). </li></ol> 3. Preparazione del campione I neutrofili umani Da capillare sanguigno <ol><li> Raccogliere il sangue capillare dal dito cazzo di volontari sani, che è in alcun immunosoppressori. Tutti i campioni sono stati ottenuti con il consenso informato scritto, e attraverso procedure approvate dal MGH e Shriners Review Boards istituzionali. <ol><li> Per dito cazzo di raccolta del sangue, lavarsi le mani con acqua e sapone e asciugare la pelle. Preparare anti-coagulant/stain soluzione stock con l&#39;aggiunta di 1 ml HBSS + 0,2% HSA e 10 microlitri Hoechst macchia (32,4 micron) di eparina tubo di raccolta del sangue (1,65 USP/50 microlitri di sangue). Pungere il dito con una lancetta di sicurezza SurgiLance (profondità 2,2 mm, 22 G), rimuovere la prima goccia di sangue e raccogliere 50 ml di sangue in provetta Eppendorf contenente lo stock anti-coagulante e la soluzione colorante fluorescente Hoechst (10 ml) e delicatamente mescolare. </li></ol></li&gt; <li> Incubare il sangue e macchia Hoechst per 10 min per permettere colorazione fluorescente nucleo. Eseguire campione entro 1 ora di raccolta del sangue. </li></ol> I neutrofili umani Da sangue venoso <ol start="3"><li> Disegna 10 ml di sangue venoso periferico in provette contenenti 33 USP eparina. </li><li> Aggiungere 50 ml di sangue venoso a media e macchia Hoechst come precedentemente descritto. Incubare il sangue e macchia Hoechst per 10 min per permettere colorazione fluorescente nucleo. Eseguire campione entro 1 ora di raccolta del sangue. </li></ol> Neutrofili Separazione Dal sangue intero - controllo positivo <ol start="5"><li> Per separare neutrofili, utilizzare tecniche sterili per isolare neutrofili dal 10 ml venoso campione di sangue intero utilizzando Hetastarch (6% w / v) gradiente di densità seguita da una selezione perlina magnetica Kit di isolamento negativo seguendo il protocollo produttori. </li><li> Risospese parte aliquota finale di neutrofili a Concentrazionezione di 4.000 cellule / mL in 1X HBSS + 0,2% di albumina sierica umana. Conservare le cellule a 37 ° C fino al momento di eseguire l&#39;esperimento. </li></ol> 4. Microscopy and Image Analysis per misure chemiotassi dei neutrofili <ol><li> Impostare la temperatura biochamber a 37 ° C, umidità al 80%, e CO 2 al 5%. </li><li> Inizia l&#39;imaging immediatamente con time-lapse imaging su un microscopio (10X o di ingrandimento superiore). </li></ol> 5. Analisi statistica <ol><li> Traccia manualmente almeno 50 neutrofili in ciascun campione. </li><li> Contare le cellule che entrano nel FCC nel tempo (Figura 2). </li><li> Calcolo velocità neutrofili nel canale tra WBLC e FCC utilizzando ImageJ (NIH) (Figura 2). </li><li> Quantificare direzionalità dei neutrofili contando il numero di cellule che passano la biforcazione e calcolando il rapporto tra il numero di cellule che trasformano verso la FCC e cellule che uscita esimoe dispositivo. Le cellule che non sono direzionali non seguono il gradiente chemiotattico e quindi migrano in numero uguale verso la FCC e il canale di uscita (figura 2). </li></ol>

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In questo lavoro, abbiamo sviluppato una piattaforma microfluidica per misurare neutrofili chemiotassi da una goccia di sangue (2 microlitri). La filtrazione on-chip meccanica dei globuli rossi da migrare attivamente neutrofili elude la necessità di metodi ingombranti separazione cellulare come la densità gradienti 10, selezione positiva 11 o selezione negativa 12, che sono inclini a introdurre artefatti attivando neutrofili. La filtrazione meccanica dei globuli rossi distingue la nost...

<html> Traffico neutrofili svolge un ruolo critico nel determinare l&#39;avanzamento e la risoluzione di molte condizioni infiammatorie, tra aterosclerosi 1, infezione batterica o sepsi 2, e ustioni 3. Per il loro importante contributo alle condizioni di salute e di malattia, conta dei neutrofili è parte dello standard di analisi del sangue spesso considerati nei laboratori clinici e di ricerca. Tuttavia, pur essendo una delle prove più diffusi, il valore di conteggio dei neutrofili nella diagnosi di infezione e sepsi è stato spesso interrogato 4. Per esempio, uno studio dei neutrofili in pazienti ustionati rivelato che conta dei neutrofili e la funzione di migrazione dei neutrofili non sono correlati; a significare che conta dei neutrofili da solo non è un indicatore accurato dello stato immunitario 3. Anche se più difficile da misurare, competenza funzionale dei neutrofili è stato proposto come più importante in una vasta gamma di condizioni. t &quot;&gt; È importante sottolineare che molti dei difetti neutrofili sono transitori e non sono attivati ​​da difetti genetici permanenti, una distinzione che è stato ampiamente trascurato nella clinica fino a poco tempo. Nell&#39;ambito di ustioni, la migrazione dei neutrofili potrebbe essere monitorato nel corso di trattamento di un paziente come indicatore di stato infiammatorio o infezione 3. saggi di migrazione tradizionali attualmente utilizzati in laboratorio (camera di Boyden, camera Dunn, micropipetta dosaggio) non può essere tradotto in un ambiente clinico perché richiedono grandi volumi di sangue ed ingombrante in termini di tempo tecniche di isolamento dei neutrofili (Tabella 1). Questi test inoltre non possono essere utilizzati per monitorare i cambiamenti transitori chemiotassi dei neutrofili nei piccoli animali da laboratorio, come topi, in quanto il volume di sangue necessario per l&#39;isolamento dei neutrofili consente solo un campione e spesso richiede anche messa in comune di sangue da più animali per un singolo test. Per esempio, uno studio condotto mcondizioni ultiple e trattamenti in più punti temporali potrebbero potenzialmente richiedere migliaia di topi utilizzando saggi di chemiotassi correnti. Questo limita la ricerca biologica di base che può essere fatto di comprendere le complesse dinamiche della funzione immunitaria nel contesto di lesioni, infezioni o bruciare spesso studiato in modelli murini 5. Per rispondere all&#39;esigenza di un saggio funzionale neutrofili che è rapido, robusto, pur richiedendo il minimo volume di sangue, abbiamo sviluppato un dispositivo microfluidico che misura la chemiotassi dei neutrofili direttamente da una piccola goccia di sangue intero. E &#39;noto che molti fattori di sangue intero, compreso il siero 6 e 7 piastrine, influenzano la funzione dei neutrofili. È quindi utile che l&#39;intero saggio microfluidico sangue minimizza trattamento del campione per mantenere il microambiente in vivo del neutrofilo quando si misurano variazioni di chemiotassi con una prova in vitro 8. Questo approccioriduce il tempo di raccolta del sangue a neutrofili test di migrazione da ore utilizzando tecniche tradizionali, a pochi minuti (Tabella 1). Dell&#39;intera piattaforma microfluidica sangue produce un gradiente chemiotattico lineare stabile per la durata dell&#39;esperimento, non ha parti in movimento, e non richiede una sorgente di pressione esterna (cioè pompa a siringa). La caratteristica fondamentale nella progettazione dell&#39;intero dispositivo microfluidica sangue è l&#39;incorporazione di un globulo rosso (RBC) pettine filtrazione che filtra meccanicamente eritrociti di entrare nel canale migrazione del dispositivo. Il diritto giri di questo pettine filtrazione evitano la necessità di filtrazione esclusione dimensioni, che sarebbe probabilmente essere intasato da globuli rossi e quindi bloccano il gradiente chemiotattico di raggiungere i neutrofili migrano attivamente nel WB. L&#39;incorporazione dell&#39;intero dispositivo microfluidica sangue in una piastra 12 o 24 pozzetti facilita la proiezione di diversi mediatori di Si chemiotassi neutrofila umana o murinamultaneamente.

<table border="0" cellpadding="0" cellspacing="0" style="width:771px;" width="771">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Name of Material/ Equipment</td>
			<td style="width:191px;">Company</td>
			<td style="width:179px;">Catalog Number</td>
			<td style="width:191px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Device Fabrication</td>
			<td style="width:191px;"></td>
			<td style="width:179px;"></td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">SU8&#160;</td>
			<td style="width:191px;">Microchem</td>
			<td style="width:179px;">Y131273</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Polydimethylsiloxane (PDMS)</td>
			<td style="width:191px;">Ellsworth Adhesives</td>
			<td style="width:179px;">Sylgard 184 1.1 lb. Kit</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Standard glass slides</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:179px;">125495</td>
			<td style="width:191px;">1 X 3 inches</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Glass-bottom plate</td>
			<td style="width:191px;">MatTek</td>
			<td style="width:179px;">P12G-1.5-14-F</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Harris Uni-Core, Tip Diameter 5.0mm</td>
			<td style="width:191px;">Ted Pella, Inc.</td>
			<td style="width:179px;">15081</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Harris Uni-Core, Tip Diameter 1.5 mm</td>
			<td style="width:191px;">Ted Pella, Inc.</td>
			<td style="width:179px;">15072</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Microfluidic Assay Preparation and Analysis</td>
			<td style="width:191px;"></td>
			<td style="width:179px;"></td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Gel-loading pipet tip</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:179px;">02-707-139</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Syringe</td>
			<td style="width:191px;">Fisher Scientfic</td>
			<td style="width:179px;">309602</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Blunt tip needle, 30g &#189; in.</td>
			<td style="width:191px;">Brico Medical Supply</td>
			<td style="width:179px;">BN3005</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Vacutainer, Heparin</td>
			<td style="width:191px;">Becton Dickinson</td>
			<td style="width:179px;"></td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;"></td>
			<td style="width:191px;"></td>
			<td style="width:179px;"></td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">HBSS&#160;</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:179px;"></td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Human serum albumin</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:179px;">A5843-5G</td>
			<td style="width:191px;">0.2% final concentration in HBSS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Fibronectin</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:179px;">F0895-1MG</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">fMLP</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:179px;">F3506-10MG</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">SurgiLance safety lancet, 2.2mm depth, 22 gauge</td>
			<td style="width:191px;"></td>
			<td style="width:179px;">SLN240</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Hoescht stain</td>
			<td style="width:191px;">Life Technologies</td>
			<td style="width:179px;">H3570</td>
			<td style="width:191px;"></td>
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			<td height="21" style="height:21px;width:211px;">Positive Control&#160;</td>
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			<td style="width:191px;"></td>
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			<td height="42" style="height:42px;width:211px;">HetaSep</td>
			<td style="width:191px;">STEMCELL Technologies Inc.</td>
			<td style="width:179px;">7906</td>
			<td style="width:191px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:211px;">EasySep Human Neutrophil Enrichment Kit</td>
			<td style="width:191px;">STEMCELL Technologies Inc.</td>
			<td style="width:179px;">19257</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;"></td>
			<td style="width:191px;"></td>
			<td style="width:179px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Equipment</td>
			<td style="width:191px;"></td>
			<td style="width:179px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:211px;">Plasma Asher</td>
			<td style="width:191px;">March Instruments</td>
			<td style="width:179px;">P-250</td>
			<td></td>
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			<td height="21" style="height:21px;width:211px;">Lindberg/Blue M Oven</td>
			<td style="width:191px;">Thermo Scientific</td>
			<td style="width:179px;">13-258-30C</td>
			<td></td>
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			<td height="42" style="height:42px;width:211px;">Stainless Steel Precision Tweezers</td>
			<td style="width:191px;">Techni Tool</td>
			<td style="width:179px;">758TW458</td>
			<td></td>
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			<td height="42" style="height:42px;width:211px;">Bel-Art Scienceware Chemical-Resistant Vacuum Desiccator</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:179px;">08-594-15A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Dataplate Digital Hot Plate</td>
			<td style="width:191px;">Alpha Multiservices</td>
			<td style="width:179px;">PMC 720&#160;</td>
			<td></td>
		</tr>
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			<td height="42" style="height:42px;width:211px;">Nikon TiE inverted microscope</td>
			<td style="width:191px;">Nikon - Micro Video Instruments Inc.</td>
			<td style="width:179px;">MEA53100</td>
			<td></td>
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			<td height="42" style="height:42px;width:211px;">CFI Plan Fluor DL 10x na 15.2wd Objective</td>
			<td style="width:191px;">Nikon - Micro Video Instruments Inc.</td>
			<td style="width:179px;">MRH20101</td>
			<td></td>
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			<td height="42" style="height:42px;width:211px;">Lumen 200 with 2 Meter Light Guide for Nikon</td>
			<td style="width:191px;">Nikon - Micro Video Instruments Inc.</td>
			<td style="width:179px;">500-L200NI2</td>
			<td></td>
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			<td height="63" style="height:63px;width:211px;">DAPI/Hoechst/AMCA Narrow Band 32mm Exciters - 25mm Emitters</td>
			<td style="width:191px;">Chroma - Micro Video Instruments Inc.</td>
			<td style="width:179px;">31013v2</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Retiga R 2000 cooled CCD Camera 1600x1200 pixels</td>
			<td style="width:191px;">Qimaging - Micro Video Instruments Inc.</td>
			<td>RET-2000R-F-M-12-C</td>
			<td></td>
		</tr>
	</tbody>
</table>

microfluidic platform for measuring neutrophil chemotaxis from unprocessed whole blood

I neutrofili hanno un ruolo essenziale nella protezione contro le infezioni e il loro numero nel sangue sono spesso misurato in clinica. Superiore conta dei neutrofili nel sangue sono di solito un indicatore di infezioni in corso, mentre una bassa conta di neutrofili sono un segnale di avvertimento per i rischi più elevati per le infezioni. Per realizzare le loro funzioni, neutrofili devono anche essere in grado di spostare efficacemente dal sangue dove trascorrono la maggior parte della loro vita, nei tessuti, dove si verificano infezioni. Di conseguenza, eventuali difetti nella capacità dei neutrofili di migrare può aumentare il rischio di infezioni, anche quando sono presenti in numero appropriato nel sangue neutrofili. Tuttavia, la misurazione dei neutrofili capacità di migrazione nella clinica è un compito impegnativo, che richiede tempo, richiede grandi quantità di sangue, e conoscenze specifiche. Per far fronte a queste limitazioni, abbiamo progettato un robusto saggi microfluidica per la migrazione dei neutrofili, che richiede una singola goccia di sangue non trasformati, circumsfiati la necessità di una separazione dei neutrofili, ed è facile da quantificare su un semplice microscopio. In questo saggio, neutrofili migrano direttamente dalla gocciolina di sangue, attraverso piccoli canali, verso la sorgente di chemiotattico. Per impedire il flusso granulare di globuli rossi attraverso gli stessi canali, abbiamo implementato filtri meccanici con virata a gomito che bloccare selettivamente l&#39;anticipo di globuli rossi. Abbiamo convalidato il test confrontando la migrazione dei neutrofili dalle gocce di sangue raccolti da dito cazzo e sangue venoso. Abbiamo anche confrontato questi sangue intero (WB) fonti di migrazione dei neutrofili da campioni di neutrofili purificati e abbiamo trovato la velocità costante e la direzionalità tra le tre fonti. Questa piattaforma microfluidica consentirà lo studio della migrazione dei neutrofili umana in clinica e l&#39;impostazione della ricerca per far progredire la nostra comprensione delle funzioni dei neutrofili nella salute e nella malattia.

Il sangue intero (WB) saggio chemiotassi dei neutrofili è stata convalidata misurando l&#39;accumulo di neutrofili verso un gradiente fMLP (Film S1). I risultati confermano che i globuli rossi sono intrappolati dal pettine filtrazione mentre neutrofili (blu) sono in grado di migrare attivamente di sangue intero (figura 3A e Movie S1). Il gradiente lineare stabile chemiotattico (verde) formato dal sangue intero dispositivo microfluidica è stata confermata usando destra...

Watch this Scientific Journal Video about Microfluidic Platform for Measuring Neutrophil Chemotaxis from Unprocessed Whole Blood at JoVE.com

Microfluidic Platform for Measuring Neutrophil Chemotaxis from Unprocessed Whole Blood

Dettagli Questo protocollo un saggio progettato per misurare la chemiotassi dei neutrofili umani da una gocciolina di sangue intero con robusto riproducibilità. Questo approccio elude la necessità di una separazione dei neutrofili e richiede solo pochi minuti di dosaggio tempo di preparazione. Il chip microfluidica consente la misura ripetuta di chemiotassi dei neutrofili nel tempo nei neonati o piccoli mammiferi, dove il volume del campione è limitata.

Piattaforma microfluidica per la misurazione dei neutrofili Chemiotassi dal Lordo Sangue intero

Neutrophils play an essential role in protection against infections and their numbers in the blood are frequently measured in the clinic. Higher ...

microfluidic-platform-for-measuring-neutrophil-chemotaxis-from

Research

JoVE Journal

Bioengineering

11.2K Views.  Massachusetts General Hospital.  Dettagli Questo protocollo un saggio progettato per misurare la chemiotassi dei neutrofili umani da una gocciolina di sangue intero con robusto riproducibilità. Questo approccio elude la necessità di una separazione dei neutrofili e richiede solo pochi minuti di dosaggio tempo di preparazione. Il chip microfluidica consente la misura ripetuta di chemiotassi dei neutrofili nel tempo nei neonati o piccoli mammiferi, dove il volume del campione è limitata. 

Video: Microfluidic Platform for Measuring Neutrophil Chemotaxis from Unprocessed Whole Blood

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

High-throughput generatore di espressione delle proteine ​​Utilizzando una piattaforma microfluidica

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

Ricerca

Bioingegneria

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

Spremere cellulare come un robusto, Microfluidic intracellulare Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need for new solutions for substance testing. Many drugs that have been removed from the market due to drug-induced liver injury released their toxic potential only after several doses of chronic testing in humans. However, a controlled microenvironment is pivotal for long-term multiple dosing experiments,&#160;as even minor alterations in extracellular conditions may greatly influence the cell physiology. We focused within our research program on the generation of a microengineered bioreactor, which can be dynamically perfused by an on-chip pump and combines at least two culture spaces for multi-organ applications. This circulatory system mimics the in vivo conditions of primary cell cultures better and assures a steadier,&#160;more quantifiable extracellular relay of signals to the cells. 
For demonstration purposes, human liver equivalents, generated by aggregating differentiated HepaRG cells with human hepatic stellate cells in hanging drop plates, were cocultured with human skin punch biopsies for up to 28 days inside the microbioreactor. The use of cell culture inserts enables the skin to be cultured at an air-liquid interface, allowing topical substance exposure. The microbioreactor system is capable of supporting these cocultures at near physiologic fluid flow and volume-to-liquid ratios, ensuring stable and organotypic culture conditions. The possibility of long-term cultures enables the repeated exposure to substances. Furthermore, a vascularization of the microfluidic channel circuit using human dermal microvascular endothelial cells yields a physiologically more relevant vascular model.

Here, we present a protocol to coculture primary cells, tissue models and punch biopsies in a microfluidic multi-organ chip for up to 28 days. Human dermal microvascular endothelial cells, liver aggregates and skin biopsies were successfully combined in a common media circulation.

Il Chip Multi-organo - Una piattaforma Microfluidic per lungo termine co-coltura Multi-tessuto

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need ...

Controlled Microfluidic Environment for Dynamic Investigation of Red Blood Cell Aggregation

Blood, as a non-Newtonian biofluid, represents the focus of numerous studies in the hemorheology field. Blood constituents include red blood cells, white blood cells and platelets that are suspended in blood plasma. Due to the abundance of the RBCs (40% to 45% of the blood volume), their behavior dictates the rheological behavior of blood especially in the microcirculation. At very low shear rates, RBCs are seen to assemble and form entities called aggregates, which causes the non-Newtonian behavior of blood. It is important to understand the conditions of the aggregates formation to comprehend the blood rheology in microcirculation. The protocol described here details the experimental procedure to determine quantitatively the RBC aggregates in microcirculation under constant shear rate, based on image processing. For this purpose, RBC-suspensions are tested and analyzed in 120 x 60 &#181;m poly-dimethyl-siloxane (PDMS) microchannels. The RBC-suspensions are entrained using a second fluid in order to obtain a linear velocity profile within the blood layer and thus achieve a wide range of constant shear rates. The shear rate is determined using a micro Particle Image Velocimetry (&#181;PIV) system, while RBC aggregates are visualized using a high speed camera. The videos captured of the RBC aggregates are analyzed using image processing techniques in order to determine the aggregate sizes based on the images intensities.

The protocol described details an experimental procedure to quantify Red Blood Cell (RBC) aggregates under a controlled and constant shear rate, based on image processing techniques. The goal of this protocol is to relate RBC aggregate sizes to the corresponding shear rate in a controlled microfluidic environment.

Ambiente controllato Microfluidic per Dynamic Indagine Globulo rosso Aggregazione

Blood, as a non-Newtonian biofluid, represents the focus of numerous studies in the hemorheology field. Blood constituents include red blood cells, white ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

Una piattaforma Microfluidic per l&#39;isolamento Single-cell high-throughput e cultura

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Fully Automated Centrifugal Microfluidic Device for Ultrasensitive Protein Detection from Whole Blood

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform for reduced sample volume and assay time as well as full automation are required for potential use in point-of-care-diagnostics. Recently, we have demonstrated ultrasensitive detection of serum proteins, C-reactive protein (CRP) and cardiac troponin I (cTnI), utilizing a lab-on-a-disc composed of TiO2 nanofibrous (NF) mats. It showed a large dynamic range with femto molar (fM) detection sensitivity, from a small volume of whole blood in 30 min. The device consists of several components for blood separation, metering, mixing, and washing that are automated for improved sensitivity from low sample volumes. Here, in the video demonstration, we show the experimental protocols and know-how for the fabrication of NFs as well as the disc, their integration and the operation in the following order: processes for preparing TiO2 NF mat; transfer-printing of TiO2 NF mat onto the disc; surface modification for immune-reactions, disc assembly and operation; on-disc detection and representative results for immunoassay. Use of this device enables multiplexed analysis with minimal consumption of samples and reagents. Given the advantages, the device should find use in a wide variety of applications, and prove beneficial in facilitating the analysis of low abundant proteins.

This protocol demonstrates how to achieve femto molar detection sensitivity of proteins in 10 &#181;L of whole blood within 30 min. This can be achieved by using electrospun nanofibrous mats integrated in a lab-on-a-disc, which offers high surface area as well as effective mixing and washing for enhanced signal-to-noise ratio.

Completamente dispositivo automatizzato centrifuga Microfluidic per il rilevamento di proteine ​​Ultrasensitive da sangue intero

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

Piattaforma microfluidica con multiplex di rilevamento elettronico per spaziale monitoraggio di particelle

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

Imaging the Root Hair Morphology of Arabidopsis Seedlings in a Two-layer Microfluidic Platform

Root hairs increase root surface area for better water uptake and nutrient absorption by the plant. Because they are small in size and often obscured by their natural environment, root hair morphology and function are difficult to study and often excluded from plant research. In recent years, microfluidic platforms have offered a way to visualize root systems at high resolution without disturbing the roots during transfer to an imaging system. The microfluidic platform presented here builds on previous plant-on-a-chip research by incorporating a two-layer device to confine the Arabidopsis thaliana main root to the same optical plane as the root hairs. This design enables the quantification of root hairs on a cellular and organelle level and also prevents z-axis drifting during the addition of experimental treatments. We describe how to store the devices in a contained and hydrated environment, without the need for fluidic pumps, while maintaining a gnotobiotic environment for the seedling. After the optical imaging experiment, the device may be disassembled and used as a substrate for atomic force or scanning electron microscopy while keeping fine root structures intact.

Imaging the Root Hair Morphology of Arabidopsis Seedlings in a Two-layer Microfluidic Platform

This article demonstrates how to culture Arabidopsis thaliana seedlings in a two-layer microfluidic platform that confines the main root and root hairs to a single optical plane. This platform can be used for real-time optical imaging of fine root morphology as well as for high-resolution imaging by other means.

La morfologia dei capelli di radice di semenzali di Arabidopsis in una piattaforma di Microfluidic di due-strato di imaging

Root hairs increase root surface area for better water uptake and nutrient absorption by the plant. Because they are small in size and often obscured by ...

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved useful as a convenient live imaging platform providing capabilities for precise control of experimental conditions in real time. In this article, we demonstrate live imaging of individual worms employing WormSpa, a previously-published custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes. To illustrate the capability, we performed proof-of-principle experiments in which worms were infected in the device with pathogenic bacteria, and the dynamics of expression of immune response genes and egg laying were monitored continuously in individual animals. The simple design and operation of this device make it suitable for users with no previous experience with microfluidic-based experiments. We propose that this approach will be useful for many researchers interested in longitudinal observations of biological processes under well-defined conditions.

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In this article, we demonstrate live imaging of individual worms employing a custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes.

Una piattaforma microfluidica per Imaging longitudinale in Caenorhabditis elegans

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved ...

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of cellular heterogeneity in biological systems. However, single-cell sequencing of large populations has been hampered by limitations in processing genomes for sequencing. In this paper, we describe a method for single-cell genome sequencing (SiC-seq) which uses droplet microfluidics to isolate, amplify, and barcode the genomes of single cells. Cell encapsulation in microgels allows the compartmentalized purification and tagmentation of DNA, while a microfluidic merger efficiently pairs each genome with a unique single-cell oligonucleotide barcode, allowing &gt;50,000 single cells to be sequenced per run. The sequencing data is demultiplexed by barcode, generating groups of reads originating from single cells. As a high-throughput and low-bias method of single-cell sequencing, SiC-seq will enable a broader range of genomic studies targeted at diverse cell populations.

Single-cell sequencing reveals genotypic heterogeneity in biological systems, but current technologies lack the throughput necessary for the deep profiling of community composition and function. Here, we describe a microfluidic workflow for sequencing &gt;50,000 single-cell genomes from diverse cell populations.

Una piattaforma di Microfluidic ad altissima velocità di trasmissione per il sequenziamento del genoma di cella singola

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of ...