Die Autoren möchten, dass Fedex Institute of Technology an der Universität von Memphis danken für ihre Unterstützung. Die Autoren bedanken sich bei Studenten der Senior Design-Projekt-Gruppe in der Maschinenbau-Abteilung an der Universität von Memphis (David Butler, Jackie Carter, Dominick Cleveland, Jacob Shaffer) anerkennen, Daniel Kohn von der Universität von Memphis Engineering Technology-Abteilung für die Motorsteuerung und Dr. Bin Teng und Frau Charlean Luellen für ihre Hilfe in der Zellkultur. Diese Arbeit wurde von K01 HL120912 (ER) und R01 HL123540 (CMW) unterstützt.

1. Aufbau der Membrane mit Well Walls für Retention von Zellkulturmedien (siehe 1D für das Endprodukt) <ol><li> Verwendung Polydimethylsiloxan (PDMS) Platten mit Collagen I beschichtet sind, schneiden die Kontur der flexiblen Membran mit einem Skalpell oder einer Düse. </li><li> Platzieren Sie jede Membran in einer 60 mm Petrischale für die Lagerung. </li><li> Erstellung von Wänden: <ol><li> Mischungs PDMS bei einem 10: 1-Gewichtsverhältnis von Elastomer A bis B (Härter) Elastomer ist. </li><li> Gießen Sie 5 ml vollständig vermischt PDMS in 50 ml Tuben. </li><li> Zeigen 50 ml Röhrchen mit nicht ausgehärteten PDMS horizontal in einem Hybridisierungsofen. </li><li> Verwenden die Rotorfunktion zur Beschichtung der Innenwände der Rohre bei 8 rpm während der Aushärtezeit. Bei RT wird das PDMS vollständig in 2 Tagen ausgehärtet werden. </li><li> Entfernen Sie die PDMS aus der Röhre in einem sterilen Zellkultur Kapuze. </li><li> Mit Hilfe einer neuen Rasierklinge, partitioniert die Zylinder des PDMS in Abschnitte 4 mm in der Höhe. </li><li> Legen Sie die Abschnitte, die als Membran wa dienen wirdlls, in eine Petrischale Behälter, ohne dass die Wände zu werden erhöht. </li></ol></li><li> Auszuwählen und in der Mitte einer Wand des PDMS an einer Membran, während die beiden in der Petrischale erhalten bleibt. </li><li> Ganz sanft ungehärteten PDMS (Verhältnis 10: 1) auf dem äußeren Umfang der Wand, als Klebstoff verwendet werden. Verhindern die Bildung von Zwischenräumen zwischen der Wand und der Membran, da es dort zu Flüssigkeit zurückzuhalten. </li><li> Platzieren Sie jede Petrischale mit den fertigen Membran und in einem Ofen bedeckt bei 70 ° C für 24 Stunden, um zu heilen. </li></ol> 2. Korrelation der Motorumdrehungen mit Clamp Verschiebung oder radiale Wachstum für Kalibrierung (Abbildung 2) <ol><li> Starten Sie die Software die Steuerung des Motors. </li><li> Verdrängen die Klemmen des Geräts mit der manuellen Einstellung der Motorsoftware. </li><li> Messen des Abstands und Aufzeichnung der Motor Zählposition zwischen gegenüberliegenden Klemmen an beiden minimalen und maximalen Verschiebung der Klammern. </li><li> Berechnen der prozentualen Änderung in der distance zwischen den Klammern in Abhängigkeit von der Motorzählung (~ 0-75k Aktivität). Dadurch wird die maximal mögliche Belastung der Membran erreicht anzuzeigen. </li></ol> 3. Anwendung der Stretch auf Maus Lung Epithelzelllinie (MLE12) <ol><li> Samen MLE12 Zellen 2,5 Millionen Zellen auf der flexiblen Membran (Schritt 1) ​​pro Vertiefung innerhalb von zwei Tagen konfluent werden. Die Aussaatdichte für unterschiedliche Zelltypen variieren. </li><li> Sicherzustellen, dass die mechanische Betätigungseinrichtung in einer vollständig entspannten Position. <ol><li> Entfernen von Zellkulturmedien. </li><li> Verwendung von 1,5 mm Biopsieausstanzungen, Punch zwei Löcher in jeder der sechs Klemm (siehe Figur 1D) Laschen der Membran. Die radiale Anordnung der Löcher bestimmt die Menge der Vorspannung der Membran zunächst Erfahrungen. Ausstanzung der Löcher auf einem Radius von 20,5 mm an den Membranzungen wenn keine Vorspannung erwünscht ist. </li><li> Positionieren Sie die Membran auf dem Keilrahmen mit den gestanzten Löchern Schlange mit den Stiften in den Klammern.Zeigen oberen Klemmen vorhanden. Ziehen Sie die Schrauben ein zu einer Zeit wechselseitig. </li><li> 1 ml Zellkulturmedium. </li></ol></li><li> Platzieren der Vorrichtung auf dem Mikroskoptisch Zentrierung der Mitte der Membran mit dem Lichtweg. </li><li> Verwenden Sie Isolierband oder Magneten (wenn möglich), um das Gerät auf die Bühne zu beheben. Sobald festen, steuern die in der Ebene (parallel zur Membran) und vertikalen (z-Richtung) der mechanischen Einrichtung mit der Tischsteuereinrichtung des Mikroskops. </li><li> Bewerben Strecke mit dem vom Hersteller durch die Position und Geschwindigkeit der Motordrehung 20 manuelle Steuerung zur Verfügung gestellte Software. </li></ol> 4. Messung der Mitochondrial reaktiven Sauerstoffspezies (ROS) <ol><li> Sobald Zellen konfluent, fügen Sie 1-2 ml RT DMEM mit mitochondrialen Superoxid-Anzeige (5 &amp; mgr; M Endkonzentration) direkt auf die Zellen. </li><li> Inkubieren für 10 min bei 37 ° C. </li><li> Wasche die Zellen vorsichtig dreimal mit Puffer erwärmt in einem Wasserbad auf37 ° C. </li><li> Sofort platzieren die flexible Membran auf dem Keilrahmen (Schritt 2.2) bereits im Rahmen einer aufrechten konfokalen Mikroskop. </li><li> 1 ml DMEM mit Phenolrot-freiem Medium und 25 mM HEPES. </li><li> Stellen Anregungs- / Emissions Filter können 510/580 nm. </li><li> Bild mehrere Felder alle 15 min, um die gewünschte Zeitverlauf der mitochondrialen Superoxid-Produktion zu schaffen. </li><li> Von aufgenommenen Bildern, Histogramme Aufzeichnung der Fluoreszenzintensität in jedem Zeitintervall unter Verwendung von Software in der Lage ist die Quantifizierung der Fluoreszenzintensität. </li></ol> 5. Anwendung der Stretch und Rasterkraftmikroskopie (AFM) Hinweis: Diese Schritte werden für eine bestimmte AFM und optischen Mikroskop Kombination (Abbildung 4 und Materialliste) versehen ist. <ol><li> Bereiten Sie das AFM für das Experiment. <ol><li> Erhöhen die Höhe des AFM Kopfes zu seiner maximalen Position in der z-Richtung. </li><li> Gesetzt Extender auf die Beine des AFMHeben Sie die Ebene, bei der AFM Cantilever Kontakten der Probe. Die Probe und das AFM Kopf muss angehoben werden, um die Höhe der mechanischen Vorrichtung aufzunehmen. </li></ol></li><li> Bereiten Sie das Lichtmikroskop (falls vorhanden). <ol><li> Entfernen Sie die AFM-Scanner Platte. Entfernen Sie das gewünschte Ziel. </li><li> Fügen Sie einen Abstandshalter auf das Ziel. Die Höhe des Abstandhalters würde die Aufgabe und die spezifische AFM Aufbau abhängen, aber es ist erforderlich, wenn optische Bildgebung erwünscht ist, da Beobachtungsebene verschiebt sich in der z-Richtung um einen Betrag gleich der Trage und Adapterhöhe (Figur sein 5A). Beachten Sie, dass die AFM bietet in der Regel geringer Vergrößerung Bildgebung aus einer optischen Pfad über dem Gerät. </li><li> Montieren Sie die gewünschte Ziel wieder in seine Lage. Stellen Sie den Scanner wieder ein, um die AFM. </li><li> Starten Sie die AFM-Software. Starten Sie alle notwendigen Lichtquellen einschließlich der Lichtquelle für Fluoreszenzmessungen. </li><li> Montieren Sie einen Chip mit einer freitragenden Balken,ist für die gewünschten Messungen geeignet. 200 pN / nm oder weniger Steifigkeit wird bevorzugt, wenn die Messung des Elastizitätsmoduls von lebenden Zellen. </li><li> Richten Sie den Laser und Kalibrierung des freitragenden Steifigkeit nach Hersteller Vorschläge auf einem Deckglas auf dem Gerät angebracht ist. </li></ol></li><li> Vorbereitung einer Membran wie in Schritt 1, jedoch mit folgenden Modifikationen. <ol><li> Unmittelbar vor der Montage der Membran an der Streckvorrichtung, schneiden die Wände bis etwa 1 mm in der Höhe. Dies verhindert, dass die Interferenz zwischen den Membranwänden und dem Last Zelle erfährt. </li><li> Halterung der Membran an der mechanischen Vorrichtung, wie beschrieben, 3.2.1-3.2.3. </li><li> Zellkulturmedium zu entfernen, um eine Beschädigung der AFM-Scanner im Falle eines Austretens verhindern. </li><li> Paar das Gerät mit einem Adapter (5A). Legen Sie die mechanische Vorrichtung mit dem Adapter auf den Scanner. </li><li> Dehnen Sie die Membran auf die gewünschte Dehnung Ebene. </li></ol></li><li&gt; Nano-Eindruck der gestreckten Zellen. <ol><li> Fügen Sie eine begrenzte (&lt;0,5 ml) Volumen von Medien auf die Zellen, um AFM Scanner oder Mikroskop Schäden zu vermeiden aufgrund eines spill. </li><li> Aktivieren Sie den Freiträger mit der Membran. </li><li> Folgen Sie dem Protokoll des jeweiligen AFM-Gerät, um Bereiche von Interesse zu scannen. </li></ol></li></ol>

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Die Autoren erklären, dass sie keine konkurrierenden finanziellen Interessen.

Ein einzigartiges Gerät für Live Cell Imaging bei mechanischer Dehnung entwickelt; und diese Vorrichtung wurde in einem Protokoll zum Lungen Epithelzellen Mechanobiologie studieren. In Voruntersuchungen wurde festgestellt, dass eine einzige statt Streck stimuliert die Produktion von Superoxid in mitochondrialen bronchiale Epithelzellen. Darüber hinaus wurde gezeigt, dass erhöhte mechanische Belastung verursacht direkte Beschädigung der Integrität einer Monoschicht von alveolären Epithelzellen. <p class="jove_...

Zellen werden gegenüber mechanischen Belastungen in vielen Geweben unterzogen, und das mechanische Stimulation wurde gezeigt, dass Veränderungen in den Mustern der Genexpression, die Freisetzung von Wachstumsfaktoren, Cytokine oder Umbau der extrazellulären Matrix zu fördern und Zytoskelett 1-4. Die intrazellulären Signale solcher mechanischen Reizen transduziert auftreten durch den Prozess der mechanotransduction 5-7. Im Atmungssystem ist ein Ergebnis der mechanotransduction die Erhöhung reaktiver Sauerstoffspezies (ROS) 8,9 und proinflammatorische Zytokine 10 in Lungenepithelzellen in Gegenwart von zyklischen Zugbelastung. Starke Beweise legen auch nahe, dass die übermäßige Zugspannung führt zu Verletzungen der Alveolarepithel direkt neben den biochemischen Reaktionen der Zellen 11-14. Obwohl der Fokus liegt hier vor allem auf die Reaktion der Lungenzellen auf mechanische Verformung, Wege durch Mechanotransduktion induzierte spielen eine Schlüsselrolle in den basic Funktion vieler Gewebe im menschlichen Körper, einschließlich der Regulation des Gefäßtonus 15 und die Entwicklung der Wachstumsplatte 16. Das wachsende Interesse an mechanotransduction hat zur Entwicklung zahlreicher Geräte für die Anwendung von physiologisch relevanten mechanischen Belastungen an kultivierte Zellen und Gewebe geführt. Insbesondere sind Geräte Anlegen einer Zugspannung, die eine häufige Form der mechanischen Belastung durch Gewebe erfahren ist, populär 11,17-19. Jedoch können viele der verfügbaren Geräte werden entweder als Bioreaktor für das Tissue Engineering-Anwendungen entwickelt oder sind nicht förderlich für eine Echtzeitabbildung mit Stretch. Als solches gibt es einen Bedarf an Werkzeugen und Verfahren, die Zellen und Gewebe unter Zugspannung zu visualisieren kann, um die Untersuchung von Wegen von mechanotransduction erleichtern. Dabei wurde eine in der Ebene mechanischen Spannvorrichtung entwickelt und Protokolle wurden entwickelt, um m geltenultiple Formen Stamm zu Geweben und Zellen und ermöglicht die Abbildung der biochemischen und mechanischen Reaktionen in Echtzeit (1A-D). Das Gerät verfügt über sechs in Umfangsrichtung angeordnet, um eine flexible Membran greifen und gelten in der Ebene liegende radiale Ausdehnung von bis zu etwa 20% (1B) gleichmäßig beabstandete Schellen. Die Betätigungsvorrichtung kann in einem Zellkulturbrutschrank über einen längeren Zeitraum gegeben werden, während der Motor (1C) außerhalb des Inkubators angeordnet ist und von proprietärer Software vom Motorlieferanten bereitgestellt gesteuert. Der Motor ist mit einem linearen Antriebsteil, der einen Innennocken dreht, fahren die sechs Trage Klemmen einheitlich in An- und Entspannung verbunden ist. Zusätzlich zu der mechanischen Vorrichtung wurden angepasst flexiblen Membranen aus im Handel erhältlichen Zellkultur bereit Membranen geschaffen, um in dem mechanischen System verwendet werden. Dann kreisförmigen Wände (mit einem Durchmesser von ca.28 mm) wurden hergestellt und auf die flexible Membran, so dass Zellen nur in diesem Bereich der gut beschriebene Spannungsprofil kultiviert werden, befestigt. Um zu bestimmen, ob die Position dieser Membranen innerhalb der Betätigungsvorrichtung würde gleichförmig und isotrop Belastung in der Mitte der flexiblen Membran bereitzustellen, wurde Finite-Elemente-Analyse unter Verwendung kommerziell erhältlicher Software (1E-F) durchgeführt. Die flexible Membran wurde mit symmetrischen Randbedingungen und die Nutzung aller vierseitigen Elemente zu dem Netz modelliert. Die konzentrischen Ringe in der Konturdiagramm der maximalen Hauptdehnung in 1F gezeigt gesehen zeigen die isotropen Verteilung der Belastung. Der Stamm von der Membran erfahren wurde durch die Aufnahme von Bildern von Markierungen durch Beladung (Figur 2) gemessen. 2D zeigt, dass der durchschnittliche Membran Dehnung in radialer und axialer Richtung gemessen wurde, war annähernd linearenin Bezug auf die angelegte Motor zählt bis zu einer maximalen linearen Dehnung von 20%. Es gab keinen signifikanten Unterschied zwischen den Dehnungsniveaus während Ausdehnung gemessen im Vergleich mit denen beim Zurückziehen wieder in die Ruheposition gemessen wird. Als nächstes wird die Verschiebung der humanen bronchialen Epithelzellen (16HBE) und ihre Kerne auf dem benutzerdefinierten flexible Membran kultiviert wurden, wurden gemessen. Fluoreszenzmarkierten (DAPI) Kerne der 16HBE Zellen wurden mit einem 20x-Objektiv unter einem konfokalen Mikroskop abgebildet wird, während ganze Zelle Verschiebung wurde mit Phasenkontrast-Aufnahmen mit einem digitalen Mikroskop aufgenommenen Mess. Wie in Figur 3 zu sehen ist, die Belastung durch die Verschiebung der Kerne gemessen wurde, war ähnlich der durch Verschieben von Markierungen auf der Membran gemessen wird, bis zu ca. 20% linear Belastung. Dies bestätigt, daß der Stamm zu den Membranen aufgebracht wurde zu den adhärenten Zellen übertragen. Die Protokolle, die die Verwendung des benutzerdefinierten Geräte auf einem herkömmlichen Mikroskop und ein Atomkraft microscope werden in den folgenden Schritten.

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	<tbody>
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			<td>SmartMotor NEMA 34: 3400 Series</td>
			<td>MOOG Animatics</td>
			<td>SM3416D</td>
			<td>Integrated motor, controller, amplifier, encoder and communications bus</td>
		</tr>
		<tr>
			<td>Flexcell Membrane (Collagen I coated)</td>
			<td>Flexcell International Corp</td>
			<td>SM2-1010C</td>
			<td>3.5x5.25x0.020&#34;</td>
		</tr>
		<tr>
			<td>Sylgard 184&#160;</td>
			<td>Dow Corning Corporation</td>
			<td></td>
			<td>10:1</td>
		</tr>
		<tr>
			<td>Hoechst 33342&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>H1399</td>
			<td>DAPI stain</td>
		</tr>
		<tr>
			<td>MitoSOX</td>
			<td>Sigma-Aldrich</td>
			<td>M36008</td>
			<td></td>
		</tr>
		<tr>
			<td>Tiron</td>
			<td>Sigma-Aldrich</td>
			<td>D7389&#160;</td>
			<td>mitochondrial superoxide label</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td></td>
			<td></td>
			<td>superoxide inhibitor</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml tubes</td>
			<td>Fisher Scientific</td>
			<td>06-443-19</td>
			<td>Any centriguge tube can be used to create an area for imaging.</td>
		</tr>
		<tr>
			<td>Hybridization oven</td>
			<td>Bellco Glass</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MLE12 Cells</td>
			<td>ATCC</td>
			<td>CRL-2110</td>
			<td>Mouse Lung Epithelial Cells&#160;</td>
		</tr>
		<tr>
			<td>16HBE cells</td>
			<td>ATCC</td>
			<td>CRL-2741</td>
			<td>Human Bronchial Epithelial Cells</td>
		</tr>
		<tr>
			<td>AFM Indentation Experiments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cantilever Beams for Nano-indentation</td>
			<td>Budget Sensors</td>
			<td>Si-Ni30</td>
			<td></td>
		</tr>
		<tr>
			<td>AFM&#160;</td>
			<td>Asylum Research</td>
			<td>MFP3D</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus microscope</td>
			<td>Olympus</td>
			<td>IX-71</td>
			<td>Inverted microscope with 20X and 40X objectives.</td>
		</tr>
		<tr>
			<td>AFM Leg Extenders</td>
			<td>Asylum Research</td>
			<td>Not available</td>
			<td>AFM microscope</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Finite Element Analyses</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ABAQUS</td>
			<td>Simulia</td>
			<td>6.12</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>NIH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscopes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Digital microscope</td>
			<td>Life Technologies</td>
			<td>EVOS XL Core</td>
			<td>Initially a self standing company, now owned by Life Technologies.</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Zeiss</td>
			<td>LSM 710</td>
			<td>2-photon upright microscope</td>
		</tr>
	</tbody>
</table>

live cell imaging during mechanical stretch

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in their capability for measuring responses in real time in live cells or viable tissue. A protocol was developed with the use of a cell actuator to distend live cells grown on or tissues attached to an elastic substrate while imaging with confocal and atomic force microscopy (AFM). Preliminary studies show that tonic stretching of human bronchial epithelial cells caused a significant increase in the production of mitochondrial superoxide. Moreover, using this protocol, alveolar epithelial cells were stretched and imaged, which showed direct damage to the epithelial cells by overdistention simulating one form of lung injury in vitro. A protocol to conduct AFM nano-indentation on stretched cells is also provided.

Reaktive Sauerstoffspezies und Deformation Frühere Studien haben Steigerungen reaktiven Sauerstoffspezies (ROS) in den Atemwegen und alveolare Epithelzellen in Reaktion auf zyklische Dehnung 21 gezeigt. Reaktive Sauerstoffspezies umfassen Moleküle und freie Radikale aus molekularem Sauerstoff mit einer hohen Reaktivität gegenüber Lipiden, Proteinen, Polysacchariden und Nukleinsäuren 22-24 abgeleitet ist. ROS dienen als gemeinsame intrazellulären Signal an...

Watch this Scientific Journal Video about Live Cell Imaging during Mechanical Stretch at JoVE.com

Live Cell Imaging during Mechanical Stretch

A novel imaging protocol was developed using a custom motor-driven mechanical actuator to allow the measurement of real time responses to mechanical strain in live cells. Relevant to mechanobiology, the system can apply strains up to 20% while allowing near real-time imaging with confocal or atomic force microscopy.

Live Cell Imaging bei Mechanical Stretch

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in ...

live-cell-imaging-during-mechanical-stretch

Research

JoVE Journal

Bioengineering

10.3K Aufrufe.  University of Tennessee Health Science Center. A novel imaging protocol was developed using a custom motor-driven mechanical actuator to allow the measurement of real time responses to mechanical strain in live cells. Relevant to mechanobiology, the system can apply strains up to 20% while allowing near real-time imaging with confocal or atomic force microscopy. 

Serie: Live Cell Imaging during Mechanical Stretch

Axon Stretch Growth: The Mechanotransduction of Neuronal Growth

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata are separated from their axon terminals due to skeletal growth of the enlarging organism (Weiss 1941; Gray, Hukkanen et al. 1992). This mechanotransduction induces a secondary mode of neuronal growth capable of accommodating continual elongation of the axon (Bray 1984; Heidemann and Buxbaum 1994; Heidemann, Lamoureux et al. 1995; Pfister, Iwata et al. 2004).
Axon Stretch Growth (ASG) is conceivably a central factor in the maturation of short embryonic processes into the long nerves and white matter tracts characteristic of the adult nervous system. To study ASG in vitro, we engineered bioreactors to apply tension to the short axonal processes of neuronal cultures (Loverde, Ozoka et al. 2011). Here, we detail the methods we use to prepare bioreactors and conduct ASG. First, within each stretching lane of the bioreactor, neurons are plated upon a micro-manipulated towing substrate. Next, neurons regenerate their axonal processes, via growth cone extension, onto a stationary substrate. Finally, stretch growth is performed by towing the plated cell bodies away from the axon terminals adhered to the stationary substrate; recapitulating skeletal growth after growth cone extension.
Previous work has shown that ASG of embryonic rat dorsal root ganglia neurons are capable of unprecedented growth rates up to 10mm/day, reaching lengths of up to 10cm; while concurrently resulting in increased axonal diameters (Smith, Wolf et al. 2001; Pfister, Iwata et al. 2004; Pfister, Bonislawski et al. 2006; Pfister, Iwata et al. 2006; Smith 2009). This is in dramatic contrast to regenerative growth cone extension (in absence of mechanical stimuli) where growth rates average 1mm/day with successful regeneration limited to lengths of less than 3cm (Fu and Gordon 1997; Pfister, Gordon et al. 2011). Accordingly, further study of ASG may help to reveal dysregulated growth mechanisms that limit regeneration in the absence of mechanical stimuli.

A unique tissue engineering method was developed to elongate numerous nerve fibers in culture by recapitulating axon stretch growth; a form of nervous system growth whereby nerves elongate in conjunction with growth of the enlarging body.

Axon Stretch Growth: The Mechanotransduktion des neuronalen Wachstums

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata ...

Forschung

Biotechnik

Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound healing, tissue regeneration, and cancer metastasis, cells must navigate through complex, three-dimensional extracellular tissue. To better understand the mechanisms behind these biological processes, it is important to examine the roles of the proteins responsible for driving cell migration. Here, we outline a protocol to study the mechanisms of cell migration using the epithelial cell line (MDCK), and a three-dimensional, fibrous, self-polymerizing matrix as a model system. This optically clear extracellular matrix is easily amenable to live-cell imaging studies and better mimics the physiological, soft tissue environment. This report demonstrates a technique for directly visualizing protein localization and dynamics, and deformation of the surrounding three-dimensional matrix. 
Examination of protein localization and dynamics during cellular processes provides key insight into protein functions. Genetically encoded fluorescent tags provide a unique method for observing protein localization and dynamics. Using this technique, we can analyze the subcellular accumulation of key, force-generating cytoskeletal components in real-time as the cell maneuvers through the matrix. In addition, using multiple fluorescent tags with different wavelengths, we can examine the localization of multiple proteins simultaneously, thus allowing us to test, for example, whether different proteins have similar or divergent roles. Furthermore, the dynamics of fluorescently tagged proteins can be quantified using Fluorescent Recovery After Photobleaching (FRAP) analysis. This measurement assays the protein mobility and how stably bound the proteins are to the cytoskeletal network.
By combining live-cell imaging with the treatment of protein function inhibitors, we can examine in real-time the changes in the distribution of proteins and morphology of migrating cells. Furthermore, we also combine live-cell imaging with the use of fluorescent tracer particles embedded within the matrix to visualize the matrix deformation during cell migration. Thus, we can visualize how a migrating cell distributes force-generating proteins, and where the traction forces are exerted to the surrounding matrix. Through these techniques, we can gain valuable insight into the roles of specific proteins and their contributions to the mechanisms of cell migration.

Cellular processes such as cell migration have traditionally been studied on two-dimensional, stiff plastic surfaces. This report describes a technique for directly visualizing protein localization and analyzing protein dynamics in cells migrating in a more physiologically relevant, three-dimensional matrix.

Live-Cell-Imaging von wandernden Zellen mit dem Ausdruck Fluoreszenz-markierten Proteinen in einer dreidimensionalen Matrix

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound ...

Monitoring the Wall Mechanics During Stent Deployment in a Vessel

Clinical trials have reported different restenosis rates for various stent designs1. It is speculated that stent-induced strain concentrations on the arterial wall lead to tissue injury, which initiates restenosis2-7. This hypothesis needs further investigations including better quantifications of non-uniform strain distribution on the artery following stent implantation. A non-contact surface strain measurement method for the stented artery is presented in this work. ARAMIS stereo optical surface strain measurement system uses two optical high speed cameras to capture the motion of each reference point, and resolve three dimensional strains over the deforming surface8,9. As a mesh stent is deployed into a latex vessel with a random contrasting pattern sprayed or drawn on its outer surface, the surface strain is recorded at every instant of the deformation. The calculated strain distributions can then be used to understand the local lesion response, validate the computational models, and formulate hypotheses for further in vivo study.

Stent-induced arterial strain distributions are characterized using an optical surface strain measurement system. This visualization technique is used to gain insights into the impact of stent implantation on the host vessel.

Die Überwachung der Wand Mechanik Während der Entfaltung des Stents in einem Gefäß

Clinical trials have reported different restenosis rates for various stent designs1. It is speculated that stent-induced strain concentrations on the ...

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 ...

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the 'larva chip'. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3rd instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush.

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Drosophila larvae are an attractive model system for live imaging due to their translucent cuticle and powerful genetics. This protocol describes how to utilize a single-layer PDMS device, called the &#39;larva chip&#39; for live imaging of cellular processes within neurons of 3rd instar Drosophila larvae.

Mit Mikrofluidik-Chips für Live-Imaging-und Studienverletzungs Antworten in<em&gt; Drosophila</em&gt; Larven

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of ...

A Microfluidic Technique to Probe Cell Deformability

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an efficient manner. Typically, data for ~102 cells can be acquired within a 1 hr experiment. An automated image analysis program enables efficient post-experiment analysis of image data, enabling processing to be complete within a few hours. Our device geometry is unique in that cells must deform through a series of micron-scale constrictions, thereby enabling the initial deformation and time-dependent relaxation of individual cells to be assayed. The applicability of this method to human promyelocytic leukemia (HL-60) cells is demonstrated. Driving cells to deform through micron-scale constrictions using pressure-driven flow, we observe that human promyelocytic (HL-60) cells momentarily occlude the first constriction for a median time of 9.3 msec before passaging more quickly through the subsequent constrictions with a median transit time of 4.0 msec per constriction. By contrast, all-trans retinoic acid-treated (neutrophil-type) HL-60 cells occlude the first constriction for only 4.3 msec before passaging through the subsequent constrictions with a median transit time of 3.3 msec. This method can provide insight into the viscoelastic nature of cells, and ultimately reveal the molecular origins of this behavior.

We demonstrate a microfluidics-based assay to measure the timescale for cells to transit through a sequence of micron-scale constrictions.

Ein Mikrofluidiktechnik to Cell Verformbarkeit Sonde

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an ...

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn&#8217;t need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on &#181;m-sized membrane regions in the micro-to-milli-second time range.

Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups.

Von Fast Fluoreszenz-Imaging, um molekulare Diffusion Gesetz über Live Cell Membrane in einem kommerziellen Mikroskop

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

Isokinetic Robotic Device to Improve Test-Retest and Inter-Rater Reliability for Stretch Reflex Measurements in Stroke Patients with Spasticity

Measuring spasticity is important in treatment planning and determining efficacy after treatment. However, the current tool used in clinical settings has been shown to be limited in inter-rater reliability. One factor in this poor inter-rater reliability is the variability of passive motion while measuring the angle of catch (AoC) measurements. Therefore, an isokinetic device has been proposed to standardize the manual joint motion; however, the benefits of isokinetic motion for AoC measurements has not been tested in a standardized manner. This protocol investigates whether isokinetic motion itself can improve inter-rater reliability for AoC measurements. For this purpose, a robotic isokinetic device was developed that is combined with surface electromyography (EMG). Two conditions, manual and isokinetic motions, are compared with the standardized method to measure the angle and subjective feeling of catch. It is shown that in 17 stroke patients with mild elbow flexor spasticity, isokinetic motion improved the intraclass correlation coefficient (ICC) for inter-rater reliability of AoC measurements to 0.890 [95% confidence interval (CI): 0.685&#8211;0.961] by the EMG criteria, and 0.931 (95% CI: 0.791&#8211;0.978) by the torque criteria, from 0.788 (95% CI: 0.493&#8211;0.920) by manual motion. In conclusion, isokinetic motion itself can improve inter-rater reliability of AoC measurements in stroke patients with mild spasticity. Given that this system may provide greater standardized angle measurements and catch of feeling, it may be a good option for the evaluation of spasticity in a clinical setting.

Using a robotic isokinetic device with electromyography (EMG) measurements, this protocol illustrates that isokinetic motion itself can improve inter-rater reliability for the angle of catch measurements in stroke patients with mild elbow flexor spasticity.

Isokinetic Robotic Device zur Verbesserung der Test-Retest und Inter-Rater Zuverlässigkeit für Stretch Reflex Messungen bei Schlaganfall-Patienten mit Spastik

Measuring spasticity is important in treatment planning and determining efficacy after treatment. However, the current tool used in clinical settings has ...

Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized in vitro stretching methods. Various available systems use 2D substrate-based stretchers, while the accessibility of 3D techniques to strain soft hydrogels, is more restricted. Here, we describe a method that allows external stretching of soft hydrogels from their circumference, using an elastic silicone strip as the sample carrier. The stretching system utilized in this protocol is constructed from 3D-printed parts and low-cost electronics, making it simple and easy to replicate in other labs. The experimental process begins with polymerizing thick (&#62;100 &#956;m) soft fibrin hydrogels (Elastic Modulus of ~100 Pa) in a cut-out at the center of a silicone strip. Silicone-gel constructs are then attached to the printed-stretching device and placed on the confocal microscope stage. Under live microscopy the stretching device is activated, and the gels are imaged at various stretch magnitudes. Image processing is then used to quantify the resulting gel deformations, demonstrating relatively homogenous strains and fiber alignment throughout the gel&#8217;s 3D thickness (Z-axis). Advantages of this method include the ability to strain extremely soft hydrogels in 3D while executing in situ microscopy, and the freedom to manipulate the geometry and size of the sample according to the user&#8217;s needs. Additionally, with proper adaptation, this method can be used to stretch other types of hydrogels (e.g., collagen, polyacrylamide or polyethylene glycol) and can allow for analysis of cells and tissue response to external forces under more biomimetic 3D conditions.

The presented method involves uniaxial stretching of 3D soft hydrogels embedded in silicone rubber while allowing live confocal microscopy. Characterization of the external and internal hydrogel strains as well as fiber alignment are demonstrated. The device and protocol developed can assess the response of cells to various strain regimes.

Kontrollierte Dehnung von 3D-Hydrogelen unter Live-Mikroskopie-Bildgebung

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized ...

A Microphysiological System to Study Leukocyte-Endothelial Cell Interaction during Inflammation

Leukocyte-endothelial cell interactions play an important role in inflammatory diseases such as sepsis. During inflammation, excessive migration of activated leukocytes across the vascular endothelium into key organs can lead to organ failure. A physiologically relevant biomimetic microfluidic assay (bMFA) has been developed and validated using several experimental and computational techniques, which can reproduce the entire leukocyte rolling/adhesion/migration cascade to study leukocyte-endothelial cell interactions. Microvascular networks obtained from in vivo images in rodents were digitized using a Geographic Information System (GIS) approach and microfabricated with polydimethylsiloxane (PDMS) on a microscope slide. To study the effect of shear rate and vascular topology on leukocyte-endothelial cell interactions, a Computational Fluid Dynamics (CFD) model was developed to generate a corresponding map of shear rates and velocities throughout the network. The bMFA enables the quantification of leukocyte-endothelial cells interactions, including rolling velocity, number of adhered leukocytes in response to different shear rates, number of migrated leukocytes, endothelial cell permeability, adhesion molecule expression and other important variables. Furthermore, by using human-related samples, such as human endothelial cells and leukocytes, bMFA provides a tool for rapid screening of potential therapeutics to increase their clinical translatability.

In this protocol, a biomimetic microfluid assay, which can reproduce a physiologically relevant microvascular environment and reproduce the entire leukocyte adhesion/migration cascade, is employed to study leukocyte-endothelial cell interactions in inflammatory disease.

Ein mikrophysiologisches System zur Untersuchung der Leukozyten-Endothelzell-Interaktion während einer Entzündung

Leukocyte-endothelial cell interactions play an important role in inflammatory diseases such as sepsis. During inflammation, excessive migration of ...