Diese Arbeit wurde vom Institut Curie, Paris, Frankreich gegründet. Die mechanische Bahre wurde von Damien Cuvelier (Institut Curie) entworfen und wird von GREM hergestellt (mecanique-grem.com). Die Strukturierung auf PDMS wurde von Ammar Azioune (Bordeaux II University) entwickelt.

1. Passivierung der PDMS <ol><li> Ein Stück des PDMS ca. 35 mm x 20 mm aus einer bereits geformten Folie (zum Beispiel GelPak, wie in der Tabelle aufgeführten Materialien). </li><li> Entfernen Sie die obere und die untere Schutzschichten aus Kunststoff (falls erforderlich) und mit Hilfe einer Pinzette, um die PDMS in einer Kunststoff legen (nicht zellkulturbehandelte) Petrischale. </li><li> Waschen Sie die PDMS mit 70% Ethanol für 5 Minuten auf einem Schüttler bei 30 Schwingungen / min. </li><li> Trocknen Sie die Oberfläche durch strömende Luft auf sie. </li><li> Illuminate mit tiefen UV (λ = 180 nm) für 5 min bei einem Abstand von den UV-Lampen von etwa 5 cm (siehe Materialien Blatt für Lampe Referenz; Parameter für verschiedene Lampen variieren). </li><li> Planen 200 ul EDC / NHS-Lösung für jedes Stück PDMS (muss unmittelbar vor der Verwendung hergestellt, da die Reaktivität der Lösung zerfällt in wenigen Stunden werden). Für 1 ml 0,05 M MES + 0,5 M NaCl-Puffer bei pH 6,0, fügen 11,5 mg Sulfo-NHS und 19,2 mg EDC. </li><li> Übertragen Sie diePDMS Blätter von der Petrischale mit dem Deckel der Petrischale, die nicht belichtet worden ist. Dadurch wird sichergestellt, dass die Umgebung des PDMS sind sehr hydrophob und ermöglichen den nächsten Schritt. </li><li> In EDC / NHS-Lösung und Inkubation für 15 min bei Raumtemperatur. </li><li> Spülen EDC / NHS mit Wasser. </li><li> Hinzufügen PLL-g-PEG-Lösung (0,5 mg / ml in 10 mM HEPES pH 8.6) und Inkubation von 3 h bis über Nacht bei Raumtemperatur. </li><li> Spülen Sie die PLL-g-PEG mit Wasser. Passi PDMS (mit PLL-g-PEG funktionalisiert) kann für mehrere Tage bei 4 ° C gelagert werden </li></ol> 2. Strukturieren der PDMS <ol><li> Nehmen Sie ein PDMS Blatt und legen Sie es auf einem synthetischen Quarzphotomaske tragen die Mikroformen zur Strukturierung (siehe Abbildung 1). Legen Sie die PDMS seitigen Lager die PLL-g-PEG mit Blick auf den Chrom Seite der Photomaske. </li><li> Beleuchten 7 Minuten durch die Photomaske in einem Abstand von der UV-Lampen von etwa 5 cm.</li><li> Fügen Sie Wasser auf die Maske + PDMS und schälen Sie die PDMS langsam aus der Maske. </li><li> Inkubieren mit Fibronectin-Lösung bei 50 mg / ml in HEPES-Puffer bei pH 8,6 für 1 Stunde bei Raumtemperatur. Es ist möglich, andere ECM-Proteine ​​zu verwenden, aber diese sind nicht mit diesem Protokoll getestet. </li><li> Spülen mit PBS. </li></ol> 3. Das Gerät montieren <ol><li> Montieren Sie die zuvor passivierten PDMS auf die Spannvorrichtung (siehe Diskussion zur Fehlersuche). <ol><li> Befestigen einer Seite des PDMS zu dem feststehenden Teil der Trage. </li><li> Befestigen Sie die andere Seite mit dem Mobilteil der Trage ohne Spann zu viel von der PDMS Blatt. </li></ol></li><li> Schneiden Sie ein Rechteck von PDMS (22 mm x 19 mm) in einer dicken PDMS Stich-und Schnitt ein weiteres Rechteck im Inneren, um einen Pool (oder Rahmen) haben, die die Zellen-und Medien behalten werden (siehe Abbildung 2). </li><li> In Silikonfett unter diese PDMS Pool und legen Sie es auf der PDMS-Blatt zu erstellen med.ium Haltebecken. Das Fett wird die Gleitfähigkeit des Pools über das PDMS Blatt beim Strecken zu ermöglichen. </li></ol> 4. Strukturieren der Zellen <ol><li> Lösen Sie die Zellen aus einer 50% Konfluenz Kulturflasche mit Versene. </li><li> Zähle die Zellen zu resuspendieren und diese in einer Konzentration von 200.000 Zellen / ml. </li><li> 1 ml der Zellsuspension in den Pool (siehe Diskussion für weitere Details). </li><li> Sei (je nach Zelltyp, RPE-1-Zellen werden 10 min und HeLa-Zellen 20 min dauern) die Zellen zu binden, um die Muster für 10-30 min. </li><li> Vorsichtig spülen Sie die schwimmenden Zellen mit ins Gleichgewicht Medium (ins Gleichgewicht mittel Inkubator). </li><li> Lassen Sie die Zellen verteilt für ein paar Stunden auf die Muster (RPE-1 ist mindestens 2 Stunden brauchen, HeLa-Zellen wird 3 Stunden brauchen). </li><li> Fügen Sie ein Deckglas auf den Pool, um Verdunstung und mittlere Leckage bei der PDMS Pausen zu vermeiden. </li><li> Legen Sie das Gerät auf einem inversen Mikroskop und starten Bildgebung. Vermeiden Sie die Verwendung von Ölimmersion objectives, da es nicht durch Fokussierung Probleme funktionieren. </li></ol> 5. Dehnung <ol><li> Die Mikrometerschraube drehen, während die Korrektur der Tischposition in x-, y-und z-Achse (hauptsächlich x). Die Bühnenposition muss korrigiert, die Erweiterung des PDMS und den Verlust des Fokus entgegenzuwirken. (Siehe &quot;Bei der Strecke&quot; Absatz in der Diskussion). </li></ol>

<ol>
	<li>Vogel, V., Sheetz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+force+and+geometry+sensing+regulate+cell+functions.">Local force and geometry sensing regulate cell functions.</a> Nat. Rev. Mol. Cell Biol. 7, 265-275 (2006).</li><li>Terenna, C. R., 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=Physical+mechanisms+redirecting+cell+polarity+and+cell+shape+in+fission+yeast.">Physical mechanisms redirecting cell polarity and cell shape in fission yeast.</a> Curr. Biol. 18 (22), 1748-1753 (2008).</li><li>Guillot, C., Lecuit, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanics+of+epithelial+tissue+homeostasis+and+morphogenesis.">Mechanics of epithelial tissue homeostasis and morphogenesis.</a> Science. 340 (6137), 1185-1189 (2013).</li><li>Bosveld, F., 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=Mechanical+control+of+morphogenesis+by+Fat/Dachsous/Four-jointed+planar+cell+polarity+pathway.">Mechanical control of morphogenesis by Fat/Dachsous/Four-jointed planar cell polarity pathway.</a> Science. 336 (6082), 724-727 (2012).</li><li>Farhadifar, R., Roper, J. C., Aigouy, B., Eaton, S., Julicher, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+cell+mechanics,+cell-cell+interactions,+and+proliferation+on+epithelial+packing.">The influence of cell mechanics, cell-cell interactions, and proliferation on epithelial packing.</a> Curr. Biol. 17, 2095-2104 (2007).</li><li>Rauzi, M., Verant, P., Lecuit, T., Lenne, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nature+and+anisotropy+of+cortical+forces+orienting+Drosophila+tissue+morphogenesis.">Nature and anisotropy of cortical forces orienting Drosophila tissue morphogenesis.</a> Nat. Cell Biol. 10, 1401-1410 (2008).</li><li>Mitrossilis, D., 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=Real-time+single-cell+response+to+stiffness.">Real-time single-cell response to stiffness.</a> Proc. Natl. Acad. Sci. U.S.A. 107 (38), 16518-16523 (2010).</li><li>Irimia, D., Charras, G., Agrawal, N., Mitchison, T., Toner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polar+stimulation+and+constrained+cell+migration+in+microfluidic+channels.">Polar stimulation and constrained cell migration in microfluidic channels.</a> Lab Chip. 12, 1783-1790 (2007).</li><li>Fink, J., 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=External+forces+control+mitotic+spindle+positioning.">External forces control mitotic spindle positioning.</a> Nat. Cell. Biol. 13 (7), 771-778 (2011).</li><li>Azioune, A., Storch, M., Bornens, M., Th&#233;ry, M., Piel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+and+rapid+process+for+single+cell+micro-patterning.">Simple and rapid process for single cell micro-patterning.</a> Lab Chip. 9 (11), 1640-1642 (2009).</li><li>Azioune, A., Carpi, N., Tseng, Q., Th&#233;ry, M., Piel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+micropatterns:+A+direct+printing+protocol+using+deep+Uvs.">Protein micropatterns: A direct printing protocol using deep Uvs.</a> Methods Cell Biol. 97, 133-146 (2010).</li><li>Azioune, A., 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=Robust+method+for+high-throughput+surface+patterning+of+deformable+substrates.">Robust method for high-throughput surface patterning of deformable substrates.</a> Langmuir. 27 (12), 7349-7352 (2011).</li><li>Carpi, N., Piel, M., Azioun, A., Cuvelier, D., Fink, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micropatterning+on+silicon+elastomer+(PDMS)+with+deep+UVs.">Micropatterning on silicon elastomer (PDMS) with deep UVs.</a> Protoc. Exch. , (2011).</li><li>Sinha, B., 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=Cells+respond+to+mechanical+stress+by+rapid+disassembly+of+caveolae.">Cells respond to mechanical stress by rapid disassembly of caveolae.</a> Cell. 144 (3), 402-413 (2011).</li></ol>

Die Autoren erklären, keine finanziellen Interessen konkurrieren.

Obwohl diese Technik wurde mehrfach verwendet und wird gründlich getestet, gibt es mehrere kritische Schritte, die zu einem fehlgeschlagenen Experiment führen kann. Über die PDMS: Für diese Arbeit GelPak ein handelsüblicher Dünn PDMS Blatt, verwendet. Alternativ PDMS Blätter können direkt von PDMS-Mischung gegossen werden. Wir empfehlen die Verwendung GelPak, weil es mehr reproduzierbar, und ist weniger wahrscheinlich zu brechen Vergleich zu maßgeschneidert...

Die Zellen Zusammensetzen eines Gewebe in höheren Organismen unterliegen mechanischen Spannungen und Dehnungskräfte kommen entweder von der äußeren Umgebung oder von umgebenden Zellen 1,2. Die Zellen müssen sich anzupassen und zu widerstehen, diese Kräfte, um die Gewebeintegrität aufrecht zu erhalten. Solche Kräfte sind auch für Gewebe-Morphogenese während der Entwicklung 3,4. Anwenden von mechanischen Kräften auf kultivierte Zellen ist ein Weg, zu imitieren, was in einem Gewebe passieren könnte, aber mit einer quantitativen und unabhängige Steuerung der Zellform und Zellverformung 5,6. Hierzu könnten verschiedene Techniken verwendet werden. Man kann auf die Zellen (die ganze Zelle oder einen Teil davon), beispielsweise mit AFM oder Derivate 7,8 oder strecken das Substrat die Zellen wachsen auf zu drücken. Die in diesem Dokument beschriebenen Verfahren zeigt, wie ein Flugzeug Substrat mit Zellen plattiert dehnen. Auf m Diese Technik wurde ursprünglich entwickelt, um beurteilen die Rolle der Streitkräfte ausgeübtitotic Säugetierzellen 9. Mitotische Zellen bleiben mit dem Substrat verbunden, durch Zurückziehen und Strecken der Fasern Membran ausgeübt wird, eine Kraft auf jene Fasern, die wiederum provozierte die Rotation der mitotischen Spindel. Das Interesse der Kombination von Klebstoff und Mikro Recken um eine unabhängige Steuerung von Kräften und Form der einzelnen Zellen zu erreichen. Es ist beispielsweise möglich, eine eiförmige Zelle in eine perfekt runde Form isotropen strecken, während einachsige Dehnung angewendet wird. Wenn die Zellen nicht auf Mikro geflochten, uniaxiales Strecken Ergebnisse in Zellstreckung, wobei die meisten Zellen, die ein mit dem Streckachse ausgerichtet langen Achse. Es ist dann schwierig, den Effekt der Ausrichtung der langen Achse und der Wirkung der Ausdehnung auf die Zellen zu trennen. Das Gerät eignet sich für alle lebenden Zellen, einschließlich Langzeitablauf Fluoreszenz-Mikroskopie, und Medikamente können während des Experiments hinzugefügt werden. Die tiefe Mikrostrukturierungsverfahren 10 UVswurde im Detail in Azioune et al. 11 Musterung auf PDMS wurde beschrieben in Azioune et al. 12 Die vorliegende Streck Protokoll ist ein Video-Version Carpi et al. 13

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">GelPak</td>
			<td style="width:71px;">GelPak</td>
			<td style="width:119px;">PF-60-X4</td>
			<td style="width:167px;">Different thickness/stickiness are available. One alternative could be to cast your PDMS yourself.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Silicon grease</td>
			<td style="width:71px;">GE Bayer Silicones</td>
			<td style="width:119px;">Baysilone-Paste</td>
			<td>This one is biocompatible</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Stretching device</td>
			<td style="width:71px;">GREM m&#233;canique</td>
			<td style="width:119px;">Stretcher 2011</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">EDC (N-(3-Dimethylaminopropyl)-N&#8242;-ethylcarbodiimide hydrochloride)</td>
			<td style="width:71px;">Sigma</td>
			<td align="right" style="width:119px;">3450</td>
			<td>Stable 6 months at -20 &#176;C</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">NHS (N-Hydroxysulfosuccinimide sodium salt)</td>
			<td style="width:71px;">Sigma</td>
			<td align="right" style="width:119px;">56485</td>
			<td>Protect from humidity</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Pll-g-peg (PLL(20)-g[3.5]-PEG(2) 20 mg)</td>
			<td style="width:71px;">SurfaceSolutions (Zurich)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Synthetic Quartz photomask</td>
			<td style="width:71px;">Toppan</td>
			<td style="width:119px;"></td>
			<td>Take standard binary photomask in Quartz</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fibronectin from bovine plasma</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">F1141</td>
			<td></td>
		</tr>
	</tbody>
</table>

stretching micropatterned cells on a pdms membrane

Mechanische Kräfte auf Zellen ausgeübt und / oder Gewebe spielen eine wichtige Rolle in zahlreichen Prozessen. Wir haben ein Gerät an Zellen auf einer Polydimethylsiloxan (PDMS)-Membran, mit bildgebenden kompatibel vernickelt strecken entwickelt. Dieses Verfahren ist reproduzierbar und vielseitig. Die PDMS-Membran, um Zellen oder Gewebe zu einem bestimmten Geometrie beschränken mikrostrukturiert werden. Der erste Schritt ist die Mikrostrukturen auf den PDMS-Membran mit einem tiefen UV-Technik gedruckt. Die PDMS-Membran wird dann auf einer mechanischen Trage befestigt. Eine Kammer ist auf der Oberseite der Membran mit biokompatiblen Fett damit Gleiten während der Strecke gebunden. Die Zellen werden ausgesät und für mehrere Stunden auf den Mikro verbreiten. Die Probe kann mehrere Male unter Verwendung einer Mikrometerschraube gespannt und ungestreckte werden. Es dauert weniger als eine Minute, um die Strecke in vollem Umfang (rund 30%) anzuwenden. Die hier vorgestellte Technik nicht eine motorisierte Vorrichtung, die für eine erforderliche umfassenpplying wiederholter Dehnungszyklen schnell und / oder Computer-kontrolliertes Recken, aber dies kann realisiert werden. Dehnen von Zellen oder Gewebe kann von Interesse für die Fragen, um die Zellkräfte, Zell-Antwort gegen mechanische Beanspruchung oder Gewebemorphogenese bezogen werden. Diese Video-Präsentation wird zeigen, wie die typischen Probleme, die, wenn Sie diese Art von scheinbar einfachen Experiment entstehen könnten.

<html> Die in diesem Video-Protokoll vorgestellte Technik erlaubt die Anwendung von Kräften auf die Rückzugs Fasern mitotischer Säugerzellen. In der Tat, während der Zellteilung, bei der mitotischen Phase, Säugetierzellen zurückziehen, um die Form einer Kugel nehmen und hinterlassen dünnen Aktin-Kabel von Membran umgeben, die auf dem Substrat befestigt sind. Diese Kabel (Rückzug Fasern), sind die Erinnerung an die Zellgeometrie, bevor er in Division. Machen Mikro mit tiefen UV durch eine Photomaske auf PDMS Dünn...

Watch this Scientific Journal Video about Stretching Micropatterned Cells on a PDMS Membrane at JoVE.com

Stretching Micropatterned Cells on a PDMS Membrane

Dieses Manuskript stellt eine Technik, um auf adhärenten Zellen oder Gewebe gelten oder freiKräfte mit unidirektionalen Stretching.

Stretching-Cells auf einer mikrostrukturierten PDMS-Membran

Mechanical forces exerted on cells and/or tissues  play a major role in numerous processes. We have developed a device to stretch  cells plated on a ...

stretching-micropatterned-cells-on-a-pdms-membrane

Research

JoVE Journal

Bioengineering

15.2K Aufrufe.  Institut Curie.  Dieses Manuskript stellt eine Technik, um auf adhärenten Zellen oder Gewebe gelten oder freiKräfte mit unidirektionalen Stretching. 

Serie: Stretching Micropatterned Cells on a PDMS Membrane

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix (ECM) components within the tumor microenvironment. Hyaluronic acid (HA) is one stromal ECM component that is known to facilitate tumor progression by enhancing invasion, growth, and angiogenesis1. Interaction of HA with its cell surface receptor CD44 induces signaling events that promote tumor cell growth, survival, and migration, thereby increasing metastatic spread2-3. HA is an anionic, nonsulfated glycosaminoglycan composed of repeating units of D-glucuronic acid and D-N-acetylglucosamine. Due to the presence of carboxyl and hydroxyl groups on repeating disaccharide units, native HA is largely hydrophilic and amenable to chemical modifications that introduce sulfate groups for photoreative immobilization 4-5. Previous studies involving the immobilizations of HA onto surfaces utilize the bioresistant behavior of HA and its sulfated derivative to control cell adhesion onto surfaces6-7. In these studies cell adhesion preferentially occurs on non-HA patterned regions.
To analyze cellular interactions with exogenous HA, we have developed patterned functionalized surfaces that enable a controllable study and high-resolution visualization of cancer cell interactions with HA. We utilized microcontact printing (uCP) to define discrete patterned regions of HA on glass surfaces. A "tethering" approach that applies carbodiimide linking chemistry to immobilize HA was used 8. Glass surfaces were microcontact printed with an aminosilane and reacted with a HA solution of optimized ratios of EDC and NHS to enable HA immobilization in patterned arrays. Incorporating carbodiimide chemistry with mCP enabled the immobilization of HA to defined regions, creating surfaces suitable for in vitro applications. Both colon cancer cells and breast cancer cells implicitly interacted with the HA micropatterned surfaces. Cancer cell adhesion occurred within 24 hours with proliferation by 48 hours. Using HA micropatterned surfaces, we demonstrated that cancer cell adhesion occurs through the HA receptor CD44. Furthermore, HA patterned surfaces were compatible with scanning electron microscopy (SEM) and allowed high resolution imaging of cancer cell adhesive protrusions and spreading on HA patterns to analyze cancer cell motility on exogenous HA.

A novel approach that allows the high-resolution analysis of cancer cell interactions with exogenous hyaluronic acid (HA) is described. Patterned surfaces are fabricated by combining carbodiimide chemistry and microcontact printing.

Mikrostrukturierten Oberflächen zum Studium Hyaluronsäure Wechselwirkungen mit Krebszellen

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix ...

Forschung

Biotechnik

Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated micro-plates. Under these conditions, cells spread and divide in all directions resulting in an inherent variability in cell shape, morphology and behavior. The high cell-to-cell variance of the overall population impedes the success of HCA, especially for drug development. The ability of micropatterns to normalize the shape and internal polarity of every individual cell provides a tremendous opportunity for solving this critical bottleneck 1-2.
To facilitate access and use of the micropatterning technology, CYTOO has developed a range of ready to use micropatterns, available in coverslip and microwell formats. In this video article, we provide detailed protocols of all the procedures from cell seeding on CYTOOchip micropatterns, drug treatment, fixation and staining to automated acquisition, automated image processing and final data analysis. With this example, we illustrate how micropatterns can facilitate cell-based assays. Alterations of the cell cytoskeleton are difficult to quantify in cells cultured on homogenous substrates, but culturing cells on micropatterns results in a reproducible organization of the actin meshwork due to systematic positioning of the cell adhesion contacts in every cell. Such normalization of the intracellular architecture allows quantification of even small effects on the actin cytoskeleton as demonstrated in these set of protocols using blebbistatin, an inhibitor of the actin-myosin interaction.

Adhesive micropatterns that normalize cellular architecture can be used to increase sensitivity in the detection of drug effects, improve reproducibility and simplify automated image acquisition and analysis. Such technology will benefit drug/siRNA screening assays, performed on conventional cell culture supports and consequently suffering from excessive cell-to-cell variability.

Verbesserte Visualisierung und quantitative Analyse von Arzneimittelwirkungen mit mikrostrukturierten Cells

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated ...

Stretching Short Sequences of DNA with Constant Force Axial Optical Tweezers

Single-molecule techniques for stretching DNA of contour lengths less than a kilobase are fraught with experimental difficulties. However, many interesting biological events such as histone binding and protein-mediated looping of DNA1,2, occur on this length scale. In recent years, the mechanical properties of DNA have been shown to play a significant role in fundamental cellular processes like the packaging of DNA into compact nucleosomes and chromatin fibers3,4. Clearly, it is then important to understand the mechanical properties of short stretches of DNA. In this paper, we provide a practical guide to a single-molecule optical tweezing technique that we have developed to study the mechanical behavior of DNA with contour lengths as short as a few hundred basepairs.
The major hurdle in stretching short segments of DNA is that conventional optical tweezers are generally designed to apply force in a direction lateral to the stage5,6, (see Fig. 1). In this geometry, the angle between the bead and the coverslip, to which the DNA is tethered, becomes very steep for submicron length DNA. The axial position must now be accounted for, which can be a challenge, and, since the extension drags the microsphere closer to the coverslip, steric effects are enhanced. Furthermore, as a result of the asymmetry of the microspheres, lateral extensions will generate varying levels of torque due to rotation of the microsphere within the optical trap since the direction of the reactive force changes during the extension.
Alternate methods for stretching submicron DNA run up against their own unique hurdles. For instance, a dual-beam optical trap is limited to stretching DNA of around a wavelength, at which point interference effects between the two traps and from light scattering between the microspheres begin to pose a significant problem. Replacing one of the traps with a micropipette would most likely suffer from similar challenges. While one could directly use the axial potential to stretch the DNA, an active feedback scheme would be needed to apply a constant force and the bandwidth of this will be quite limited, especially at low forces.
We circumvent these fundamental problems by directly pulling the DNA away from the coverslip by using a constant force axial optical tweezers7,8. This is achieved by trapping the bead in a linear region of the optical potential, where the optical force is constant-the strength of which can be tuned by adjusting the laser power. Trapping within the linear region also serves as an all optical force-clamp on the DNA that extends for nearly 350 nm in the axial direction. We simultaneously compensate for thermal and mechanical drift by finely adjusting the position of the stage so that a reference microsphere stuck to the coverslip remains at the same position and focus, allowing for a virtually limitless observation period.

We illustrate the use of a constant force axial optical tweezers to explore the mechanical properties of short DNA molecules. By stretching DNA axially, we minimize steric hindrances and artifacts arising in conventional lateral manipulation, allowing us to study DNA molecules as short as ~100 nm.

Stretching kurze DNA-Sequenzen mit Constant Force Axial Optische Pinzetten

Single-molecule techniques for stretching DNA of contour lengths  less than a kilobase are fraught with experimental difficulties. However, many ...

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 ...

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range of 10-100 &#197;. By measuring the distances under various ligated conditions, conformational changes of the protein can be easily assessed. With LRET, a lanthanide, most often chelated terbium, is used as the donor fluorophore, affording advantages such as a longer donor-only emission lifetime, the flexibility to use multiple acceptor fluorophores, and the opportunity to detect sensitized acceptor emission as an easy way to measure energy transfer without the risk of also detecting donor-only signal. Here, we describe a method to use LRET on membrane proteins expressed and assayed on the surface of intact mammalian cells. We introduce a protease cleavage site between the LRET fluorophore pair. After obtaining the original LRET signal, cleavage at that site removes the specific LRET signal from the protein of interest allowing us to quantitatively subtract the background signal that remains after cleavage. This method allows for more physiologically relevant measurements to be made without the need for purification of protein.

We describe here an improved Luminescence Resonance Energy Transfer (LRET) method where we introduce a protease cleavage site between the donor and acceptor fluorophore sites. This modification allows us to obtain specific LRET signals arising from membrane proteins of interest, allowing for the study of membrane proteins without protein purification.

Lumineszenz-Resonanzenergietransfer zu Konformationsänderungen in Membranproteinen in Säugerzellen exprimiert Studieren

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range ...

Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(&#949;-caprolactone) Electrospun Yarns

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell number. While researchers commonly reference their seeding density this does not necessarily indicate the actual number of cells that have adhered to the material in question. This is particularly the case for materials, or scaffolds, that do not cover the base of standard cell culture well plates. This study investigates the initial attachment of human mesenchymal stem cells seeded at a known number onto electrospun poly(&#949;-caprolactone) yarn after 4 hr in culture. Electrospun yarns were held within several different set-ups, including bioreactor vessels rotating at 9 rpm, cell culture inserts positioned in low binding well plates and polytetrafluoroethylene (PTFE) troughs placed within petri dishes. The latter two were subjected to either static conditions or positioned on a shaker plate (30 rpm). After 4 hr incubation at 37&#160;oC, 5% CO2, the location of seeded cells was determined by cell DNA assay. Scaffolds were removed from their containers and placed in lysis buffer. The media fraction was similarly removed and centrifuged &#8211; the supernatant discarded and pellet broken up with lysis buffer. Lysis buffer was added to each receptacle, or well, and scraped to free any cells that may be present. The cell DNA assay determined the percentage of cells present within the scaffold, media and well fractions. Cell attachment was low for all experimental set-ups, with greatest attachment (30%) for yarns held within cell culture inserts and subjected to shaking motion. This study raises awareness to the actual number of cells attaching to scaffolds irrespective of the stated cell seeding density.

Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(&amp;#949;-caprolactone) Electrospun Yarns

This article describes a range of set-ups for seeding human mesenchymal stem cells onto materials, in this case electrospun yarns, that do not cover the base of standard culture well plates in order to maximize and quantify the number of cells that initially attach compared to the known seeding density.

Optimierung der Befestigung der humanen mesenchymalen Stammzellen auf Poly (ε-caprolacton) Elektrogesponnene Garne

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell ...

Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. The cell spheroids are prepared in a culture system on micropatterned plates coated with a thermosensitive polymer. A number of spheroids are formed on the plates, corresponding to the cell adhesion areas of 100 &#181;m diameter that are regularly arrayed in a two-dimensional manner, surrounded by non-adhesive areas that are coated by a polyethylene glycol (PEG) matrix. The spheroids can be easily recovered as a liquid suspension by lowering the temperature of the plates, and their structure is well maintained by passing them through injection needles with a sufficiently large caliber (over 27 G). Genetic modification is achieved by gene transfection using the original non-viral gene carrier, polyplex nanomicelle, which is capable of introducing genes into cells without disrupting the spheroid structure. For primary hepatocyte spheroids transfected with a luciferase-expressing gene, the luciferase is sustainably obtained in transplanted animals, along with preserved hepatocyte function, as indicated by albumin expression. This system can be applied to a variety of cell types including mesenchymal stem cells.

This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

Gentransfektion in Richtung Spheroid Zellen auf mikrostrukturierten Kulturplatten für genetisch veränderte Zelltransplantation

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. ...

Microfluidic Genipin Deposition Technique for Extended Culture of Micropatterned Vascular Muscular Thin Films

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular behaviors on disease-relevant time scales. Previously, microcontact printing has been used to fabricate two-dimensional functional arterial mimics through patterning of extracellular matrix protein as guidance cues for tissue organization. Vascular muscular thin films utilized these mimics to assess functional contractility. However, the microcontact printing fabrication technique used typically incorporates hydrophobic PDMS substrates. As the tissue turns over the underlying extracellular matrix, new proteins must undergo a conformational change or denaturing in order to expose hydrophobic amino acid residues to the hydrophobic PDMS surfaces for attachment, resulting in altered matrix protein bioactivity, delamination, and death of the tissues.
Here, we present a microfluidic deposition technique for patterning of the crosslinker compound genipin. Genipin serves as an intermediary between patterned tissues and PDMS substrates, allowing cells to deposit newly-synthesized extracellular matrix protein onto a more hydrophilic surface and remain attached to the PDMS substrates. We also show that extracellular matrix proteins can be patterned directly onto deposited genipin, allowing dictation of engineered tissue structure. Tissues fabricated with this technique show high fidelity in both structural alignment and contractile function of vascular smooth muscle tissue in a vascular muscular thin film model. This technique can be extended using other cell types and provides the framework for future study of chronic tissue- and organ-level functionality.

We present a method for microfluidic deposition of patterned genipin and fibronectin on PDMS substrates, allowing extended viability of vascular smooth muscle cell-dense tissues. This tissue fabrication method is combined with previous vascular muscular thin film technology to measure vascular contractility over disease-relevant time courses.

Mikrofluidischen Genipin Abscheidetechnik für Extended Kultur von mikrostrukturierten Vascular Muscular Thin Films

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular ...

Double Emulsion Generation Using a Polydimethylsiloxane (PDMS) Co-axial Flow Focus Device

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper presents a polydimethylsiloxane (PDMS) microfluidic device capable of generating water/oil/water double emulsions using a coaxial flow focusing geometry that can be fabricated entirely using soft lithography. Similar to emulsion devices using glass capillaries, double emulsions can be formed in channels with uniform wettability and with dimensions much smaller than the channel sizes. Three dimensional flow focusing geometry is achieved by casting a pair of PDMS slabs using two layer soft lithography, then mating the slabs together in a clamshell configuration. Complementary locking features molded into the PDMS slabs enable the accurate registration of features on each of the slab surfaces. Device testing demonstrates formation of double emulsions from 14 &#181;m to 50 &#181;m in diameter while using large channels that are robust against fouling and clogging.

Double Emulsion Generation Using a Polydimethylsiloxane PDMS Co-axial Flow Focus Device

Microfluidic double emulsions generation typically involves devices with patterned wettability or custom-fabricated glass components. Here we describe the fabrication and testing of an all polydimethylsiloxane (PDMS) double emulsion generator that does not require surface treatment or complicated fabrication processes, and is capable of producing double emulsions down to 14 &#181;m.

Doppelemulsion Erstellung Mit einem Polydimethylsiloxan (PDMS) Co-Axial Flow Fokus Geräte-

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper ...

Treatment of Ligament Constructs with Exercise-conditioned Serum: A Translational Tissue Engineering Model

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, and translation of findings is often poor. Thus, there is ample opportunity for alternative experimental approaches. Here we present an approach in which human cells are isolated from human anterior cruciate ligament tissue remnants, expanded in culture, and used to form engineered ligaments. Exercise alters the biochemical milieu in the blood such that the function of many tissues, organs and bodily processes are improved. In this experiment, ligament construct culture media was supplemented with experimental human serum that has been &#39;conditioned&#39; by exercise. Thus the intervention is more biologically relevant since an experimental tissue is exposed to the full endogenous biochemical milieu, including binding proteins and adjunct compounds that may be altered in tandem with the activity of an unknown agent of interest. After treatment, engineered ligaments can be analyzed for mechanical function, collagen content, morphology, and cellular biochemistry. Overall, there are four major advantages versus traditional monolayer culture and animal models, of the physiological model of ligament tissue that is presented here. First, ligament constructs are three-dimensional, allowing for mechanical properties (i.e., function) such as ultimate tensile stress, maximal tensile load, and modulus, to be quantified. Second, the enthesis, the interface between boney and sinew elements, can be examined in detail and within functional context. Third, preparing media with post-exercise serum allows for the effects of the exercise-induced biochemical milieu, which is responsible for the wide range of health benefits of exercise, to be investigated in an unbiased manner. Finally, this experimental model advances scientific research in a humane and ethical manner by replacing the use of animals, a core mandate of the National Institutes of Health, the Center for Disease Control, and the Food and Drug Administration.

We present a model of ligament tissue in which three-dimensional constructs are treated with the human exercise-conditioned serum and analyzed for collagen content, function, and cellular biochemistry.

Behandlung von Ligamentenkonstrukten mit ausüberschüssigem Serum: Ein Translations-Gewebe-Engineering-Modell

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, ...