Die Autoren danken Ronald F. Renzi und Michael S. Bartsch für ihre Beiträge zur Gestaltung und Entwicklung von DMF-Geräten und dem DMF-Hub. Diese Forschung wurde vollständig durch das Laboratory Directed Forschungs-und Entwicklungsprogramm bei Sandia National Laboratories unterstützt. Sandia ist ein Multi-Programm-Labor verwaltet und von Sandia Corporation, eine hundertprozentige Tochtergesellschaft von Lockheed Martin Corporation, betrieben für das US Department of National Nuclear Energy Security Administration unter Vertrag DE-AC04-94AL85000.

Diese allgemeine Protokoll wurde entwickelt, um Anwendung der Plattform für eine Vielzahl von Studien zu unterstützen; Aspekte spezifisch für die repräsentative Studie in diesem Bericht beschrieben sind in Klammern getrennt Abbildung 2 zeigt eine repräsentative Studie durchgeführt unter Verwendung des Protokolls.. Beachten Sie, dass in dem Protokoll, alle &quot;anweisen&quot;-Befehle durch die Verwendung von vorgegebenen Script automatisiert. Beachten Sie auch, dass Schritt 2 kann in parallel mit Schritt 1 (z. B. während der Schritte 1.7 und 1.8) durchgeführt werden, und dass Schritt 2.1 ist oft umgangen (weil gemustert / beschichtete DMF Gerät Platten können gewaschen und wieder verwendet werden, siehe Schritt 5.2) . 1. Montieren und Bestücken Sie die Zelle Perfusionskammern <ol><li> Bestreichen Sie die Innenflächen sterilisiert (autoklaviert) Mikrokapillaren (Fused-Silica; 679 od um, 534 um id; 15 cm lang) mit Fibronektin, durch Anweisen der Spritzenpumpe, um eine sterile Fibronektin-Lösung (30 ul von 50 ug / ml) zu ziehen in jedemmikrokapillaren, Inkubation (37 ° C für 2 Stunden), instruiert die Spritzenpumpe um die Lösung zu entziehen, und lassen die Mikrokapillaren trocken (Raumtemperatur (RT) für 6 Stunden). </li><li> Verbinden Sie jeden fibronektinbeschichtete mikrokapillaren (Perfusionskammer) zu Polycarbonat-Schlauch (500 um od 100 um ID) und der Schlauch mit dem Multi-Port-Spritzenpumpe mit CapTite Armaturen (6 Perfusionskammern, 8-Port-Spritzenpumpe). </li><li> Mit Klebeband, sichern den Körper jedes Perfusionskammer zu einem Heizblock auf gewünschte Temperatur (37 ° C). </li><li> Tauchen die offenen Enden der Perfusionskammern in einem Behälter (microcentifuge Rohr oder Mikrotiterplatte) enthaltenden Zellen (P388D.1 murine Makrophagen) in frischem Wachstumsmedium suspendiert (RPMI-1640, 10% FBS, 500 Einheiten / ml Penicillin, 500 ug / ml Streptomycin) bei gewünschten Konzentration (10 6 Zellen / ml). </li><li> Weisen Sie die Spritzenpumpe an Zellen (10 ul, 10 4 Zellen) in jede Perfusionskammer von genügend ai gefolgt ziehenr, um die Flüssigkeit Stecker (~ 4,5 cm) auf den Abschnitt, der an der Wärme Block bewegen. </li><li> Lassen Sie die Zellen an der mit Fibronectin beschichteten Innenflächen der Perfusionskammern (37 ° C für 1 - 2 Stunden) zur Verfügung. In der Zwischenzeit, übertragen die offenen Enden der Perfusionskammern aus den Zellen Reservoir zu einem neuen Behälter mit frischem Wachstumsmedium. </li><li> Nach dem Festhalten Zeitraum, weisen die Spritzenpumpe, um die Flüssigkeit Stecker (konditionierten Medien und ungebunden Zellen) zu senden, um zu verschwenden, zurückzutreten frischem Medium (10 ul), und folgen Sie mit genügend Luft, um die neuen flüssigen Steckern (frisches Medium) über die Haftposition Zellen. </li><li> Weiter zur Durchführung von mittel-Börsen (dh wiederholen Sie Schritt 1.7) in regelmäßigen Abständen (alle 2 Stunden), bis die Zellpopulationen ausreichend äquilibriert und erweiterte (16 - 24 h von Mikro-Maßstab Kultur erzeugten Populationen von ~ 1.000 Zellen / Kammer) sind. </li></ol> 2. Fabricate die DMF Geräte-und zusammenbauen Hub Architektur <ol><li&gt; Wie zuvor 14,29, beschrieben Muster der Bodenplatte des DMF Gerät mit sechsundvierzig Indium-Zinn-Oxid (ITO)-Elektroden (Treiber von Tröpfchen Betätigung) durch Photolithographie und Ätzen und Mantel mit 5 um von Parylene-C und 50 nm von Teflon-AF. Bilden die obere Platte des DMF Vorrichtung durch Beschichten einer unstrukturierten ITO Glassubstrat mit Teflon-AF, wie oben beschrieben. </li><li> Mit Metalldruckfeder Rahmen fixieren DMF Platten in eine Polymergussfolie 14,30 mit Ausnehmungen, die eine 400 um Abstand zwischen den Platten zu halten; dieser Abstand mit dem Aktivierungselektrode Größe (2,5 mm 2) kombiniert, definiert das Tropfenvolumen (2,5 ul pro Elektrode). Beachten Sie, dass DMF Montage mit einfacheren Mitteln 24 erreicht werden kann. </li><li> Einfügen teflonbeschichteten Übertragung Mikrokapillaren (360 um od, 100 um id; 3.5 - 4.0 cm) in den Raum zwischen den Platten DMF, Positionieren jeder an den Rand der zugehörigen Betätigung Elektrode erstrecken. </li><li> Um die entgegengesetzte end von jedem Transfer mikrokapillaren Anbringung eines CapTite Montage. </li><li> Rasten Sie die elektrischen Anschlüsse nach Spannung an die ITO-Elektroden liefern. Elektrode Aktivierung Sequenzen durch Verwendung eines computergesteuerten elektronischen Schnittstelle ausgeführt vorbestimmten Script automatisiert. </li><li> Optional: Den zusammengesetzten DMF Nabe auf die Translationsbewegung eines Mikroskops (SZ-6145TR (Olympus, Japan)) mit einer Charge-Coupled Device (CCD)-Kamera (DCR-HC96 (Sony, Japan)) ausgestattet, um zu ermöglichen Tracking von Tröpfchen . 31. </li></ol> 3. Verbinden Sie die Perfusionskammern der DMF Hub und Stimulieren der Zellpopulationen <ol><li> Entfernen Sie die offenen Enden der Perfusionskammern aus dem Medium Reservoir (siehe Schritt 1.6) und fügen Sie sie in die CapTite Armaturen angebracht, um die Enden der Übertragung Mikrokapillaren der DMF-Hub (siehe Schritt 2.4). </li><li> Weisen Sie die Spritzenpumpe, die Flüssigkeit Stecker (konditionierten Medien) in den Perfusionskammern zu verschwenden senden. </li><li> Inst.ruct die DMF-Hub an Reiz ziehen (E. coli Biopartikel bei 100 ug / ml in frischem Medium mit 0,1% Pluronic F127 w / v) aus einer On-Board-Speicher und liefern ein Tröpfchen von geeigneter Größe (10 ul) zu jedem Transfer Mikrokapillare . Die On-Board-Reservoire von zylinderförmigen Kammern (1,5 x 1,5 cm je) besteht und mit dem DMF Hub über einen Schlauch. </li><li> Weisen Sie die Spritzenpumpe zu zeichnen der Stimulus-Stecker in den Transfer Mikrokapillaren und folgen mit genügend Luft, um die Stecker in den Zellpopulationen in den Perfusionskammern positionieren. </li><li> Inkubieren der Zellen mit Stimulus (37 ° C für 1 - 4 Stunden). Optional: Exchange für neue Impulse (dh wiederholen Sie die Schritte 3.2-3.4) in regelmäßigen Abständen. </li></ol> 4. Beenden Stimulation und Recover Zelllysate für Analysis <ol><li> Optional: Wenn ein Post-Stimulation Zeitraum erforderlich ist, tauschen die Stimulus-haltigen Flüssigkeit Stecker für frisches Medium (dh wiederholen Sie die Schritte 3,2-3.4, Substitution frischem Medium für Stimulus) und weiter inkubieren und nach Bedarf austauschen. </li><li> Austausch der Flüssigkeit in den Stecker Perfusionskammern für Waschpuffer (Prelude Direkte Lysis Buffer Modul B) (dh wiederholen Sie die Schritte 3,2-3,4, Substitution Waschpuffer für Stimulus) und inkubieren nach Bedarf (5 min). </li><li> Tauschen Sie die Flüssigkeit Stecker (Waschpuffer) für die Lyse-Lösung (Prelude Direkte Lysemodul Puffer A mit 0,1% Pluronic F127 w / v) (dh wiederholen Sie die Schritte 3,2-3,4, Substitution Lyselösung für Stimulus) und inkubieren nach Bedarf (5 min) . </li><li> Weisen Sie die Spritzenpumpe, die Flüssigkeit Stecker (Zelllysate) mit dem DMF-Hub senden. </li><li> Demontieren Sie die DMF-Hub, entfernen Sie die obere Platte des DMF-Gerät (mit Pflege nicht auf die Platte seitlich kippen, da dies in Tropfenform Durchmischung führen kann), und sammeln Sie die Lysate mit einem Pipetman oder Spritze für off-Plattform Analyse (Genexpressionsanalysen über qPCR). </li></ol> 5. Saubere Plattform für HardwareRe-use <ol><li> Tauchen Sie den Transfer Mikrokapillaren und CapTite Armaturen in 10% Bleichmittel (v / v in Wasser) für 10 min bei RT, gut mit deionisiertem Wasser abspülen und trocken unter Verwendung von Stickstoffgas. </li><li> Tauchen Sie die DMF Gerät Platten in 10% Bleichmittel für 10 min bei RT, gründlich mit Isopropanol gefolgt von deionisiertem Wasser und trocken unter Verwendung von Stickstoffgas durch Backen bei 160 ° C für 10 min folgte. </li><li> Die Spritze Pumpe Polycarbonat Schläuche, und der DMF Geräts Polymer Besetzung und Kompression Rahmen kann ohne Reinigung wieder verwendet werden. Die Perfusionskammer Mikrokapillaren werden nach jedem Experiment verworfen. </li></ol>

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E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+immunoassays+as+rapid+saliva-based+clinical+diagnostics.">Microfluidic immunoassays as rapid saliva-based clinical diagnostics.</a> Proceedings of the National Academy of Sciences of the United States of America. 104, 5268-5273 (2007).</li></ol>

Die Autoren erklären, dass sie keine finanziellen Interessen haben.

Wir haben eine einfache und vielseitige automatisierte Plattform für die Mikro-Maßstab Zellkultur und Stimulation Experimente entwickelt. Die Plattform ermöglicht es uns, mit kleinen Mengen und Kultur Zellpopulationen (1 - 20 ul und 100 - 2.000 Zellen pro Kammer) arbeiten; Kultur Größen weiter durch Verwendung von Mikrokapillaren von kleineren Durchmesser reduziert werden konnte. Arbeiten bei diesen Skalen reduziert die Kosten für Routine-Untersuchungen und Studien, die machbar macht Gebrauch von wertvoll...

Das Studium der Zellen in Kultur gehalten (in vitro-Analyse) hat einen wertvollen Einblick in die grundlegenden Prinzipien und molekularen Mechanismen, die komplexen biologischen Systemen und der menschlichen Gesundheit zur Verfügung gestellt. Die herkömmlichen Methoden für die Kultur, Anregung und Sammlung von Zellen für die Analyse, die Petrischalen und Mikrotiterplatten nutzen, wurden für die Studie von großen Populationen von Zellen (≥ 10 5) in Milliliter-Maßstab Kulturvolumina (≥ 0,1 ml) ausgelegt. Es gibt jedoch viele Fälle, in denen nur geringe Mengen von Zellen zur Verfügung stehen (z. B. primäre Zellen) oder kleine Zellpopulationen sind wünschenswert (zB Zell-zu-Zell-Variabilität in der Bevölkerung zu verringern), oder erforderlichen Reagenzien sind schwierig zu erhalten oder unerschwinglich teuer (zB gereinigte Zellen sezernierten Faktoren). Solche Probleme erfolgreich durch Verkleinerung Kultur Größe angesprochen werden, hat das den zusätzlichen Vorteil der Reduzierung des Verbrauchs allerReagenzien für die in-vitro-Analyse 1,2 erforderlich. Leider herkömmliche Geräte und Methoden unterstützen nicht präzise und reproduzierbare Manipulation von Mikro-Maßstab Kulturen und Mikrofluidik-basierte automatisierte Systeme, die derzeit zur Verfügung 3-11 sind zu komplex und spezialisiert für den routinemäßigen Einsatz von den meisten Labors. In diesem Bericht beschreiben wir die Montage und Verwendung eines einfachen und vielseitigen Technologie-Plattform für die automatisierte Kultur, Anregung und Wiederherstellung von kleinen Populationen von Zellen (100 - 2.000 Zellen) in Mikro-Maßstab Bände (1 - 20 ul). Die Plattform-Architektur (Abbildung 1) ist modular aufgebaut: eine Reihe von fibronektinbeschichtete Mikrokapillaren (&quot;cell Perfusionskammern&quot; Modul) dient als Standort für die Errichtung, Wartung und Stimulation von Mikro-Maßstab Kulturen, und ein digitaler Mikrofluidik (DMF ) 12,13 Gerät mit &quot;Transfer&quot; Mikrokapillaren (&quot;zentrale Drehscheibe&quot; Modul) 14,15 Routen Zellen ausgestattetund Reagenzien zu und von den Perfusionskammern. DMF ermöglicht dem Anwender individuell ansprechen mehrere Tröpfchen gleichzeitig und zu ändern oder neu zu ordnen die Manipulationen (dh neu Probenaufbereitung Züge), ohne dass die Geräte-Hardware. Seine enorme Flexibilität in seiner jüngsten Entstehung als Schlüsseltechnologie in einer breiten Palette von Anwendungen, einschließlich der Zellkultur 16,17, Enzymassays 18,19, 20,21 Immunoassays, DNA-Analyse 22,23, Eiweiß Verarbeitung, 24,25 evident und klinischen Probe Verarbeitung. 26,27 Unsere zentrale Drehscheibe nutzt die inhärente Flexibilität DMF Geräte und weiter verbessert sie durch die Zugabe von mikrokapillaren Schnittstellen, die Möglichkeit zur Durchführung einer Teilmenge von Manipulationen (zB Zellkultur) in spezialisierten peripheren bietet Module, und nicht auf der DMF Gerät. Kompartimentierung der Verarbeitung Züge auf diese Weise vereinfacht auch Design der plaTForm Architektur (keine Notwendigkeit, ein Gerät, das DMF durchführen können alle Bearbeitungsschritte zu bauen) und erleichtert seine Entwicklung als neue Funktionen erforderlich sind (einfach Integration neuer Peripherie-Module je nach Bedarf). Transport von Zellen und Reagenzien innerhalb der zentralen Nabe wird durch Elektrobenetzung Kräfte durch sequentielle Aktivierung der Elektroden innerhalb des DMF Gerät erzeugt 13,28 angetrieben, Verkehr nach, von und innerhalb der Perfusionskammern wird von Druckänderungen, die durch eine hochpräzise Spritze erzeugten Strom versorgt Pumpe. Alle diese Bewegungen Fluid über eine einfache elektronische Schnittstelle gesteuert und automatisiert durch Verwendung von vorbestimmten Script. Als repräsentatives Beispiel zeigen wir die Verwendung der Plattform für die Untersuchung der Transkriptionsaktivität hervorgerufenen Reaktionen in Immunzellen auf Provokation mit Bakterien (Abbildung 2). Die Durchführung dieser Experimente auf der Plattform konnten wir mit einer kleinen Anzahl von Zellen (~ 1.000 pro experimentell arbeitental Bedingung), zu minimieren Experiment zu Experiment Variabilität, konservieren Reagenzien und re-direct hands-on Arbeit. Angesichts der Vorteile, die es überträgt, sowie die Erreichbarkeit und die Vielseitigkeit, sollte diese Plattform in einer Vielzahl von Laboratorien und Anwendungen finden und doch besonders nützlich erleichtert Analyse von Zellen und Impulse, die in nur geringe Mengen sind.

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Prelude Direct Lysis Module</td>
 <td>NuGEN</td>
 <td>1400-24</td>
 <td></td>
 </tr>
 <tr>
 <td>Trypan Blue (0.4% w/v)</td>
 <td>GIBCO</td>
 <td>15250-061</td>
 <td></td>
 </tr>
 <tr>
 <td>Cell Stripper</td>
 <td>Cellgro</td>
 <td>25-056-C1</td>
 <td></td>
 </tr>
 <tr>
 <td>Ovation PicoSL WTA</td>
 <td>NuGEN</td>
 <td>3310-048</td>
 <td></td>
 </tr>
 <tr>
 <td>Agencourt RNAClean XP</td>
 <td>Beckman Coulter Genomics</td>
 <td>A63987</td>
 <td></td>
 </tr>
 <tr>
 <td>pHrodo BioParticles</td>
 <td>Invitrogen</td>
 <td>P35361</td>
 <td></td>
 </tr>
 <tr>
 <td>CCL4 TaqMan qRT-PCR assay</td>
 <td>Applied Biosystems</td>
 <td>Mm00443111_m1</td>
 <td></td>
 </tr>
 <tr>
 <td>CCL5 TaqMan qRT-PCR assay</td>
 <td>Applied Biosystems</td>
 <td>Mm01302428_m1</td>
 <td></td>
 </tr>
 <tr>
 <td>PTGS2 TaqMan qRT-PCR assay</td>
 <td>Applied Biosystems</td>
 <td>Mm00478374_m1</td>
 <td></td>
 </tr>
 <tr>
 <td>TNF TaqMan qRT-PCR assay</td>
 <td>Applied Biosystems</td>
 <td>Mm00443258_m1</td>
 <td></td>
 </tr>
 <tr>
 <td>GAPDH TaqMan qRT-PCR assay</td>
 <td>Applied Biosystems</td>
 <td>Mm99999915_g1</td>
 <td></td>
 </tr>
 <tr>
 <td>Pluronic F127</td>
 <td>Sigma Chemical</td>
 <td>2594628</td>
 <td></td>
 </tr>
 <tr>
 <td>Fluorinert FC-40</td>
 <td>Sigma Chemical</td>
 <td>51142-49-5</td>
 <td></td>
 </tr>
 <tr>
 <td>Parylene C dimer</td>
 <td>Specialty Coating Systems</td>
 <td>28804-46-8</td>
 <td></td>
 </tr>
 <tr>
 <td>Teflon-AF</td>
 <td>DuPont</td>
 <td>AF1600</td>
 <td></td>
 </tr>
 <tr>
 <td>Polyimide tape</td>
 <td>ULINE</td>
 <td>S-11928</td>
 <td></td>
 </tr>
 <tr>
 <td>Indium tin oxide (ITO) coated glass substrates </td>
 <td>Delta Technologies </td>
 <td>CB-40IN-1107</td>
 <td></td>
 </tr>
 <tr>
 <td>DMF hub Teflon-coated fused-silica microcapillaries</td>
 <td>Polymicro Technologies</td>
 <td>TSU100375</td>
 <td></td>
 </tr>
 <tr>
 <td>Perfusion chamber microcapillaries</td>
 <td>Polymicro Technologies</td>
 <td>TSP530700</td>
 <td></td>
 </tr>
 <tr>
 <td>Tubing and microcapillary fittings</td>
 <td>Sandia National Laboratories</td>
 <td>&nbsp;</td>
 <td></td>
 </tr>
 <tr>
 <td>Polycarbonate tubing</td>
 <td>Paradigm Optics</td>
 <td>CTPC100-500-5</td>
 <td></td>
 </tr>
 <tr>
 <td>8-port precision syringe pump equipped with 30 mm (500 &mu;l capacity) syringes</td>
 <td>Hamilton Company</td>
 <td>54848-01</td>
 <td></td>
 </tr>
 <tr>
 <td>Parylene-C vapor deposition instrument</td>
 <td>Specialty Coating Systems</td>
 <td>PDS 2010 Labcoter 2</td>
 <td></td>
 </tr>
 <tr>
 <td>High-voltage function generator</td>
 <td>Trek</td>
 <td>615A-1 615-3</td>
 <td></td>
 </tr>
 <tr>
 <td>MVX10 microscope</td>
 <td>Olympus</td>
 <td></td>
 <td>Optional (facilitates tracking of droplets on DMF hub)</td>
 </tr>
 <tr>
 <td>QIClick digital CCD camera</td>
 <td>QImaging</td>
 <td>QIClick-F-CLR-12</td>
 <td>Optional (facilitates tracking of droplets on DMF hub)</td>
 </tr>
</tbody>
</table>

a versatile automated platform for micro-scale cell stimulation experiments

Studium der Zellen in Kultur (in vitro-Analyse) hat wichtige Einblicke in komplexe biologische Systeme zur Verfügung gestellt. Herkömmliche Verfahren und Anlagen für die in vitro-Analyse sind gut geeignet, um einer großen Anzahl von Zellen (≥ 10 5) in Milliliter-Skala Volumina (≥ 0,1 ml) zu untersuchen. Es gibt jedoch viele Fälle, in denen es notwendig oder wünschenswert sein, Kultur Größe verkleinern, um den Verbrauch der Zellen von Interesse und / oder Reagenzien für die Kultur, Stimulation oder der Verarbeitung zu reduzieren. Leider haben konventionelle Ansätze nicht unterstützt präzise und reproduzierbare Manipulation von Mikro-Maßstab Kulturen, und die Mikrofluidik-basierte automatisierte Systeme, die derzeit verfügbar sind zu komplex und spezialisiert für den routinemäßigen Einsatz von den meisten Labors. In Mikro-Maßstab Volumen - Um dieses Problem anzugehen, haben wir eine einfache und vielseitige Technologie-Plattform für die automatisierte Kultur, Anregung und Wiederherstellung von kleinen Populationen von Zellen (2000 Zellen 100) entwickelts (1 - 20 ul). Die Plattform besteht aus einer Reihe von fibronektinbeschichtete Mikrokapillaren (&quot;cell Perfusionskammern&quot;), in dem Mikro-Maßstab Kulturen etabliert, werden beibehalten und angeregt, eine digitale Mikrofluidik (DMF)-Gerät mit &quot;Transfer&quot; Mikrokapillaren (&quot;zentrale Drehscheibe ausgestattet &quot;), die Strecken Zellen und Reagenzien zu und von den Perfusionskammern; eine hochpräzise Spritzenpumpe, die Befugnisse Transport von Materialien zwischen den Perfusionskammern und der zentralen Nabe und eine elektronische Schnittstelle, die Kontrolle über den Transport von Materialien bietet, das ist koordiniert und automatisiert via vorgegebenen Skripten. Als Beispiel haben wir die Plattform zur Untersuchung der transkriptionellen Reaktionen hervorgerufen in Immunzellen auf Herausforderung mit Bakterien zu erleichtern. Die Nutzung der Plattform konnten wir den Verbrauch von Zellen und Reagenzien zu reduzieren, minimieren Experiment zu Experiment Variabilität und re-direct hands-on Arbeit. Angesichts der Vorteile, die es verleiht, sowie die Erreichbarkeit und die Vielseitigkeit, our Plattform sollte den Einsatz in einer Vielzahl von Laboren und Anwendungen zu finden, und als besonders nützlich erweisen bei der Erleichterung der Analyse von Zellen und Stimuli, die in nur begrenzten Mengen sind.

Als Demonstration der automatisierten Plattform, haben wir es für die Durchführung einer Studie, in der kleinen Populationen von Immunzellen im Mikro-Maßstab gezüchtet wurden, mit Bakterien herausgefordert, und lysiert für off-Plattform Analyse von pro-inflammatorischen Reaktionen (Abbildung 2 ). Jeder der sechs Zellen Perfusionskammern wurde mit 10 4 Immunzellen (P388D.1 murine Makrophagen) in 10 ul Wachstumsmedium resuspendiert ausgesät. Nach einer Einhalt...

Watch this Scientific Journal Video about A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments at JoVE.com

A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

Wir haben eine automatisierte Zellkultur und Verhör Plattform für Mikro-Skala-Zell-Stimulation Experimente entwickelt. Die Plattform bietet eine einfache, vielseitige und präzise Kontrolle in den Anbau und die Förderung kleiner Populationen von Zellen und Gewinnen Lysate für molekulare Analysen. Die Plattform wird auch auf Studien, die kostbaren Zellen und / oder Reagenzien geeignet.

Eine vielseitige automatisierte Plattform für Mikro-Skala Handy Stimulation Experimente

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in ...

a-versatile-automated-platform-for-micro-scale-cell-stimulation

Research

JoVE Journal

Bioengineering

10.5K Aufrufe.  Sandia National Laboratories. Wir haben eine automatisierte Zellkultur und Verhör Plattform für Mikro-Skala-Zell-Stimulation Experimente entwickelt. Die Plattform bietet eine einfache, vielseitige und präzise Kontrolle in den Anbau und die Förderung kleiner Populationen von Zellen und Gewinnen Lysate für molekulare Analysen. Die Plattform wird auch auf Studien, die kostbaren Zellen und / oder Reagenzien geeignet.

Serie: A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and metastasis of tumor cells. Migration has been studied on glass coverslips in order to make cellular dynamics amenable to investigation by light microscopy. However, it has become clear that many aspects of cell migration depend on features of the local environment including its elasticity, protein composition, and pore size, which are not faithfully represented by rigid two dimensional substrates such as glass and plastic 1. Furthermore, interaction with other cell types, including stromal fibroblasts 2 and immune cells 3, has been shown to play a critical role in promoting the invasion of cancer cells. Investigation at the molecular level has increasingly shown that molecular dynamics, including response to drug treatment, of identical cells are significantly different when compared in vitro and in vivo 4. 
Ideally, it would be best to study cell migration in its naturally occurring context in living organisms, however this is not always possible. Intermediate tissue culture systems, such as cell derived matrix, matrigel, organotypic culture (described here) tissue explants, organoids, and xenografts, are therefore important experimental intermediates. These systems approximate certain aspects of an in vivo environment but are more amenable to experimental manipulation such as use of stably transfected cell lines, drug treatment regimes, long term and high-resolution imaging. Such intermediate systems are especially useful as proving grounds to validate probes and establish parameters required to image the dynamic response of cells and fluorescent reporters prior to undertaking imaging in vivo 5. As such, they can serve an important role in reducing the need for experiments on living animals.

A method is described for the preparation of a 3-dimensional matrix consisting of collagen type I and primary human fibroblasts. This organotypic gel serves as a useful substrate to assess invasive cell migration because it mimics basic features of tissue stroma and is amenable to many forms of microscopy.

Organotypische Collagen I Assay: A Temperguss Platform to Cell Verhalten in 3-Dimensional Kontext beurteilen

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

Forschung

Biotechnik

GENPLAT: an Automated Platform for Biomass Enzyme Discovery and Cocktail Optimization

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid transportation fuels such as ethanol. We have developed an integrated high throughput platform, called GENPLAT, for the discovery and development of novel enzymes and enzyme cocktails for the release of sugars from diverse pretreatment/biomass combinations. GENPLAT comprises four elements: individual pure enzymes, statistical design of experiments, robotic pipeting of biomass slurries and enzymes, and automated colorimeteric determination of released Glc and Xyl. Individual enzymes are produced by expression in Pichia pastoris or Trichoderma reesei, or by chromatographic purification from commercial cocktails or from extracts of novel microorganisms. Simplex lattice (fractional factorial) mixture models are designed using commercial Design of Experiment statistical software. Enzyme mixtures of high complexity are constructed using robotic pipeting into a 96-well format. The measurement of released Glc and Xyl is automated using enzyme-linked colorimetric assays. Optimized enzyme mixtures containing as many as 16 components have been tested on a variety of feedstock and pretreatment combinations.
GENPLAT is adaptable to mixtures of pure enzymes, mixtures of commercial products (e.g., Accellerase 1000 and Novozyme 188), extracts of novel microbes, or combinations thereof. To make and test mixtures of &tilde;10 pure enzymes requires less than 100 &mu;g of each protein and fewer than 100 total reactions, when operated at a final total loading of 15 mg protein/g glucan. We use enzymes from several sources. Enzymes can be purified from natural sources such as fungal cultures (e.g., Aspergillus niger, Cochliobolus carbonum, and Galerina marginata), or they can be made by expression of the encoding genes (obtained from the increasing number of microbial genome sequences) in hosts such as E. coli, Pichia pastoris, or a filamentous fungus such as T. reesei. Proteins can also be purified from commercial enzyme cocktails (e.g., Multifect Xylanase, Novozyme 188). An increasing number of pure enzymes, including glycosyl hydrolases, cell wall-active esterases, proteases, and lyases, are available from commercial sources, e.g., Megazyme, Inc. (www.megazyme.com), NZYTech (www.nzytech.com), and PROZOMIX (www.prozomix.com).
Design-Expert software (Stat-Ease, Inc.) is used to create simplex-lattice designs and to analyze responses (in this case, Glc and Xyl release). Mixtures contain 4-20 components, which can vary in proportion between 0 and 100%. Assay points typically include the extreme vertices with a sufficient number of intervening points to generate a valid model. In the terminology of experimental design, most of our studies are "mixture" experiments, meaning that the sum of all components adds to a total fixed protein loading (expressed as mg/g glucan). The number of mixtures in the simplex-lattice depends on both the number of components in the mixture and the degree of polynomial (quadratic or cubic). For example, a 6-component experiment will entail 63 separate reactions with an augmented special cubic model, which can detect three-way interactions, whereas only 23 individual reactions are necessary with an augmented quadratic model. For mixtures containing more than eight components, a quadratic experimental design is more practical, and in our experience such models are usually statistically valid.
All enzyme loadings are expressed as a percentage of the final total loading (which for our experiments is typically 15 mg protein/g glucan). For "core" enzymes, the lower percentage limit is set to 5%. This limit was derived from our experience in which yields of Glc and/or Xyl were very low if any core enzyme was present at 0%. Poor models result from too many samples showing very low Glc or Xyl yields. Setting a lower limit in turn determines an upper limit. That is, for a six-component experiment, if the lower limit for each single component is set to 5%, then the upper limit of each single component will be 75%. The lower limits of all other enzymes considered as "accessory" are set to 0%. "Core" and "accessory" are somewhat arbitrary designations and will differ depending on the substrate, but in our studies the core enzymes for release of Glc from corn stover comprise the following enzymes from T. reesei: CBH1 (also known as Cel7A), CBH2 (Cel6A), EG1(Cel7B), BG (&beta;-glucosidase), EX3 (endo-&beta;1,4-xylanase, GH10), and BX (&beta;-xylosidase).
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GENPLAT (GLBRC Enzyme Platform) is an automated platform for discovery and optimization of enzyme cocktails for biomass degradation. It can be adapted to multiple feedstocks and mixtures of enzymes containing multiple components.

GENPLAT: eine automatisierte Plattform für Biomasse Enzym-Entdeckung und Cocktail-Optimierung

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid ...

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 ...

A Novel Stretching Platform for Applications in Cell and Tissue Mechanobiology

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have contributed dramatically to the understanding of basic mechanobiology. These techniques have been extensively used to demonstrate how the onset and progression of various diseases are heavily influenced by mechanical cues. This article presents a multi-functional biaxial stretching (BAXS) platform that can either mechanically stimulate single cells or quantify the mechanical stiffness of tissues. The BAXS platform consists of four voice coil motors that can be controlled independently. Single cells can be cultured on a flexible substrate that can be attached to the motors allowing one to expose the cells to complex, dynamic, and spatially varying strain fields. Conversely, by incorporating a force load cell, one can also quantify the mechanical properties of primary tissues as they are exposed to deformation cycles. In both cases, a proper set of clamps must be designed and mounted to the BAXS platform motors in order to firmly hold the flexible substrate or the tissue of interest. The BAXS platform can be mounted on an inverted microscope to perform simultaneous transmitted light and/or fluorescence imaging to examine the structural or biochemical response of the sample during stretching experiments. This article provides experimental details of the design and usage of the BAXS platform and presents results for single cell and whole tissue studies. The BAXS platform was used to measure the deformation of nuclei in single mouse myoblast cells in response to substrate strain and to measure the stiffness of isolated mouse aortas. The BAXS platform is a versatile tool that can be combined with various optical microscopies in order to provide novel mechanobiological insights at the sub-cellular, cellular and whole tissue levels.

We present in this article a novel stretching platform that can be used to investigate single cell responses to complex anisotropic biaxial mechanical deformation and quantify the mechanical properties of biological tissues.

A Novel Stretching-Plattform für Anwendungen in der Zell-und Gewebe Mechanobiologie

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have ...

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

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

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

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 ...

Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential mechanosensing characteristics. Engineering and analysis of these tissue types remain a challenge. Layered hydrogel constructs provide an opportunity for investigating the interactions among multiple cell populations within single constructs. Alginate hydrogels are both biocompatible and allow for easy isolation of cells after experimentation. Here, we describe a method for the development of small sized dual layered alginate hydrogel discs. This process maintains high cell viability of human mesenchymal stem cells during the formation process and these layered discs can withstand unconfined cyclic compression, commonly used for stimulation of hMSCs undergoing chondrogenesis. These layered constructs can potentially be scaled up to include additional levels, and also be used to segregate cell populations initially after layering. This dual layer alginate hydrogel culture platform can be used for many different applications including engineering and analysis of cells of load bearing tissues and co-cultures of other cell types.

Engineering and analysis of load bearing tissues with heterogeneous cell populations are still a challenge. Here, we describe a method for creating bi-layered alginate hydrogel discs as a platform for co-culture of diverse cell populations within one construct.

Layered Alginate Konstrukten: Eine Plattform für Co-Kultur von heterogenen Zellpopulationen

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential ...

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 Versatile Method of Patterning Proteins and Cells

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning techniques work well only with simple shapes, small areas and selected bio-materials. This article describes a method to distribute cell suspensions as well as substrate solutions into complex, long, closed (dead-end) polydimethylsiloxane (PDMS) microchannels using negative pressure. This method enables researchers to pattern multiple substrates including fibronectin, collagen, antibodies (Sal-1), poly-D-lysine (PDL), and laminin. Patterning of substrates allows one to indirectly pattern a variety of cells. We have tested C2C12 myoblasts, the PC12 neuronal cell line, embryonic rat cortical neurons, and amphibian retinal neurons. In addition, we demonstrate that this technique can directly pattern fibroblasts in microfluidic channels via brief application of a low vacuum on cell suspensions. The low vacuum does not significantly decrease cell viability as shown by cell viability assays. Modifications are discussed for application of the method to different cell and substrate types. This technique allows researchers to pattern cells and proteins in specific patterns without the need for exotic materials or equipment and can be done in any laboratory with a vacuum.

This report describes a simple, easy to perform technique, using low pressure vacuum, to fill microfluidic channels with cells and substrates for biological research.

Eine vielseitige Methode von Patterning Proteinen und Zellen

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning ...

DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of flexible polymers nor of rigid rods. Underlying filaments remain outstretched due to their non-vanishing backbone stiffness, which is quantified via the persistence length (lp), but they are also subject to strong thermal fluctuations. Their finite bending stiffness leads to unique, non-trivial collective mechanics of bulk networks, enabling the formation of stable scaffolds at low volume fractions while providing large mesh sizes. This underlying principle is prevalent in nature (e.g., in cells or tissues), minimizing the high molecular content and thereby facilitating diffusive or active transport. Due to their biological implications and potential technological applications in biocompatible hydrogels, semiflexible polymers have been subject to considerable study. However, comprehensible investigations remained challenging since they relied on natural polymers, such as actin filaments, which are not freely tunable. Despite these limitations and due to the lack of synthetic, mechanically tunable, and semiflexible polymers, actin filaments were established as the common model system. A major limitation is that the central quantity lp cannot be freely tuned to study its impact on macroscopic bulk structures. This limitation was resolved by employing structurally programmable DNA nanotubes, enabling controlled alteration of the filament stiffness. They are formed through tile-based designs, where a discrete set of partially complementary strands hybridize in a ring structure with a discrete circumference. These rings feature sticky ends, enabling the effective polymerization into filaments several microns in length, and display similar polymerization kinetics as natural biopolymers. Due to their programmable mechanics, these tubes are versatile, novel tools to study the impact of lp on the single-molecule as well as the bulk scale. In contrast to actin filaments, they remain stable over weeks, without notable degeneration, and their handling is comparably straightforward.

Semiflexible polymers display unique mechanical properties that are extensively applied by living systems. However, systematic studies on biopolymers are limited since properties such as polymer rigidity are inaccessible. This manuscript describes how this limitation is circumvented by programmable DNA nanotubes, enabling experimental studies on the impact of filament rigidity.

DNA-Nanoröhren als ein vielseitiges Werkzeug, semiflexiblen Polymere zu studieren

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of ...

A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic regulatory circuits. The analysis of synthetic genetic regulatory networks in vivo is time consuming and suffers from a lack of environmental control, with exogenous synthetic components interacting with host processes resulting in undesired behavior. To overcome these issues, cell-free characterization of novel circuitry is becoming more prevalent. In vitro transcription and translation (IVTT) mixtures allow the regulation of the experimental environment and can be optimized for each unique system. The protocols presented here detail the fabrication of a multilayer microfluidic device that can be utilized to sustain IVTT reactions for prolonged durations. In contrast to batch reactions, where resources are depleted over time and (by-) products accumulate, the use of microfluidic devices allows the replenishment of resources as well as the removal of reaction products. In this manner, the cellular environment is emulated by maintaining an out-of-equilibrium environment in which the dynamic behavior of gene circuits can be investigated over extended periods of time. To fully exploit the multilayer microfluidic device, hardware and software have been integrated to automate the IVTT reactions. By combining IVTT reactions with the microfluidic platform presented here, it becomes possible to comprehensively analyze complex network behaviors, furthering our understanding of the mechanisms that regulate cellular processes.

The fabrication process of a PDMS-based, multilayer, microfluidic device that allows in vitro transcription and translation (IVTT) reactions to be performed over prolonged periods is described. Furthermore, a comprehensive overview of the hardware and software required to automate and maintain these reactions for prolonged durations is provided.

Eine multilayer mikrofluidische Plattform für die Leitung einer längeren zellfreien Genexpression

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic ...