Die Autoren danken den Einsatz der Oberflächenanalyse Laboratory an der Johns Hopkins, als Teil des Materials Science and Engineering Center durch die National Science Foundation finanziert. LED ist ein IGERT Praktikant und ein National Science Foundation Graduate Fellow. Diese Arbeit wurde teilweise durch NIH U54CA143868 unterstützt.

1. Standard-Photolithographie für mikrostrukturierten Stempel Fabrication <ol><li> Spülen Sie ein neues Silizium-Wafer mit Ethanol und trocken mit Luftstrom. Verwenden einer Pinzette, um Wafer Griff und verhindern Oberflächendefekte während des gesamten Prozesses. </li><li> Transfer-Wafer-Coater Spin und decken Waferoberfläche mit SU-2025 negativer Photoresist. Abdeckung von mindestens 80% des Wafers mit Fotolack. Spin Mantel für 10 Sekunden bei 600U/min, um 30 Sekunden bei 3000rpm gefolgt. Aufgrund der Lichtempfindlichkeit der Fotolack, schalten Sie Raumbeleuchtung von diesem Punkt an. </li><li> Transfer Fotolack beschichteten Silizium-Wafer zu heißen Platte für &quot;Soft-bake&quot; bei 95 ° C für 3 Minuten. Remove from Heizplatte und ermöglichen 1 Minute abkühlen lassen. </li><li> Legen Sie eine vorgefertigte Maske mit dem gewünschten Muster auf der Oberseite des Fotolack bedeckt Silizium-Wafer und setzen mit ultraviolettem (UV) Bestrahlung (350-450 nm) für 20 Sekunden. </li><li> Transfer-Silizium-Wafer zu heißen Platte für &quot;hard-backen&quot; bei 110 ° C für 6 Minuten. </li><li> Schalten Sie die Beleuchtung ein und entfernen Wafer aus Heizplatte. Verschiedene Muster sichtbar sein soll an dieser Stelle. </li><li> Sorgfältig abspülen Silizium-Wafer mit SU-8 Fotolack-Entwickler. Nach 3 Spülungen mit Entwickler-Lösung, mit Ethanol spülen. Wenn ein weißer Rückstand vorhanden ist, wiederholen Sie gründlich mit Fotolack Entwicklerlösung, oder einfach nur tauchen die Wafer in Entwickler-Lösung für 2 Minuten und fahren wieder mit Ethanol spülen. Sofort mit Wasser und der Luft trocknen. </li><li> Mix Polydimethylsiloxan (PDMS) Elastomer-Lösung und Härter im Verhältnis 10:1 Gewichtsverhältnis. Zentrifugation bei 900rpm für 5 Minuten, um Luftblasen zu entfernen. </li><li> Ort strukturierten Wafer in Petrischale und gieße PDMS auf Silizium-Wafer. Wenn es Blasen, in Exsikkator und Vakuum für ein paar Minuten, bis Blasen verschwinden. </li><li> Cure über Nacht bei Raumtemperatur (RT) mit einem komplementären elastomere Stempel zu bilden. Wenn PDMS ist nicht ganz nach 12 Stunden, im Ofen gehärtet bei 60 ° C für 30 Minuten. </li><li> Entfernen Sie vorsichtig PDMS aus Silizium-Wafer. Verwenden des Messers Schneide, um Stempel auf die gewünschte Größe geschnitten. Mit Ultraschall in Ethanol für 15 Minuten. Waffeln kann mehrfach genutzt werden, um neue elastomere Stempel erstellen. </li></ol> 2. HA Mikrostrukturierung <ol><li> Plasma sauberen Glasobjektträger für 5 Minuten. Legen Sie alle Dias in die Kammer und Vakuum für 5 Minuten. Dann können geringe Zufuhr von Sauerstoff in die Kammer betreten. Schalten Sie Plasma-Reiniger auf 5 Minuten. Entfernen Sie aus dem Plasma-Reiniger und in einer Petrischale mit einer Pinzette. </li><li> Während Plasma-Reinigung, beschallen PDMS-Stempel in Ethanol für 15 Minuten. </li><li> Bereiten Sie eine neue 3% v / v Lösung von 3-Aminopropyltrimethoxysilan (APTMS) in 95% v / v Ethanol in einem 15 mL Zentrifugenröhrchen. Lassen Sie für 5 Minuten bei RT reagieren, um eine Silanol bilden. </li><li> Legen Sie PDMS-Stempel Muster-up auf Spin Coater Futter und Deckung strukturierte Oberfläche mit APTMS Lösung. Spincoat bei 3500rpm für 30 Sekunden. Berühren Sie nicht den Stempel Oberfläche, nur die Seiten des PDMS-Stempel. </li><li> Transfer APTMS Lösung, um das Plasma zu reinigen Glasflächen: set Stempel nach unten auf ein Ende des strukturierten Seite und sanft gleiten Drücken PDMS so dass es vollständig in Kontakt mit Glasoberfläche für 1 Minute. </li><li> Entfernen Sie vorsichtig PDMS-Stempel und damit Ornamentglas Folie in RT für 30 Minuten inkubieren. Spülen gemusterten Oberflächen mit Ethanol und an der Luft trocknen. Ultraschallbad PDMS für 15 Minuten, um überschüssige APTMS entfernen Stempel. </li><li> Heizflächen im Ofen oder auf einer Heizplatte für 1 Stunde bei 115 ° C. Spülen mit Ethanol und trocken mit Luft. </li><li> Bereiten Sie frische Polyethylenglykol (PEG)-Silan-Lösung von 20% v / v 2 - [Methoxy (Polyethylenoxy) propyl] trimethoxysilan in Toluol. Dies wird als nicht klebend Region um Muster dienen. </li><li> Transfer-gemusterten Objektträger aus Glas, ein Glas-Petrischale und inkubieren in PEG-Silan-Lösung für 45 Minuten bei 75 ° C auf einer Heizplatte. Spülen mit Toluol, Wasser und trocken mit Luft. </li><li> Sterilisieren für 1 Stunde mit UV-Bestrahlung keimtötende in einem biologischen Sicherheitsschrank. Gehen Sie in biologischen Sicherheitswerkbank von diesem Punkt an, um die Sterilität zu erhalten. </li><li> Bereiten wässrige sterile HA-Lösung. 10mm 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimid (EDC) 5mm von N-Hydroxy- 50 Mikrogramm / ml Fluorescein-markierten HA </li><li> Bewerben HA Lösung für die strukturierte Glassubstrate unter sterilen Bedingungen und vor Licht geschützt. Gemusterte Substrate sollten ungestört 16-24 Stunden. </li><li> Absaugen HA-Lösung, mit Wasch-Phosphat-gepufferter Salzlösung (PBS) und Deckfläche mit 1% Rinderserumalbumin (BSA) in PBS für 1 Stunde. Die Oberfläche ist nun bereit für die Zellkultur. </li></ol> 3. Cell Culture auf HA mikrostrukturierten Oberflächen <ol><li> Expand MDA-MB-231 Brustkrebszellen und LS174T kolorektalen Karzinom-Zellen, nach den Anweisungen des Herstellers. </li><li> Seed einzelnen Zellsuspensionen von Krebszellen mit Dichten zwischen 0,4 und 1x10 <sup&gt; 6 Krebszellen pro 18,75 cm 2 Glasfläche und auf HA immobilisierten Oberflächen. Inkubieren in befeuchteten Inkubator (37 ° C) in Atmosphären mit 5% CO 2 gehalten. Zelladhäsion sollte innerhalb von 24 Stunden auftreten und beobachtet mit invertierten Lichtmikroskop werden </li><li> Zellkultur Oberflächen können bis zu 5 Tage in Standard-Inkubator Bedingungen eingehalten werden. Ändern Medien jeden zweiten Tag. Zellmorphologie und Wachstum zu beobachten mit invertierten Lichtmikroskop werden. Zusätzliche zellulären Assays angewendet, um Antworten auf die HA-Oberfläche analysieren. </li></ol> 4. Repräsentative Ergebnisse: Die Carbodiimid-Chemie verwendet werden, um kovalent zu immobilisieren HA APTMS Ornamentglas Dias ist in Abbildung 1A dargestellt. EDC ist ein Null-Länge Vernetzer, die mit HA Carboxylgruppen reagiert auf Amin-reaktiven Zwischenstufen zu bilden. Da dieser Zwischenstufe ist hydrolyseempfindlich ist NHS hinzugefügt Carbodiimidreaktion Effizienz zu steigern. Als HA-Lösung interagiert mit APTMS Mikrostrukturen, eine stabile Amidbindung bildet sich zwischen HA Zwischen-und primären Amin ATPMS. Ein Beispiel für HA gemusterte Oberfläche visualisiert mit Hilfe eines Fluoreszenz-Mikroskops und einer ImageJ Fluoreszenzintensität Analyse zeigen die strukturierte Immobilisierung von HA dargestellt (Abbildung 1B-C). Die HA mikrostrukturierten Oberflächen ermöglichen die Untersuchung von Krebszellen Interaktionen mit expgenous HA. Beide MDA-MB-231 menschlichen Brust und LS174T menschlichen Dickdarmkrebs-Zelllinien bevorzugt an HA mikrostrukturierten Regionen (Abb. 2) zu halten. Hochauflösende Bildgebung mit Hilfe der Rasterelektronenmikroskopie können verwendet werden, um Wechselwirkungen von kultivierten Zellen mit HA immobilisierten Oberflächen (Abbildung 3) zu analysieren. <img src="/files/ftp_upload/2413/2413fig1.jpg" alt="Abbildung 1" /> Abbildung 1 HA mikrostrukturierten Oberflächen (A) Schematische Beschreibung der Entwicklung der HA funktionale Oberflächen, (B) Fluoreszenzbild von Fluorescein (grün)-markiertem HA mikrostrukturierten Oberfläche. (C) 3D Pixelverteilung Karte mit ImageJ Software zeigt die diskrete HA-Muster. Maßstab = 100 um. Geändert von 9 mit Genehmigung von Elsevier. <img src="/files/ftp_upload/2413/2413fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Cancer Zelladhäsion auf mikrostrukturierten Oberflächen HA. Beide Doppelpunkt (A) und Brust (B) Karzinom bevorzugt auf HA-Patches mit 24 Stunden Kultur eingehalten werden. Geändert von 9 mit Genehmigung von Elsevier. <img src="/files/ftp_upload/2413/2413fig3.jpg" alt="Abbildung 3" /> Abbildung 3. Cancer Zell-Interaktion mit HA. (A) Rasterelektronenmikroskopie Bilder von Darmkrebs-Zellen (A) kultiviert auf HA Oberflächen bei geringer Vergrößerung (links) und hoher Vergrößerung (rechts; der Quadrate auf der linken Seite) gezeigt und von Brustkrebszellen (B) auf HA Oberflächen gezeigt, bei kultivierten geringer Vergrößerung (links) und hoher Vergrößerung (rechts; der Quadrate auf der linken Seite).

<ol>
	<li>Toole, B. P., Wight, T. N., Tammi, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyaluronan-cell+interactions+in+cancer+and+vascular+disease.">Hyaluronan-cell interactions in cancer and vascular disease.</a> Journal of Biological Chemistry. 277, 4593-4596 (2002).</li><li>Hamilton, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Hyaluronan+Receptors+CD44+and+Rhamm+(CD168)+Form+Complexes+with+ERK1,2+That+Sustain+High+Basal+Motility+in+Breast+Cancer+Cells.">The Hyaluronan Receptors CD44 and Rhamm (CD168) Form Complexes with ERK1,2 That Sustain High Basal Motility in Breast Cancer Cells.</a> Journal of Biological Chemistry. 282, 16667-16680 (2007).</li><li>G&#246;tte, M., Yip, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heparanase,+Hyaluronan,+and+CD44+in+Cancers:+A+Breast+Carcinoma+Perspective.">Heparanase, Hyaluronan, and CD44 in Cancers: A Breast Carcinoma Perspective.</a> Cancer Research. 66, 10233-10237 (2006).</li><li>Lord, M. S., Pasqui, D., Barbucci, R., Milthorpe, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+adsorption+on+derivatives+of+hyaluronic+acid+and+subsequent+cellular+response.">Protein adsorption on derivatives of hyaluronic acid and subsequent cellular response.</a> Journal of Biomedical Materials Research. Part A 91, 635-646 (2009).</li><li>Barbucci, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modification+of+hyaluronic+acid+by+sulphate+groups+insertion+to+obtain+a+heparin-like+molecule.">Modification of hyaluronic acid by sulphate groups insertion to obtain a heparin-like molecule.</a> Gazz. Chim. Ital. 125, 169-180 (1995).</li><li>Morra, M., Cassinelli, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-fouling+properties+of+polysaccharide-coated+surfaces.">Non-fouling properties of polysaccharide-coated surfaces.</a> Journal of Biomaterials Science, Polymer Edition. 10, 1107-1124 (1999).</li><li>Khademhosseini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Layer-by-layer+deposition+of+hyaluronic+acid+and+poly-L-lysine+for+patterned+cell+co-cultures.">Layer-by-layer deposition of hyaluronic acid and poly-L-lysine for patterned cell co-cultures.</a> Biomaterials. 25, 3583-3592 (2004).</li><li>Ibrahim, S., Joddar, B., Craps, M., Ramamurthi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+surface-tethered+model+to+assess+size-specific+effects+of+hyaluronan+(HA)+on+endothelial+cells.">A surface-tethered model to assess size-specific effects of hyaluronan (HA) on endothelial cells.</a> Biomaterials. 28, 825-835 (2007).</li><li>Dickinson, L. E., Ho, C. C., Wang, G. M., Stebe, K. J., Gerecht, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+surfaces+for+high-resolution+analysis+of+cancer+cell+interactions+on+exogenous+hyaluronic+acid.">Functional surfaces for high-resolution analysis of cancer cell interactions on exogenous hyaluronic acid.</a> Biomaterials. 31, 5472-5478 (2010).</li><li>Gao, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyaluronan+oligosaccharides+promote+excisional+wound+healing+through+enhanced+angiogenesis.">Hyaluronan oligosaccharides promote excisional wound healing through enhanced angiogenesis.</a> Matrix Biology. 29, 107-116 (2010).</li><li>Slevin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyaluronan-mediated+angiogenesis+in+vascular+disease:+Uncovering+RHAMM+and+CD44+receptor+signaling+pathways.">Hyaluronan-mediated angiogenesis in vascular disease: Uncovering RHAMM and CD44 receptor signaling pathways.</a> Matrix Biology. 26, 58-68 (2007).</li></ol>

Video schematische Abbildung mit Genehmigung vervielfältigt, Copyright Wiley-VCH Verlag GmbH &amp; Co. KGaA. Dickinson LE, Kusuma S, Gerecht S. Rekonstruktion der Differenzierung Nische von embryonalen Stammzellen mit Biomaterialien. Macromol. Biosci. 2010; 21 Oktober. [Epub ahead of print].

Die HA Mikrostrukturierung vorgestellte Methode ermöglicht die Untersuchung von Zell-Interaktionen mit exogenen HA. HA ist bekannt, eine wichtige Rolle in der Tumorprogression 1 spielen jedoch gab es nur Studien zur Untersuchung der Wechselwirkung von Krebszellen auf zweidimensionalen HA gemusterten Oberflächen worden. Ein steuerbarer Studie über exogenen HA Mikrostrukturen ermöglicht hochauflösende Visualisierung von Krebs Zelladhäsion, das Wachstum und die Migration und kann weitere Grundlagen von Kre...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
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<tr>
<td>SU-2025 photoresist</td>
<td>&nbsp;</td>
<td>MicroChem Corp.</td>
<td>Y111069</td>
<td>&nbsp;</td>
<tr>
<td>SU-8 developer</td>
<td>&nbsp;</td>
<td>MicroChem Corp.</td>
<td>Y020100</td>
<td>&nbsp;</td>
<tr>
<td>Sylgard 184</td>
<td>&nbsp;</td>
<td>Dow Corning</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>3-aminopropyltrimethoxysilane (APTMS)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>281778</td>
<td>&nbsp;</td>
<tr>
<td>2- [methoxy(polyethyleneoxy) propyl] trimethoxysilane (Peg-silane)</td>
<td>&nbsp;</td>
<td>Gelest Inc</td>
<td>SIM6492.7</td>
<td>&nbsp;</td>
<tr>
<td>1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>22980</td>
<td>&nbsp;</td>
<tr>
<td>N-hydroxysuccinimide (NHS)</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>24500</td>
<td>&nbsp;</td>
<tr>
<td>Fluorescein labeled hyaluronic acid (FL-HA)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrick</td>
<td>F1177</td>
<td>Reconstitute with 10ml of DI water </td>
</tr>
<tr>
<td>MDA-MB-231 breast carcinoma cells</td>
<td>&nbsp;</td>
<td>ATCC</td>
<td>HTB-26</td>
<td>&nbsp;</td>
<tr>
<td>LS174t colon carcinoma cells</td>
<td>&nbsp;</td>
<td>ATCC</td>
<td>Cl-188</td>
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micropatterned surfaces to study hyaluronic acid interactions with cancer cells

Cancer Invasion und Progression beinhaltet eine bewegliche Zelle Phänotyp, die unter komplexen Regulation durch Wachstumsfaktoren / Zytokine und extrazelluläre Matrix (ECM)-Komponenten in der Tumor-Mikroumgebung ist. Hyaluronsäure (HA) ist einer stromalen ECM-Komponente, die bekanntermaßen Tumorprogression durch Verbesserung der Invasion, Wachstum und Angiogenese 1 zu erleichtern wird. Interaktion von HA mit ihren Zelloberflächen-Rezeptor CD44 induziert Signal-Ereignisse, das Wachstum von Tumorzellen, das Überleben und die Migration zu fördern, wodurch Metastasierung 2-3. HA ist ein anionisches, nicht sulfatierten Glycosaminoglycan von sich wiederholenden Einheiten von D-Glucuronsäure und DN-Acetylglucosamin zusammen. Durch die Anwesenheit von Carboxyl-und Hydroxyl-Gruppen auf die Wiederholung Disaccharideinheiten, native HA weitgehend hydrophilen und zugänglich chemische Modifikationen, die Sulfatgruppen für photoreative Immobilisierung 4-5 einzuführen. Frühere Studien, die die Immobilisierung von HA auf Oberflächen nutzen die bioresistenten Verhalten der HA und seine sulfatierte Derivat Zelladhäsion auf Oberflächen 6-7 steuern. In diesen Studien Zelladhäsion bevorzugt auf Nichtspieler-HA strukturierten Gebieten. Zur Analyse zellulärer Interaktionen mit exogenen HA, haben wir gemusterten funktionalisierten Oberflächen, die ein steuerbares Studie und hochauflösende Visualisierung der Krebszelle Interaktionen mit HA ermöglichen entwickelt. Wir verwendeten Mikrokontaktdrucken (UCP) zu diskreten strukturierten Gebieten HA auf Glasflächen zu definieren. A &quot;Tethering&quot;-Ansatz, dass Carbodiimid Verknüpfung Chemie gilt für HA immobilisieren wurde 8 verwendet. Glasflächen wurden Mikrokontakt mit einem Aminosilan gedruckt und reagierte mit einer HA-Lösung optimiert Verhältnis von EDC und NHS zum HA Immobilisierung in gemusterten Arrays ermöglichen. Die Einbeziehung Carbodiimid-Chemie mit MCP konnte die Immobilisierung von HA auf definierte Bereiche, die Schaffung Oberflächen geeignet für in-vitro-Anwendungen. Beide Dickdarmkrebszellen und Brustkrebszellen implizit mit der HA mikrostrukturierten Oberflächen interagiert. Krebs Zelladhäsion innerhalb von 24 Stunden mit der Proliferation von 48 Stunden eingetreten ist. Mit HA mikrostrukturierten Oberflächen haben wir gezeigt, dass Krebs Zelladhäsion erfolgt durch die HA-Rezeptor CD44. Darüber hinaus wurden HA gemusterten Oberflächen kompatibel mit Rasterelektronenmikroskopie (SEM) und erlaubt hochauflösende Bildgebung der Krebszelle Klebstoff Vorsprünge und Verteilen auf HA-Muster zu Krebs Zellbewegung auf exogene HA analysieren.

Watch this Scientific Journal Video about Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells at JoVE.com

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Ein neuartiger Ansatz, dass die hochauflösende Analyse von Krebs Zell-Interaktionen ermöglicht mit exogenen Hyaluronsäure (HA) beschrieben wird. Gemusterten Oberflächen werden durch die Kombination Carbodiimid Chemie und Mikrokontaktdrucken hergestellt.

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 ...

micropatterned-surfaces-to-study-hyaluronic-acid-interactions-with

Research

JoVE Journal

Bioengineering

15.0K Aufrufe.  Johns Hopkins University. Ein neuartiger Ansatz, dass die hochauflösende Analyse von Krebs Zell-Interaktionen ermöglicht mit exogenen Hyaluronsäure (HA) beschrieben wird. Gemusterten Oberflächen werden durch die Kombination Carbodiimid Chemie und Mikrokontaktdrucken hergestellt.

Serie: Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

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 ...

Forschung

Biotechnik

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

Entwurf eines zyklischen Druck Bioreaktor für die Ex Vivo Studium der Aorten-Herzklappen

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

Advances in microfabrication techniques have enabled the production of inexpensive and reproducible microfluidic systems for conducting biological and biochemical experiments at the micro- and nanoscales 1,2. In addition, microfluidics have also been specifically used to quantitatively analyze hematologic and microvascular processes, because of their ability to easily control the dynamic fluidic environment and biological conditions3-6. As such, researchers have more recently used microfluidic systems to study blood cell deformability, blood cell aggregation, microvascular blood flow, and blood cell-endothelial cell interactions6-13.However, these microfluidic systems either did not include cultured endothelial cells or were larger than the sizescale relevant to microvascular pathologic processes. A microfluidic platform with cultured endothelial cells that accurately recapitulates the cellular, physical, and hemodynamic environment of the microcirculation is needed to further our understanding of the underlying biophysical pathophysiology of hematologic diseases that involve the microvasculature.
Here, we report a method to create an "endothelialized" in vitro model of the microvasculature, using a simple, single mask microfabrication process in conjunction with standard endothelial cell culture techniques, to study pathologic biophysical microvascular interactions that occur in hematologic disease. This "microvasculature-on-a-chip" provides the researcher with a robust assay that tightly controls biological as well as biophysical conditions and is operated using a standard syringe pump and brightfield/fluorescence microscopy. Parameters such as microcirculatory hemodynamic conditions, endothelial cell type, blood cell type(s) and concentration(s), drug/inhibitory concentration etc., can all be easily controlled. As such, our microsystem provides a method to quantitatively investigate disease processes in which microvascular flow is impaired due to alterations in cell adhesion, aggregation, and deformability, a capability unavailable with existing assays.

A method to culture an endothelial cell monolayer throughout the entire inner 3D surface of a microfluidic device with microvascular-sized channels (&lt;30 &mu;m) is described. This in vitro microvasculature model enables the study of biophysical interactions between blood cells, endothelial cells, and soluble factors in hematologic diseases.

Endothelialisierten Mikrofluidik für ein Studium Mikrovaskuläre Wechselwirkungen in Hämatologische Krankheiten

Advances in  microfabrication techniques have enabled the production of inexpensive and  reproducible microfluidic systems for conducting biological and ...

Stretching Micropatterned Cells on a PDMS Membrane

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 PolyDiMethylSiloxane (PDMS) membrane, compatible with imaging. This technique is reproducible and versatile. The PDMS membrane can be micropatterned in order to confine cells or tissues to a specific geometry. The first step is to print micropatterns onto the PDMS membrane with a deep UV technique. The PDMS membrane is then mounted on a mechanical stretcher. A chamber is bound on top of the membrane with biocompatible grease to allow gliding during the stretch. The cells are seeded and allowed to spread for several hours on the micropatterns. The sample can be stretched and unstretched multiple times with the use of a micrometric screw. It takes less than a minute to apply the stretch to its full extent (around 30%). The technique presented here does not include a motorized device, which is necessary for applying repeated stretch cycles quickly and/or computer controlled stretching, but this can be implemented. Stretching of cells or tissue can be of interest for questions related to cell forces, cell response to mechanical stress or tissue morphogenesis. This video presentation will show how to avoid typical problems that might arise when doing this type of seemingly simple experiment.

This manuscript presents a technique to apply or release forces on adherent cells or tissues using unidirectional 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 ...

Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and invasion. Rat tail type I collagen and/or matrix derived from Engelbreth-Holm-Swarm mouse sarcoma cells have been traditionally employed as the substrates to model the matrix or stromal microenvironment into which mesenchymal cells (usually fibroblasts) are populated. Although experiments using such matrices are very informative, it can be argued that due to an overriding presence of a single protein (such as in type I Collagen) or a high content of basement membrane components and growth factors (such as in matrix derived from mouse sarcoma cells), these substrates do not best reflect the contribution to matrix composition made by the stromal cells themselves. To study native matrices produced by primary dermal fibroblasts isolated from patients with a tumor prone, genetic blistering disorder (recessive dystrophic epidermolysis bullosa), we have adapted an existing native matrix protocol to study tumor cell invasion. Fibroblasts are induced to produce their own matrix over a prolonged period in culture. This native matrix is then detached from the culture dish and epithelial cells are seeded onto it before the entire coculture is raised to the air-liquid interface. Cellular differentiation and/or invasion can then be assessed over time. This technique provides the ability to assess epithelial-mesenchymal cell interactions in a 3D setting without the need for a synthetic or foreign matrix with the only disadvantage being the prolonged period of time required to produce the native matrix. Here we describe the application of this technique to assess the ability of a single molecule expressed by fibroblasts, type VII collagen, to inhibit tumor cell invasion.

Tissue engineered fibroblast-derived native matrix is an emerging tool to generate a stromal substrate which supports epithelial cell proliferation and differentiation. Here a protocol applying this methodology to assess the impact of different stromal cell types on tumor cell biology is presented.

Tissue Engineering von Tumor-Stroma-Mikromilieu mit Anwendung auf die Invasion von Krebszellen

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and ...

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. ...

Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the Langmuir-Schaefer method. Amphiphilic block copolymers composed of polystyrene and PIPAAm (St-IPAAms) were synthesized by reversible addition-fragmentation chain transfer (RAFT) radical polymerization. A chloroform solution of St-IPAAm molecules was gently dropped into a Langmuir-trough apparatus, and both barriers of the apparatus were moved horizontally to compress the film to regulate its density. Then, the St-IPAAm Langmuir film was horizontally transferred onto a hydrophobically modified glass substrate by a surface-fixed device. Atomic force microscopy images clearly revealed nanoscale sea-island structures on the surface. The strength, rate, and quality of cell adhesion and detachment on the prepared surface were modulated by changes in temperature across the lower critical solution temperature range of PIPAAm molecules. In addition, a two-dimensional cell structure (cell sheet) was successfully recovered on the optimized surfaces. These unique PIPAAm surfaces may be useful for controlling the strength of cell adhesion and detachment.

Nanoscaled sea-island surfaces composed of thermoresponsive block copolymers were fabricated by the Langmuir-Schaefer method for controlling spontaneous cell adhesion and detachment. Both the preparation of the surface and the adhesion and detachment of cells on the surface were visualized.

Herstellung von Thermoresponsive nanostrukturierte Oberflächen für Tissue Engineering

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

Herstellung von 3D-Kollagen-Gel und Mikrokanäle für das Studium der 3D-Interaktionen<em&gt; In Vivo</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

A Facile and Eco-friendly Route to Fabricate Poly(Lactic Acid) Scaffolds with Graded Pore Size

Over the recent years, functionally graded scaffolds (FGS) gaineda crucial role for manufacturing of devices for tissue engineering. The importance of this new field of biomaterials research is due to the necessity to develop implants capable of mimicking the complex functionality of the various tissues, including a continuous change from one structure or composition to another. In this latter context, one topic of main interest concerns the design of appropriate scaffolds for bone-cartilage interface tissue. In this study, three-layered scaffolds with graded pore size were achieved by melt mixing poly(lactic acid) (PLA), sodium chloride (NaCl) and polyethylene glycol (PEG). Pore size distributions were controlled by NaCl granulometry and PEG solvation. Scaffolds were characterized from a morphological and mechanical point of view. A correlation between the preparation method, the pore architecture and compressive mechanical behavior was found. The interface adhesion strength was quantitatively evaluated by using a custom-designed interfacial strength test. Furthermore, in order to imitate the human physiology, mechanical tests were also performed in phosphate buffered saline (PBS) solution at 37 &#176;C. The method herein presented provides a high control of porosity, pore size distribution and mechanical performance, thus offering the possibility to fabricate three-layered scaffolds with tailored properties by following a simple and eco-friendly route.

A Facile and Eco-friendly Route to Fabricate PolyLactic Acid Scaffolds with Graded Pore Size

In this work, poly(lactic acid)/polyethylene glycol (PLA/PEG) scaffolds were prepared by using a combination of melt mixing and selective leaching. The method herein discussed permitted to develop three-layer scaffolds by highly controlling both porosity and pore size. The mechanical properties were also evaluated in a physiological environment.

Ein Facile und umweltfreundliche Weg zu fabrizieren Poly (Milchsäure) Scaffolds mit Graded Porengröße

Over the recent years, functionally graded scaffolds (FGS) gaineda crucial role for manufacturing of devices for tissue engineering. The importance of ...

Site-Directed Immobilization of Bone Morphogenetic Protein 2 to Solid Surfaces by Click Chemistry

Different therapeutic strategies for the treatment of non-healing long bone defects have been intensively investigated. Currently used treatments present several limitations that have led to the use of biomaterials in combination with osteogenic growth factors, such as bone morphogenetic proteins (BMPs). Commonly used absorption or encapsulation methods require supra-physiological amounts of BMP2, typically resulting in a so-called initial burst release effect that provokes several severe adverse side effects. A possible strategy to overcome these problems would be to covalently couple the protein to the scaffold. Moreover, coupling should be performed in a site-specific manner in order to guarantee a reproducible product outcome. Therefore, we created a BMP2 variant, in which an artificial amino acid (propargyl-L-lysine) was introduced into the mature part of the BMP2 protein by codon usage expansion (BMP2-K3Plk). BMP2-K3Plk was coupled to functionalized beads through copper catalyzed azide-alkyne cycloaddition (CuAAC). The biological activity of the coupled BMP2-K3Plk was proven in vitro and the osteogenic activity of the BMP2-K3Plk-functionalized beads was proven in cell based assays. The functionalized beads in contact with C2C12 cells were able to induce alkaline phosphatase (ALP) expression in locally restricted proximity of the bead. Thus, by this technique, functionalized scaffolds can be produced that can trigger cell differentiation towards an osteogenic lineage. Additionally, lower BMP2 doses are sufficient due to the controlled orientation of site-directed coupled BMP2. With this method, BMPs are always exposed to their receptors on the cell surface in the appropriate orientation, which is not the case if the factors are coupled via non-site-directed coupling techniques. The product outcome is highly controllable and, thus, results in materials with homogeneous properties, improving their applicability for the repair of critical size bone defects.

Biomaterials doped with Bone Morphogenetic Protein 2 (BMP2) have been used as a new therapeutic strategy to heal non-union bone fractures. To overcome side effects resulting from an uncontrollable release of the factor, we propose a new strategy to site-directly immobilize the factor, thus creating materials with improved osteogenic capabilities.

Site-verwiesene Immobilisierung von Knochen morphogenetische Protein 2 an feste Oberflächen durch Click-Chemie

Different therapeutic strategies for the treatment of non-healing long bone defects have been intensively investigated. Currently used treatments present ...