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

Hinweis: Die Fotomasken-Designs für unsere Mikrofluidik-Vorrichtung Fertigung wurden gezeichnet von einem Computer Aided Design (CAD) Software. Die Entwürfe wurden dann verwendet, um Chrom Photomasken herzustellen Kommerzielle Dienst. Die PDMS - Geräte wurden unter Verwendung von weichen Lithographietechniken hergestellt. 11 1. Herstellung von Urformen von Lithography <ol><li> Vor dem photolithographischen Prozeß 12, verwenden , um die 4-Zoll - Siliziumwafer als Substrat und entwässern um die Wafer in einem konventionellen Ofen bei 120 ° C für 10 min. </li><li> Reinigen Sie die dehydriert Silizium-Wafern durch Sauerstoffplasmabehandlung bei 100 Watt für 30 Sekunden in einem Plasma-Reiniger. </li><li> Vorwärmen zwei Kochplatten bei 65 ° C und 95 ° C, jeweils für die nachfolgenden Backvorgang. </li><li> Mantel 5 g negativer Photoresist (PR) auf den gereinigten Siliziumwafer durch eine Schleuderbeschichtungsvorrichtung; Spin bei 1200 Umdrehungen pro Minute (SU-8 50) für 30 Sekunden die Mikrokanalschicht zu erzeugen. </li><li> Legen Sie die PRbeschichteten Wafers auf eine vorgeheizte Heizplatte bei 65 ° C für 12 min und übertragen sie für 33 min bei 95 ° C auf einen anderen vorgewärmte Heizplatte (100 &amp; mgr; m dicken Muster) einen weichen Bake-Verfahren durchzuführen. </li><li> Nach dem Backen, legen Sie die PR beschichteten Silizium-Wafer auf dem Halter eines halbautomatischen Mask Aligner und richten Sie sie auf eine 25.400 dpi Auflösung Transparenz Photomaske. </li><li> Setzen Sie den PR beschichteten Silizium - Wafers mit UV - Licht (365 nm) in einer Dosis von 500 mJ / cm 2 , um die PR - Muster auf dem Silizium - Wafer zu schaffen. </li><li> Entfernen Sie den Wafer von dem Ausrichter und legen Sie es auf einer Heizplatte für die Zeit nach dem Backen bei 95 ° C für 12 min. </li><li> Weichen Sie die Wafer in SU-8-Entwickler (Propylenglykolmonomethylether Acetat, PGMEA) -Lösung nicht vernetzten PR für 12 min zu waschen und sanft mit Stickstoffgas trocken die Ausrichtungsmarken zu belichten. </li><li> Wiederum Mantel 5 g negativen Photoresists auf den Wafern durch eine Schleuderbeschichtungsvorrichtung; Spin bei 700 Umdrehungen pro Minute (SU-8 100) für 30 Sekunden und 1200 Umdrehungen pro Minute (SU-8 10) 30 s für 300 &amp;# 181; m dicke Muster und 27 &amp; mgr; m dicken Muster jeweils die Mikrowell-Schicht zu machen. </li><li> Platzieren Sie den PR beschichtete Wafer auf einer Heizplatte bei 65 ° C für 4 min und bei 95 ° C für 8 min (27 &amp; mgr; m tief capture-well layer); und bei 65 ° C für 40 min und bei 95 ° C für 110 min (bei 300 &amp; mgr; m tief Kultur-well layer). </li><li> Nach dem Abkühlen legen Sie die PR-beschichteten Silizium-Wafer auf dem Mask Aligner mit UV-Licht ausgestattet. </li><li> Setzen Sie den PR beschichteten Silizium - Wafer mit dem UV - Licht (365 nm) in einer Dosis von 250 mJ / cm 2 (27 &amp; mgr; m dicken Muster) und 700 mJ / cm 2 (300 &amp; mgr; m dicken Muster). </li><li> Backen Sie die Wafer bei 95 ° C für 5 min (27 &amp; mgr; m dicken Muster) und 30 min (300 &amp; mgr; m dicken Muster) sind. </li><li> Waschen Sie die nicht vernetzten PR durch die PGMEA für 6 min (27 &amp; mgr; m dicken Muster) und 25 min (300 &amp; mgr; m dicken Muster) ab, bzw., und dann trocknen Sie sie mit Stickstoffgas. </li><li> Messen Sie die Höhe der Muster-Funktionen aufder Wafer mit einem Scanning-Laser-Profilometer, indem der Wafer auf dem XY-Tisch eines abtastenden Laser-Profilometer platzieren. <ol><li> Stellen Sie die Brennebene deutlich das Muster zeigen, auf dem Wafer verfügt mit dem &quot;Kameraansicht&quot; Beobachtungsmodus unter einem 20X Objektiv. </li><li> Schalten Sie den Beobachtungsmodus von &quot;Kameraansicht&quot; auf &quot;Laser-Ansicht&quot;, und stellen Sie die oberen und unteren Positionen der Merkmale. </li><li> Stellen Sie den Messmodus, Bereich, Qualität und z-Pitch bis transparent (oben), 1 Zeile (1.024 x 1), mit hoher Genauigkeit und 0,5 &amp; mgr; m sind, und drücken Sie dann die Start unten, um die Messung zu beginnen. </li></ol></li></ol> 2. Herstellung von PDMS-Geräte für Einzelzell-Isolierung <ol><li> Vor PDMS Gießen, silanisieren die Urformen mit Trichlorsilan eine hydrophobe Oberfläche zu schaffen, die es leichter zu schälen die PDMS Repliken von den Urformen macht. <ol><li> Legen Sie die Urformen und einen Gewichtungs Schiffchen mit200 &amp; mgr; l von Trichlorsilan in einem Exsikkator und gelten Vakuum (-85 kPa) für 15 min. </li><li> Stoppen Sie das Vakuum und dann die Stammformen im Exsikkator verlassen für mindestens 1 Stunde die Urformen bei Raumtemperatur silanisieren. </li><li> Entfernen Sie die Urformen aus dem Exsikkator und platzieren Sie jede Master-Form in einer 10 cm Petrischale. </li></ol></li><li> Mischen Sie eine Gesamtmenge von 17,6 g PDMS-Polymer-Kit eine Basis und einen Härter in einem Verhältnis von 10: 1 enthält, und dann gießen Sie die PDMS auf die Urform in der Petrischale. </li><li> Legen Sie die Petrischale in einem Exsikkator und gelten Vakuum (-85 kPa) für 1 h Luftblasen in den PDMS zu entfernen. </li><li> Entfernen Sie die Petrischale aus dem Exsikkator und legen Sie sie in einem herkömmlichen Ofen bei 65 ° C für 3-6 Stunden die PDMS zu heilen. </li><li> Entfernen des ausgehärteten PDMS Replikate aus den Stammformen und Punch zwei Löcher als Einlass und einen Auslass an den beiden Enden des Mikrokanals auf der capture-Well Array PDMS Replik durch eine Stanze mit einem Innendurchmesser von 0.75 mm für den fluidischen Kanal (2A). </li><li> Verwenden Sie Klebeband um die Oberfläche der PDMS-Repliken zu reinigen, und dann die PDMS-Repliken in einem Plasma-Reiniger für kurze Sauerstoffplasmabehandlung (100 Watt für 14 sec). </li><li> Entfernen Sie die PDMS-Repliken aus der Sauerstoffplasma-Maschine. </li><li> Richten Sie eine Top (mit den Capture-Mikrotitervertiefungen) und eine untere PDMS (mit den Kultur Mikrotiterstreifen) Replik von Hand unter einem Stereomikroskop und bringt sie in Kontakt. </li><li> Platzieren der ausgerichteten PDMS Replikate in einem Ofen bei 65 ° C für 24 h dauerhafte Verbindung zwischen den PDMS Repliken zu erreichen, um die endgültige Vorrichtung zu bilden. </li><li> Genießen Sie die PDMS-Gerät in einem deionisierten (DI) -Wasser gefüllten Behälter und stellen Sie den Behälter in einem Exsikkator unter Vakuum (-85 kPa) für 15 Minuten Luft aus dem Mikrokanal des PDMS-Gerät zu entfernen. </li><li> Legen Sie das DI-Wasser gefüllten PDMS-Gerät in einer Gewebekultur Haube und verwenden UV-Licht (Wellenlänge des Lichts: 254 nm) der Vorrichtung zu sterilisieren 30 min. </li><li> Ersetzen den DI-Wasser in der PDMS-Gerät mit einer 5% Rinderserumalbumin (BSA) in 1x PBS-Lösung und Inkubation für 30 min bei 37 ° C-Zellen zu verhindern, auf die Oberfläche PDMS kleben. Hinweis: Die BSA-Beschichtung kritisch ist die Übertragungseffizienz von isolierten Zellen von der Aufnahme-gut zu kultur gut zu verbessern. </li><li> Ersetzen Sie die 5% BSA-Lösung in der PDMS-Gerät mit sterilisiertem 1x PBS-Lösung. </li></ol> 3. Herstellung von Einzelzellsuspension <ol><li> Kultur neurale Stammzelle KT98 und menschliche Karzinomzell A549 und MDA-MB-435 wir in Petrischale in konventionellen Zellkulturbrutschrank (37 ° C, 5% CO 2 und 95% Luftfeuchtigkeit) Zellen für die PDMS Vorrichtungszelle Experiment vorzubereiten . </li><li> Entfernen und entsorgen Sie die verbrauchte Kulturmedium (DMEM Basismedium versorgt mit 10% fötalem Rinderserum und 1% Antibiotika) aus der Kulturschale mit Zellen auf 70 gewachsen - 80% Konfluenz. </li><li> Waschen Sie sich leicht die Zellen mit sterilisiertem PBS dreimal. &lt;/ Li&gt; <li> Entfernen und entsorgen Sie die PBS und 2 ml eines rekombinanten Enzymmischung mit proteolytischen, kollagenolytischer und DNase Aktivitäten (bitte die Materialliste für die detaillierte Informationen). </li><li> Inkubieren der Kulturschale bei Raumtemperatur für 5 min und dann auf die Kulturschale Abgriffzelle Ablösung zu erleichtern. </li><li> 4 ml sterilisiertem PBS Zellen zu dispergieren, und dann übertragen die Zellsuspension in ein 15 ml konischen Röhrchen. </li><li> Zentrifugieren Sie die Röhrchen bei 300 g für 3 Minuten und entfernen Sie den Überstand. </li><li> Resuspendieren vorsichtig das Zellpellet in 1 ml sterilisiertem PBS und zählen Sie die Live - Zellzahl 13 den Standard Trypanblau Ausschlussverfahren. Hinweis: Dieser Aufwirbelung Schritt zur Herstellung von gut distanzierte Einzelzellsuspension kritisch ist es, die Einzelzellisolierung Effizienz zu verbessern. </li></ol> 4. Single-Zell-Isolierung und Geklonte Kultur <ol><li> Last 50 ul Zellsuspension in einer Konzentration von 2,2 bis 2,5 x10 <sup&gt; 6 Zellen / ml in den Mikrokanal des PDMS - Gerät über die Austrittsöffnung des Gerätes mit einer Handpipette. Hinweis: Die Zellsuspension Lade Pipette werden muss angehalten und an der ersten Stoppposition gehalten zu vermeiden Luftblasen in den Mikrokanal eingeführt wird. </li><li> Laden Sie weitere 50 ul Zellsuspension durch das Einlassloch gleichmäßig den gesamten Mikrokanal mit Zellen füllen. </li><li> Verschließen Sie die Austrittsöffnung mit einem Stopfen (3 mm lang, 1 mm Durchmesser geschnitten Nylon Angelschnur) um den Fluss zu vermeiden, induziert durch hydrostatischen Druck aus den Tröpfchen auf Eintritts- und Austrittslöcher. Hinweis: Die Stecker UV in einer Gewebekultur Haube sterilisiert waren vor der Verwendung. </li><li> Füllen Sie eine 1 ml sterile Spritze mit Kulturmedium nach oben, werfen Luftblasen aus es und setzte es auf eine Spritzenpumpe auf. </li><li> Schließen Sie das Medium geladen Spritze mit dem Einlassloch des PDMS-Gerät über einen 23 G stumpfe Nadel und einem Poly-tetrafluorethen (PTFE) Schläuche. </li><li> Ziehen Sie den Stecker aus der Steckdose Loch und alleow eine 2-Minuten Zeitintervall, damit die Zellen in die Single-Cell-Capture-Brunnen durch die Schwerkraft zu regeln. </li><li> Waschen der nicht erfaßten Zellen mit 300 &amp; mgr; l Kulturmedium mit einer Strömungsgeschwindigkeit von 600 ul / min entfernt von der Spritzenpumpe angetrieben. </li><li> Warten für 2 min um das Gerät zu stabilisieren, und Abdichten der Eintritts- und Austrittsöffnungen mit Stopfen einen geschlossenen Kultursystem zu bilden. </li><li> Drehen Sie das Gerät mit der Hand die aufgenommenen Einzelzellen auf die Kultur Mikrotitervertiefungen zu übertragen. </li><li> Legen Sie die PDMS-Gerät in einem 100-mm-Gewebekulturschale, und dann werden 10 ml sterilisiertem PBS um das Gerät Kulturmedium Verdampfen aus dem Mikrokanal zu vermeiden. </li><li> Bewegen der Kulturschale mit einem herkömmlichen Zellkulturbrutschrank (37 ° C, 5% CO 2 und 95% Luftfeuchtigkeit) für klonale Kultur der einzelnen Zellen. </li></ol> 5. Kulturmedium Replenishment <ol><li> Nach 1 d der Kultur, ersetzen Sie das Kulturmedium in der PDMS-Gerät mit frischem Medium Zelle pr zu verbessernoliferation. </li><li> Platzieren zwei Tröpfchen von Kulturmedium auf der Oberseite des PDMS-Gerät in der Nähe der Eintritts- und Austrittsöffnungen Bereiche Einführen von Luftblasen in den Mikrokanal zu vermeiden, während der nächste Schritt durchgeführt wird. </li><li> Stanzen zwei Löcher von der Oberseite der PDMS Vorrichtung in der Nähe der beiden Enden des Mikrokanals als Einlass und einem Auslass für den Mikrokanal. Hinweis: Verwenden Sie nicht durch die ganze zwei Schichten des PDMS Gerät Punch; statt, Punsch nur durch die oberste Schicht des PDMS. </li><li> Schließen Sie eine 1-ml-Plastikspritze mit frischem Medium mit dem Einlass über eine 23 G stumpfe Nadel und PTFE-Schlauch. </li><li> Strom 120 &amp; mgr; l frisches Medium in das Gerät für 5 min das alte Medium zu ersetzen. </li><li> Legen Sie zwei Stecker den Ein- und Austrittslöcher abzudichten, und schicken Sie das Gerät an die Zellkultur-Inkubator. </li><li> Aktualisieren das Kulturmedium alle 2 d während der Dauer der Kultur durch die Verschlussstopfen zu entfernen, gefolgt von den Schritten von 5,4 bis 5,5 zu wiederholen. </li></ol>

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The authors declare that they have no competing financial interests.

Mikrowell-basierten Gerätesysteme 6,14 wurden für die Einzelzell - Manipulation und Analyse, wie beispielsweise großflächige Einzelzelle verwendet und 6 einzigen hämatopoetischen Stammzellproliferation Trapping 15. Obwohl gut Größe, Anzahl und Form können für spezifische Anwendungen angepasst werden, die Einzelzellisolierung Effizienz wird immer gefährdet , wenn die Größe des Bohrlochs erhöht. 9,15 Um diese Einschränkung zu überwinde...

Derzeit Einzelzellen einzeln in einem Kulturraum platziert wird üblicherweise durch Verwendung limitierende Verdünnung oder fluoreszenzaktivierte Zellsortierung (FACS) erzielt. Für viele Laboratorien limitierende Verdünnung ist ein zweckmßiges Verfahren, da es nur eine Pipette und Gewebekulturplatten benötigt, die leicht verfügbar sind. In diesem Fall wird eine Zellsuspension zu einer geeigneten Zelldichte seriell verdünnt und dann in Kulturvertiefungen gegeben durch eine manuelle Pipette. Diese abgeteilten Einzelzellen werden dann für die Zellanalyse verwendet werden, wie genetische Heterogenität Screenen 1 und Koloniebildung 2. Jedoch ist dieses Verfahren mit niedrigem Durchsatz und arbeitsintensiv, ohne einen Roboterarm zur Unterstützung verwendet wird , weil die Poisson - Verteilung der Natur des Verfahrens limitierende Verdünnungseinzelzellereignisse bis zu einer maximalen Wahrscheinlichkeit von 37% 3 einschränkt. FACS-Maschinen mit einem integrierten Roboterarm kann die Begrenzung der Poisson-Verteilung zu überwinden, indem genau placing einer Einzelzelle in einer Kultur auch in einer Zeit 4. Die hohe mechanische Scherspannung jedoch (so senkte die Lebensfähigkeit der Zellen) 5 und Maschinenkauf und Betriebskosten haben seine Verwendung in vielen Labors beschränkt. Um die oben genannten Einschränkungen zu überwinden, mikroskaligen Geräte wurden entwickelt , um sehr effizient einzelne Zellen in Mikrovertiefungen 6 laden. Jedoch bieten die Mikrotitervertiefungen nicht ausreichend Platz für die beladenen Zellen zu proliferieren, aufgrund der Notwendigkeit der Herstellung der Größe jedes der von einer einzigen Zelle schließen Mikrotiterplatten-Einzelzelllade Wahrscheinlichkeit zu maximieren. Als Kulturassays in vielen zellbasierten Anwendungen erforderlich sind (zB Klonogentest 7), größere Kavitäten (90 bis 650 &amp; mgr; m im Durchmesser oder in Seitenlänge) wurden ebenfalls verwendet worden , für längere Zellkulturen zu ermöglichen. Jedoch, wie das Grenzverdünnungsverfahren, sie besitzen auch eine niedrige einzelne Zelle Ladewirkungsgrade im Bereich von 10 -. 30% 89 Zuvor haben wir ein Hochdurchsatz - mikrofluidischen Plattform zu isolieren Einzelzellen in den einzelnen Kavitäten entwickelt und zeigen ihre Anwendung in klonogenen Assay der isolierten Zellen. Die Vorrichtung 10 mit poly-dimethylsiloxan (PDMS) hergestellt wurde, und weist zwei Sätze von Mikrovertiefungsarrays mit verschiedenen Mikrovertiefungsgrößen, was die Effizienz in dem Laden einer einzelnen Zelle in einer Mikrovertiefung, deren Größe wesentlich größer als die Zelle weitgehend verbessern. Bemerkenswert ist, ermöglicht diese &quot;dual-well&quot; -Konzept die Größe des Kulturbereich flexibel, ohne dass die Einzelzellen-Abscheidungseffizienz zu beeinflussen eingestellt werden, so dass es einfach, die Konstruktion der Vorrichtung zur Einstellung verschiedener Zelltypen und Anwendungen anzupassen. Diese hocheffiziente Methode sollte für die langfristige Zellkulturexperimente für die Zell Heterogenität Studien und monoklonale Zelllinie Einrichtung nützlich sein.

<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.

Die mikrofluidische Plattform für Einzelzellisolierung und Kultur umfasst einen Mikrokanal (200 &amp; mgr; m in der Höhe) mit zwei Sätzen von Mikrovertiefungsanordnungen (2A). Die zwei Sätze von Mikrovertiefungsarrays werden als capture-Well (25 um Durchmesser und 27 um Tiefe) und Kultur-Well (285 &amp; mgr; m im Durchmesser und 300 &amp; mgr; m in der Tiefe) bezeichnet für die Einzelzell-Isolierung und Kultur sind und jeweils Capture-gut ist in der Mitte einer Kult...

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.

Ein Mikrofluidik-Plattform für Hochdurchsatz-Einzelzell-Isolierung und Kultur

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

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Research

JoVE Journal

Bioengineering

11.2K Aufrufe.  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. 

Serie: 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 Protein Expression Generator mit einem mikrofluidischen Plattform

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

Forschung

Biotechnik

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-Einzel-Zell-und Multiple-Zelle Mikroverkapselung

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: Eine Hochdurchsatz-96-Well-Plattform für Mess DNA-Schäden in menschlichen Zellen Microarray

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.

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.

Zellzusammendrücken als Robust, Mikrofluidik Intrazelluläre 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.

Mikrofluidik-Plattform mit Multiplexed elektronische Sensoren für Raumverfolgung von Partikeln

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.

Ein Hochdurchsatz-Zellarray-Plattform für korrelative Analyse von Zelldifferenzierung und Zugkräften

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.

Mikrofluidische Plattform für längs-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 ...

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.

Eine höchste Durchsatz mikrofluidischen Plattform für einzellige Genom-Sequenzierung

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.

Eine mikrofluidische Plattform zur Stimulierung von Chondrozyten mit dynamischer Kompression

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