This work was supported by a grant from the National Health Research Institutes (03-A1 BNMP11-014).

Nota: I disegni fotomaschere per la nostra fabbricazione di dispositivi microfluidica sono stati elaborati utilizzando un software di progettazione assistita da computer (CAD). I disegni sono stati poi utilizzati per fabbricare fotomaschere Chrome con un servizio commerciale. I dispositivi PDMS sono state effettuate utilizzando tecniche di litografia morbide. 11 1. Realizzazione di Maestro Stampi per litografia <ol><li> Prima che il processo di fotolitografia 12, utilizzare i wafer di silicio da 4 pollici come substrato e disidratare i wafer in un forno convenzionale a 120 ° C per 10 min. </li><li> Pulire i wafer di silicio disidratati mediante trattamento al plasma di ossigeno a 100 watt per 30 secondi in un pulitore plasma. </li><li> Preriscaldare due piastre a 65 ° C e 95 ° C, rispettivamente, per il seguente processo di cottura. </li><li> Cappotto 5 g di photoresist negativo (PR) sui wafer di silicio pulite da un dispositivo a induzione di spin; centrifuga a 1200 rpm (SU-8 50) per 30 sec per produrre lo strato di microcanali. </li><li> Posizionare il PRrivestito wafer su una piastra preriscaldato a 65 ° C per 12 min e trasferirlo in un&#39;altra piastra preriscaldato a 95 ° C per 33 min (per 100 micron di spessore modelli) per eseguire un processo di cottura morbida. </li><li> Dopo la cottura, posizionare il wafer di silicio rivestito PR sul supporto di una maschera allineatore semi-automatico e allinearlo a una trasparenza fotomaschera 25.400 dpi di risoluzione. </li><li> Esporre il rivestito PR wafer di silicio a luce UV (365 nm) ad una dose di 500 mJ / cm 2 per creare il modello PR sul wafer di silicio. </li><li> Rimuovere il wafer dal allineatore e posizionarlo su una piastra per la post-cottura a 95 ° C per 12 min. </li><li> Mettere a bagno i wafer di SU-8 sviluppatore della soluzione (glicole propilenico monometiletere acetato, PGMEA) per lavare via PR non reticolato per 12 min e asciugare delicatamente con azoto per esporre i segni di allineamento. </li><li> Ancora una volta, cappotto 5 g di fotoresist negativo sui wafer da un coater centrifuga; centrifuga a 700 rpm (SU-8 100) per 30 sec e 1.200 rpm (SU-8 10) per 30 sec per 300 &amp;# 181; m modello di spessore e 27 micron modello di spessore, rispettivamente, per rendere lo strato micropozzetto. </li><li> Posizionare il wafer PR rivestito su una piastra riscaldante a 65 ° C per 4 min ed a 95 ° C per 8 min (per 27 micron strato profondo cattura-well); ed a 65 ° C per 40 minuti ea 95 ° C per 110 min (per 300 micron strato profondo cultura-well). </li><li> Dopo raffreddamento, posizionare il wafer di silicio rivestito PR sul allineatore maschera dotata di luce UV. </li><li> Esporre il rivestito PR wafer di silicio alla luce UV (365 nm) ad una dose di 250 mJ / cm 2 (per 27 micron di spessore pattern) e 700 mJ / cm 2 (per 300 micron di spessore pattern). </li><li> Cuocere i wafer a 95 ° C per 5 min (27 micron di spessore pattern) e 30 min (300 micron di spessore pattern), rispettivamente. </li><li> Lavare il PR non reticolato dal PGMEA per 6 min (27 micron di spessore pattern) e 25 min (300 micron di spessore pattern), rispettivamente, e poi asciugare con azoto. </li><li> Misurare l&#39;altezza del modello dispone suil wafer con un profilometro scansione laser posizionando il wafer sul xy fase di un profilometro laser a scansione. <ol><li> Regolare il piano focale per mostrare chiaramente il modello presenta sul wafer utilizzando la modalità di osservazione &quot;telecamera&quot; sotto una lente obiettivo 20X. </li><li> Cambiare la modalità di osservazione da &quot;telecamera&quot; a &quot;vista laser&quot;, e impostare le posizioni superiori ed inferiori delle caratteristiche. </li><li> Impostare la modalità di misurazione, zona, la qualità, e Z-passo fino a trasparente (in alto), 1 linea (1.024 x 1), ad alta precisione e 0,5 micron, rispettivamente, e quindi premere il fondo di partenza per iniziare la misurazione. </li></ol></li></ol> 2. Preparazione di dispositivi PDMS per l&#39;isolamento Single-cell <ol><li> Prima di PDMS casting, silanizzare gli stampi di perfezionamento con triclorosilano per creare una superficie idrofobica, che rende più facile per staccare il PDMS repliche dagli stampi maestri. <ol><li> Mettere gli stampi master e una barca ponderazione contenente200 ml di triclorosilano in essiccatore e applicare il vuoto (-85 kPa) per 15 min. </li><li> Arrestare il vuoto e poi lasciare gli stampi di master in un essiccatore per silanizzare gli stampi maestri a temperatura ambiente per almeno 1 ora. </li><li> Rimuovere gli stampi maestri dal essiccatore e posizionare ogni stampo master in un 10 centimetri Petri. </li></ol></li><li> Mescolare un totale di 17,6 g di polimero kit PDMS contenente una base e un agente indurente in un rapporto di 10: 1, e quindi versare il PDMS sullo stampo master nel piatto Petri. </li><li> Posizionare la piastra di Petri in un essiccatore e applicare il vuoto (-85 kPa) per 1 ora per rimuovere le bolle d&#39;aria nei PDMS. </li><li> Rimuovere la piastra di Petri dal essiccatore e metterla in un forno tradizionale a 65 ° C per 3-6 ore per curare il PDMS. </li><li> Rimuovere le repliche PDMS indurito dagli stampi master e perforare due fori come un ingresso e una uscita alle due estremità del microcanale sull&#39;array PDMS replica capture-bene da un perforatore con interno-diametro di 0.75 mm per il canale fluidica (Figura 2A). </li><li> Utilizzare nastro per pulire la superficie delle repliche PDMS, e quindi posizionare le repliche PDMS un detersivo plasma per breve trattamento al plasma di ossigeno (100 watt per 14 sec). </li><li> Rimuovere le repliche PDMS dalla macchina plasma di ossigeno. </li><li> Allineare un top (contenente i pozzetti di cattura) e un PDMS fondo (che contengono i pozzetti di coltura) replica a mano allo stereomicroscopio e metterli in contatto. </li><li> Posizionare i PDMS repliche allineate in un forno a 65 ° C per 24 ore per ottenere l&#39;unione permanente tra le PDMS repliche per formare il dispositivo finale. </li><li> Bagnare il dispositivo PDMS in un deionizzata (DI) -acqua contenitore riempito e posizionare il contenitore in un essiccatore sotto vuoto (-85 kPa) per 15 minuti per eliminare l&#39;aria dalla microcanali del dispositivo PDMS. </li><li> Posizionare il dispositivo PDMS piena d&#39;acqua DI in una cappa coltura tissutale e utilizzando luce UV (lunghezza d&#39;onda: 254 nm) per sterilizzare il dispositivo per 30 min. </li><li> Sostituire l&#39;acqua deionizzata nel dispositivo PDMS con un siero albumina bovina 5% (BSA) in soluzione PBS 1x e incubare a 37 ° C per 30 minuti per impedire alle cellule di attaccarsi alla superficie PDMS. Nota: Il rivestimento BSA è essenziale per migliorare l&#39;efficienza di trasferimento di cellule isolate dalla cattura pozzetti per coltura pozzetti. </li><li> Sostituire la soluzione BSA 5% nel dispositivo PDMS con una soluzione di PBS 1x sterilizzata. </li></ol> 3. Preparazione di cella singola sospensione <ol><li> Culture Neural staminali KT98 cella e A549 cellule di carcinoma umano MDA-MB-435 che in piastra di Petri in coltura cellulare convenzionale incubatore (37 ° C, 5% di CO 2, e 95% di umidità) per preparare le cellule per l&#39;esperimento PDMS cella periferica . </li><li> Rimuovere e scartare il terreno di coltura esaurito (DMEM medio basale in dotazione con il 10% di siero fetale bovino e 1% di antibiotici) dal piatto di coltura con cellule coltivate a 70 - 80% di confluenza. </li><li> Lavare delicatamente le cellule con sterilizzati PBS per tre volte. &lt;/ Li&gt; <li> Rimuovere e scartare il PBS e aggiungere 2 ml di una miscela di enzimi proteolitici ricombinante con, collagenolitica, e le attività di DNasi (si veda l&#39;elenco dei materiali per le informazioni dettagliate). </li><li> Incubare il piatto cultura a temperatura ambiente per 5 minuti, quindi toccare il piatto cultura per facilitare il distacco delle cellule. </li><li> Aggiungere 4 ml di PBS sterile per disperdere le cellule, e poi trasferire la sospensione cellulare in un tubo da 15 ml. </li><li> Centrifugare la provetta a 300 xg per 3 min e rimuovere il surnatante. </li><li> Risospendere delicatamente il pellet di cellule in 1 ml sterilizzati PBS e contare il numero di cellulare dal vivo con il metodo standard di esclusione Trypan Blue 13. Nota: questo passaggio risospensione è fondamentale per la preparazione di ben dissociato-cella singola sospensione per migliorare l&#39;efficienza di isolamento cella singola. </li></ol> 4. Isolamento Single-cell e cultura clonale <ol><li> Carico 50 ml di sospensione cellulare ad una concentrazione di 2,2 - 2.5 x10 <sup&gt; 6 cellule / ml nel microcanali del dispositivo PDMS tramite foro di uscita del dispositivo con una pipetta palmare. Nota: Il caricamento della pipetta sospensione cellulare deve essere fermato e tenuto alla prima posizione di arresto per evitare l&#39;introduzione di bolle d&#39;aria nel microcanali. </li><li> Caricare altri 50 ml di sospensione cellulare attraverso il foro di ingresso per riempire in modo uniforme l&#39;intero microcanali con cellule. </li><li> Sigillare il foro di uscita con un tappo (a lunghezza 3 mm, 1 mm di diametro nylon taglio lenza) per evitare il flusso indotto dalla pressione idrostatica dalle goccioline di fori di ingresso e di uscita. Nota: le spine erano UV sterilizzato all&#39;interno di una cappa coltura tissutale prima dell&#39;uso. </li><li> Riempire una siringa da 1 ml sterile con terreno di coltura, espellere le bolle d&#39;aria da esso, e impostarlo su una pompa a siringa. </li><li> Collegare il supporto caricato siringa al foro di ingresso del dispositivo PDMS tramite un ago G smussata 23 ed una poli-tetrafluoroethene tubazione (PTFE). </li><li> Togliere la spina dalla presa di corrente e tutto il foro diow un intervallo di tempo di 2 minuti per consentire alle cellule di stabilirsi nella cella singola cattura-pozzi di forza gravitazionale. </li><li> Lavare via le cellule non catturate con 300 ml di terreno di coltura ad una portata di 600 ml / min spinto dalla pompa a siringa. </li><li> Attendere 2 minuti per stabilizzare il dispositivo, e sigillare i fori di ingresso e di uscita con spine per formare un sistema di coltura chiuso. </li><li> Capovolgere il dispositivo a mano per trasferire i catturati singole cellule ai pozzetti di coltura. </li><li> Posizionare il dispositivo PDMS in un piatto di coltura di tessuti da 100 mm, e aggiungere 10 ml di PBS sterile intorno al dispositivo per evitare l&#39;evaporazione cultura media dal microcanali. </li><li> Spostare il piatto cultura di un incubatore tradizionale coltura cellulare (37 ° C, 5% di CO 2, e 95% di umidità) per la cultura clonale delle singole cellule. </li></ol> 5. terreno di coltura Replenishment <ol><li> Dopo 1 d della cultura, sostituire il terreno di coltura nel dispositivo PDMS con terreno fresco per migliorare pr celluleoliferation. </li><li> Inserire due gocce di mezzo di coltura sulla parte superiore del dispositivo PDMS vicino alle aree fori di ingresso e di uscita per evitare l&#39;introduzione di bolle d&#39;aria nel microcanale durante l&#39;esecuzione della fase successiva. </li><li> Punch due fori dalla parte superiore del dispositivo PDMS prossimità delle due estremità del microcanale come un ingresso e un&#39;uscita per il microcanali. Nota: non perforare attraverso l&#39;intero due strati del dispositivo PDMS; invece, proprio pugno attraverso lo strato superiore delle PDMS. </li><li> Collegare una siringa di plastica da 1 ml contenente mezzo fresco per l&#39;ingresso attraverso un ago G smussata 23 e tubi in PTFE. </li><li> Portata media 120 microlitri fresca nel dispositivo per 5 minuti per sostituire il vecchio mezzo. </li><li> Inserire due tappi per sigillare entrata e uscita fori, e restituire il dispositivo per l&#39;incubatore di coltura cellulare. </li><li> Aggiornare il terreno di coltura ogni 2 d durante il periodo di coltura rimuovendo le spine di tenuta, seguito ripetendo i punti 5.4 al 5.5. </li></ol>

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The authors declare that they have no competing financial interests.

Sistemi di dispositivi di pozzetti a base di 6,14 sono stati utilizzati per la manipolazione a cella singola e di analisi, come ad esempio su larga scala singola cellula intrappolando 6 e la proliferazione delle cellule staminali ematopoietiche singolo 15. Sebbene dimensioni ben, il numero e la forma può essere regolata per applicazioni specifiche, l&#39;efficienza di isolamento singola cella è sempre compromessa quando si aumenta la dimensione del pozzo. 9,15 <p class="...

Attualmente posizionando le celle singole singolarmente in uno spazio culturale è comunemente raggiunto utilizzando la diluizione limitando o cella a fluorescenza-attivato (FACS). Per molti laboratori, diluizione limite è un metodo conveniente, in quanto richiede solo una pipetta e piastre di coltura di tessuto, che sono prontamente disponibili. In questo caso, una sospensione cellulare viene diluito serialmente ad una densità cellulare appropriata, e quindi posto in pozzetti di coltura utilizzando una pipetta manuale. Queste cellule singoli compartimenti sono poi utilizzati per l&#39;analisi delle cellule, come ad esempio lo screening eterogeneità genetica 1 e colonia di formazione 2. Tuttavia, questo metodo è basso throughput e intensità di lavoro, senza utilizzare un braccio robotico per l&#39;assistenza, perché la natura distribuzione di Poisson del metodo di diluizione limitante limita eventi monocellulari ad una probabilità massima del 37% 3. macchine FACS con un braccio robotico integrato in grado di superare la limitazione della distribuzione di Poisson con precisione placing una singola cella in una cultura ben alla volta 4. Tuttavia, l&#39;elevato sforzo di taglio meccanico (quindi abbassato vitalità cellulare) 5 ed acquisto della macchina e dei costi operativi hanno limitato il suo utilizzo in molti laboratori. Per superare le limitazioni di cui sopra, i dispositivi microscala sono stati sviluppati per caricare altamente efficiente singole cellule nei pozzetti 6. Tuttavia, i pozzetti non forniscono spazio sufficiente per le cellule caricate a proliferare, a causa della necessità di rendere le dimensioni di ciascun pozzetto vicino a quello di una singola cella di massimizzare il carico probabilità cella singola. Come test di cultura sono necessari in molte applicazioni basati su celle (ad esempio, test clonogenica 7), pozzetti più grandi (90-650 micron di diametro o in lunghezza del lato) sono stati utilizzati per consentire colture cellulari estesi. Tuttavia, come il metodo di diluizione limitante, possiedono anche bassi singole efficienze di carico delle cellule, che vanno da 10 -. 30% 8, 9 In precedenza, abbiamo sviluppato una piattaforma microfluidica high-throughput per isolare singole cellule nei singoli pozzetti e dimostrare la sua applicazione nel saggio clonogenica delle cellule isolate. 10 Il dispositivo è stato realizzato con poli-dimetilsiloxano (PDMS), e comprende due serie di array di pozzetti con differenti formati micropozzetti, che può sostanzialmente migliorare l&#39;efficienza nel caricare una singola cella in un micropozzetto cui dimensione è significativamente più grande della cella. In particolare, questo concetto &quot;dual-well&quot; permette la dimensione dell&#39;area culturale di regolare in modo flessibile senza compromettere l&#39;efficienza di cattura cella singola, rendendo semplice per regolare la progettazione del dispositivo per soddisfare diversi tipi di cellule e applicazioni. Questo metodo ad alta efficienza dovrebbe essere utile per esperimenti di coltura cellulare a lungo termine per gli studi eterogeneità delle cellule e monoclonale stabilimento linea cellulare.

<table border="0" cellpadding="0" cellspacing="0" width="1323">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">AutoCAD software</td>
			<td style="width:225px;">Autodesk</td>
			<td style="width:235px;">AutoCAD LT 2011</td>
			<td style="width:463px;">Part No. 057C1-74A111-1001</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silicon wafer&#160;</td>
			<td>Eltech corperation</td>
			<td style="width:235px;">SPE0039</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Conventional oven</td>
			<td>YEONG-SHIN company</td>
			<td style="width:235px;">ovp45</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plasma cleaner</td>
			<td>Nordson</td>
			<td style="width:235px;">AP-300</td>
			<td>Bench-Top Plasma Treatment System</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SU-8 50 negative photoresist</td>
			<td>MicroChem</td>
			<td>Y131269</td>
			<td style="width:463px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SU-8 100 negative photoresist</td>
			<td>MicroChem</td>
			<td>Y131273</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Spin coater</td>
			<td>Synrex Co., Ltd.</td>
			<td>SC-HMI 2&#34; ~ 6&#34;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hotplate</td>
			<td>YOTEC company</td>
			<td>YS-300S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Msak aligner</td>
			<td>Deya Optronic CO.</td>
			<td>A1K-5-MDA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SU-8 developer</td>
			<td>Grand Chemical Companies</td>
			<td>GP5002-000000-72GC</td>
			<td>Propylene glycol monomethyl ether acetate</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scanning laser profilometer</td>
			<td>KEYENCE</td>
			<td>VK-X 100</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Trichlorosilane</td>
			<td>Gelest, Inc</td>
			<td>SIT8174.0</td>
			<td style="width:463px;">TRIDECAFLUORO-1,1,2,2-TETRAHYDROOCTYL.&#160; Hazardous. Corrosive to the respiratory tract., reacts violently with water.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Desiccator</td>
			<td>Bel-Art Products&#160;</td>
			<td>F42020-0000</td>
			<td>SPACE SAVER VACUUM DESICCATOR 190MM WHITE BASE</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polydimethylsiloxane (PDMS) kit</td>
			<td>Dow corning</td>
			<td>Sylgard 184</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Harris Uni-Core puncher</td>
			<td>Ted Pella Inc.</td>
			<td>15072</td>
			<td>with 0.75 mm inner-diameter</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Removable tape</td>
			<td>3M Company</td>
			<td>Scotch Removable Tape 811</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Stereomicroscope</td>
			<td>Leica Microsystems</td>
			<td>Leica E24</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine serum albumin (BSA)</td>
			<td>Bersing Technology</td>
			<td>ALB001.500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM basal medium</td>
			<td>Gibco</td>
			<td style="width:235px;">12800-017</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal bovine serum</td>
			<td>Thermo Hyclone</td>
			<td>SH30071.03HI</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Antibiotics</td>
			<td>Biowest</td>
			<td>L0014-100</td>
			<td>Glutamine-Penicillin-Streptomycin</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Recombinant enzyme mixture</td>
			<td>Innovative cell technology</td>
			<td style="width:235px;">AM-105</td>
			<td>Accumax</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DiIC12(3) cell membrane dye</td>
			<td>BD Biosciences</td>
			<td style="width:235px;">354218</td>
			<td>Used as a cell tracker</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe pump</td>
			<td>Harvard Apparatus</td>
			<td style="width:235px;">703007</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plastic syringe (1 mL)</td>
			<td>BD Biosciences</td>
			<td style="width:235px;">309659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">23 gauge blunt needles</td>
			<td>Ever Sharp Technology, Inc.</td>
			<td style="width:235px;">TD21</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Poly-tetrafluoroethene (PTFE) tubing</td>
			<td>Ever Sharp Technology, Inc.</td>
			<td style="width:235px;">TFT-23T</td>
			<td>&#160;inner diameter, 0.51 mm; outer diameter, 0.82 mm</td>
		</tr>
	</tbody>
</table>

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.

La piattaforma microfluidica per l&#39;isolamento e la coltura singola cella comprende un microcanale (200 micron di altezza) con due serie di matrici di micropozzetti (Figura 2A). Le due serie di array di pozzetti sono definiti come la cattura-bene (25 micron di diametro e 27 micron di profondità) e la cultura-bene (285 micron di diametro e 300 micron di profondità) per l&#39;isolamento a cella singola e cultura, rispettivamente, e ogni cattura-well è posizionata al ...

Watch this Scientific Journal Video about A Microfluidic Platform for High-throughput Single-cell Isolation and Culture at JoVE.com

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

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 ...

a-microfluidic-platform-for-high-throughput-single-cell-isolation

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

11.2K Views.  National Health Research Institutes, Taiwan. 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. 

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

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

High Throughput Single-cell and Multiple-cell Micro-encapsulation

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement from a bulk fluid environment with applications in high throughput screening, cytometry, and mass spectrometry. We describe a method to not only encapsulate single cells, but to repeatedly capture a set number of cells (here we demonstrate one- and two-cell encapsulation) to study both isolation and the interactions between cells in groups of controlled sizes. By combining drop generation techniques with cell and particle ordering, we demonstrate controlled encapsulation of cell-sized particles for efficient, continuous encapsulation. Using an aqueous particle suspension and immiscible fluorocarbon oil, we generate aqueous drops in oil with a flow focusing nozzle. The aqueous flow rate is sufficiently high to create ordering of particles which reach the nozzle at integer multiple frequencies of the drop generation frequency, encapsulating a controlled number of cells in each drop. For representative results, 9.9 &mu;m polystyrene particles are used as cell surrogates. This study shows a single-particle encapsulation efficiency Pk=1 of 83.7% and a double-particle encapsulation efficiency Pk=2 of 79.5% as compared to their respective Poisson efficiencies of 39.3% and 33.3%, respectively. The effect of consistent cell and particle concentration is demonstrated to be of major importance for efficient encapsulation, and dripping to jetting transitions are also addressed. 
Introduction 
Continuous media aqueous cell suspensions share a common fluid environment which allows cells to interact in parallel and also homogenizes the effects of specific cells in measurements from the media. High-throughput encapsulation of cells into picoliter-scale drops confines the samples to protect drops from cross-contamination, enable a measure of cellular diversity within samples, prevent dilution of reagents and expressed biomarkers, and amplify signals from bioreactor products. Drops also provide the ability to re-merge drops into larger aqueous samples or with other drops for intercellular signaling studies.1,2 The reduction in dilution implies stronger detection signals for higher accuracy measurements as well as the ability to reduce potentially costly sample and reagent volumes.3 Encapsulation of cells in drops has been utilized to improve detection of protein expression,4 antibodies,5,6 enzymes,7 and metabolic activity8 for high throughput screening, and could be used to improve high throughput cytometry.9 Additional studies present applications in bio-electrospraying of cell containing drops for mass spectrometry10 and targeted surface cell coatings.11 Some applications, however, have been limited by the lack of ability to control the number of cells encapsulated in drops. Here we present a method of ordered encapsulation12 which increases the demonstrated encapsulation efficiencies for one and two cells and may be extrapolated for encapsulation of a larger number of cells.
To achieve monodisperse drop generation, microfluidic "flow focusing" enables the creation of controllable-size drops of one fluid (an aqueous cell mixture) within another (a continuous oil phase) by using a nozzle at which the streams converge.13 For a given nozzle geometry, the drop generation frequency f and drop size can be altered by adjusting oil and aqueous flow rates Qoil and Qaq. As the flow rates increase, the flows may transition from drop generation to unstable jetting of aqueous fluid from the nozzle.14 
When the aqueous solution contains suspended particles, particles become encapsulated and isolated from one another at the nozzle. For drop generation using a randomly distributed aqueous cell suspension, the average fraction of drops Dk containing k cells is dictated by Poisson statistics, where Dk = &lambda;k exp(-&lambda;)/(k!) and &lambda; is the average number of cells per drop. The fraction of cells which end up in the "correctly" encapsulated drops is calculated using Pk = (k x Dk)/&Sigma;(k' x Dk'). The subtle difference between the two metrics is that Dk relates to the utilization of aqueous fluid and the amount of drop sorting that must be completed following encapsulation, and Pk relates to the utilization of the cell sample. As an example, one could use a dilute cell suspension (low &lambda;) to encapsulate drops where most drops containing cells would contain just one cell. While the efficiency metric Pk would be high, the majority of drops would be empty (low Dk), thus requiring a sorting mechanism to remove empty drops, also reducing throughput.15
Combining drop generation with inertial ordering provides the ability to encapsulate drops with more predictable numbers of cells per drop and higher throughputs than random encapsulation. Inertial focusing was first discovered by Segre and Silberberg16 and refers to the tendency of finite-sized particles to migrate to lateral equilibrium positions in channel flow. Inertial ordering refers to the tendency of the particles and cells to passively organize into equally spaced, staggered, constant velocity trains. Both focusing and ordering require sufficiently high flow rates (high Reynolds number) and particle sizes (high Particle Reynolds number).17,18 Here, the Reynolds number Re =uDh/&nu; and particle Reynolds number Rep =Re(a/Dh)2, where u is a characteristic flow velocity, Dh [=2wh/(w+h)] is the hydraulic diameter, &nu; is the kinematic viscosity, a is the particle diameter, w is the channel width, and h is the channel height. Empirically, the length required to achieve fully ordered trains decreases as Re and Rep increase. Note that the high Re and Rep requirements (for this study on the order of 5 and 0.5, respectively) may conflict with the need to keep aqueous flow rates low to avoid jetting at the drop generation nozzle. Additionally, high flow rates lead to higher shear stresses on cells, which are not addressed in this protocol. The previous ordered encapsulation study demonstrated that over 90% of singly encapsulated HL60 cells under similar flow conditions to those in this study maintained cell membrane integrity.12 However, the effect of the magnitude and time scales of shear stresses will need to be carefully considered when extrapolating to different cell types and flow parameters. The overlapping of the cell ordering, drop generation, and cell viability aqueous flow rate constraints provides an ideal operational regime for controlled encapsulation of single and multiple cells.
Because very few studies address inter-particle train spacing,19,20 determining the spacing is most easily done empirically and will depend on channel geometry, flow rate, particle size, and particle concentration. Nonetheless, the equal lateral spacing between trains implies that cells arrive at predictable, consistent time intervals. When drop generation occurs at the same rate at which ordered cells arrive at the nozzle, the cells become encapsulated within the drop in a controlled manner. This technique has been utilized to encapsulate single cells with throughputs on the order of 15 kHz,12 a significant improvement over previous studies reporting encapsulation rates on the order of 60-160 Hz.4,15 In the controlled encapsulation work, over 80% of drops contained one and only one cell, a significant efficiency improvement over Poisson (random) statistics, which predicts less than 40% efficiency on average.12
In previous controlled encapsulation work,12 the average number of particles per drop &lambda; was tuned to provide single-cell encapsulation. We hypothesize that through tuning of flow rates, we can efficiently encapsulate any number of cells per drop when &lambda; is equal or close to the number of desired cells per drop. While single-cell encapsulation is valuable in determining individual cell responses from stimuli, multiple-cell encapsulation provides information relating to the interaction of controlled numbers and types of cells. Here we present a protocol, representative results using polystyrene microspheres, and discussion for controlled encapsulation of multiple cells using a passive inertial ordering channel and drop generation nozzle.

Combining monodisperse drop generation with inertial ordering of cells and particles, we describe a method to encapsulate a desired number of cells or particles in a single drop at kHz rates. We demonstrate efficiencies twice exceeding those of unordered encapsulation for single- and double-particle drops.

High Throughput singola cellula e cellula Multiple-Micro-incapsulamento

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement ...

CometChip: A High-throughput 96-Well Platform for Measuring DNA Damage in Microarrayed Human Cells

DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular metabolites. Ironically, at high doses DNA damaging agents are also used to treat cancer. The ability to quantify DNA damage responses is thus critical in the public health, pharmaceutical and clinical domains. Here, we describe a novel platform that exploits microfabrication techniques to pattern cells in a fixed microarray. The &#8216;CometChip&#8217; is based upon the well-established single cell gel electrophoresis assay (a.k.a. the comet assay), which estimates the level of DNA damage by evaluating the extent of DNA migration through a matrix in an electrical field. The type of damage measured by this assay includes abasic sites, crosslinks, and strand breaks. Instead of being randomly dispersed in agarose in the traditional assay, cells are captured into an agarose microwell array by gravity. The platform also expands from the size of a standard microscope slide to a 96-well format, enabling parallel processing. Here we describe the protocols of using the chip to evaluate DNA damage caused by known genotoxic agents and the cellular repair response followed after exposure. Through the integration of biological and engineering principles, this method potentiates robust and sensitive measurements of DNA damage in human cells and provides the necessary throughput for genotoxicity testing, drug development, epidemiological studies and clinical assays.

We describe here a platform that allows comet assay detection of DNA damage with unprecedented throughput. The device patterns mammalian cells into a microarray and enables parallel processing of 96 samples. The approach facilitates analysis of base level DNA damage, exposure-induced DNA damage and DNA repair kinetics.

CometChip: A High-throughput di 96-Well Piattaforma per la misurazione dei danni cagionati DNA in cellule umane Microarrayed

DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular ...

High Throughput Microfluidic Rapid and Low Cost Prototyping Packaging Methods

In this work, 3 different packaging and assembly techniques are presented. They can be classified into two categories: one-time use and reusable packaging techniques.
The one-time use packaging technique employs UV-based and temperature curing epoxies to connect microtubes to access holes, wire-bonding for integrated circuit connections, and silver epoxy for electrical connections. This method is based on a robust assembly technique that can support relatively high pressure close to 1 psi and does not need any support to strengthen the microfluidic architecture.
Reusable packaging techniques consist of PDMS-based microtube interconnectors and anisotropic adhesive films for electrical connections. These devices are more sensitive and fragile. Consequently, Plexiglas support is added to the microfluidic structure to improve the electrical contact when anisotropic adhesive films are used, and also to strengthen the microfluidic architecture. In addition, a micromanipulator is needed to maintain tubes while using a thin PDMS layer to connect them to the access holes. Different PDMS layer thicknesses, ranging from 0.45-3 mm, are tested to compare the best adherence versus injection rates. Applied injection rates are varied from 50-300 &#956;l/hr for 0.45-3 mm PDMS layers, respectively. These techniques are mainly applicable for low-pressure applications. However, they can be extended for high-pressure ones through plasma-oxygen process to permanently seal the PDMS to glass substrates. The main advantage of this technique, besides the fact that it is reusable, consists of keeping the device observable when the microchannel length is very short (in the range of 3 mm or lower).

In this article we describe different techniques for microfluidic rapid prototyping platforms. The proposed techniques are based on ultraviolet (UV) sensitive and temperature curing epoxies, polydimethylsiloxane (PDMS) based tubing, wire-bonding, and anisotropic adhesive films. The assembling procedures presented are developed for both one-time use devices as well as reusable microfluidic systems.

High Throughput Microfluidic rapido ed economico prototipazione metodi a basso Packaging

In this work, 3 different packaging and assembly techniques are presented. They can be classified into two categories: one-time use and reusable packaging ...

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 ...

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 ...

A High-throughput Cell Microarray Platform for Correlative Analysis of Cell Differentiation and Traction Forces

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly controlled, high-throughput engineered system for measuring the impact of arrayed biochemical signals on cell differentiation. Recent efforts using cell microarrays have demonstrated their utility for combinatorial studies in which many microenvironmental factors are presented in parallel. However, these efforts have focused primarily on investigating the effects of biochemical cues on cell responses. Here, we present a cell microarray platform with tunable material properties for evaluating both cell differentiation by immunofluorescence and biomechanical cell&#8211;substrate interactions by traction force microscopy. To do so, we have developed two different formats utilizing polyacrylamide hydrogels of varying Young&#39;s modulus fabricated on either microscope slides or glass-bottom Petri dishes. We provide best practices and troubleshooting for the fabrication of microarrays on these hydrogel substrates, the subsequent cell culture on microarrays, and the acquisition of data. This platform is well-suited for use in investigations of biological processes for which both biochemical (e.g., extracellular matrix composition) and biophysical (e.g., substrate stiffness) cues may play significant, intersecting roles.

Cell differentiation is regulated by a host of microenvironmental factors, including both matrix composition and substrate material properties. We describe here a technique utilizing cell microarrays in conjunction with traction force microscopy to evaluate both cell differentiation and biomechanical cell&#8211;substrate interactions as a function of microenvironmental context.

Una piattaforma cellulare microarray high-throughput per l&#39;analisi correlativa cellulare Differenziazione e forze di trazione

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly ...

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 ...

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 ...

Una piattaforma Microfluidic per l'isolamento Single-cell high-throughput e cultura

Riepilogo

Esplora Altri Video

Capitoli in questo video

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

High Throughput singola cellula e cellula Multiple-Micro-incapsulamento

CometChip: A High-throughput di 96-Well Piattaforma per la misurazione dei danni cagionati DNA in cellule umane Microarrayed

High Throughput Microfluidic rapido ed economico prototipazione metodi a basso Packaging

Spremere cellulare come un robusto, Microfluidic intracellulare Delivery Platform

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

Una piattaforma cellulare microarray high-throughput per l'analisi correlativa cellulare Differenziazione e forze di trazione

Una piattaforma microfluidica per Imaging longitudinale in Caenorhabditis elegans

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

Una piattaforma microfluidica per stimolare i condrociti con la compressione dinamica

Una piattaforma Microfluidic per l'isolamento Single-cell high-throughput e cultura

Riepilogo

Esplora Altri Video

Capitoli in questo video

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

High Throughput singola cellula e cellula Multiple-Micro-incapsulamento

CometChip: A High-throughput di 96-Well Piattaforma per la misurazione dei danni cagionati DNA in cellule umane Microarrayed

High Throughput Microfluidic rapido ed economico prototipazione metodi a basso Packaging

Spremere cellulare come un robusto, Microfluidic intracellulare Delivery Platform

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

Una piattaforma cellulare microarray high-throughput per l'analisi correlativa cellulare Differenziazione e forze di trazione

Una piattaforma microfluidica per Imaging longitudinale in Caenorhabditis elegans

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

Una piattaforma microfluidica per stimolare i condrociti con la compressione dinamica

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