Funding for this work, GTK, TK, and JDM were provided by the Wisconsin Institute for Discovery and the Wisconsin Alumni Research Foundation.

1. Gene Elastomere Briefmarken <ol style="margin-left: 40px;"><li> Um Silizium Meister der PDMS-Stempel zu erzeugen, Design-Merkmal-Muster der Photomaske ist mit Computer-Aided-Design-Software. <ol><li> Gestalten Sie das erste Muster als 20 x 20 Anordnung von Kreisringen mit 300 &amp; mgr; m Innendurchmesser (ID) und 600 &amp; mgr; m OD mit 1200 &amp; mgr; m von Mitte zu Mitte-Abstand. </li><li> Gestalten Sie das zweite Muster als 20 x 20 Anordnung von Kreisringen mit 600 um ID und 900 &amp; mgr; m OD mit 1200 &amp; mgr; m von Mitte zu Mitte-Abstand. </li><li> Zusätzlich legen 1 x 1 mm 2 Quadratreferenzmarken an allen vier Ecken jedes Array-Design Abstand 1.200 &amp; mgr; m von Mitte zu Mitte von der Ecke Muster in einem 45 ° -Winkel. </li><li> Fabrizieren die Silizium-Master für die Verwendung in diesem Experiment mit 1: 1-Seitenverhältnisse, in Korrelation zu einem 300 um Feature Tiefe mit Standardlithographietechniken anderswo 15 oder in Zusammenarbeit mit einem mikrofluidischen Gießerei detailliert. HINWEIS: Feature Tiefen von weniger als 100 &amp; mgr; m können zu abnorm Verformung Briefmarken vor mit Substratoberflächen kontaktieren führen. HINWEIS: Dieses Protokoll beginnt mit mit bereits erhaltenen Silizium-Master mit den beschriebenen Muster aus Fotolack, die Spezialausrüstung und saubere Zimmer zu schaffen erfordert. Am besten ist es mit einem Verarbeiter oder Core Facility, diese strukturiert Master-Dokumente erstellen zu konsultieren. </li></ol></li><li> Silanisieren Silizium Meister O / N durch Inkubation mit (Tridecafluor-1,1,2,2-tetrahydroocty) Trichlorsilan Dampf. HINWEIS: Silandampf ist sehr giftig und sollte nur in einer chemischen Abzugshaube gehandhabt werden. </li><li> Erstellen inverse Repliken der Silizium Meister durch Härten einer 10: 1-Verhältnis von PDMS-Prepolymer und PDMS Härter auf der Oberseite der silanisierten Silizium Meister in einer Petrischale O / N bei 60 ° C. </li><li> Entfernen Sie die PDMS-Stempel aus den Silizium Meister und binden die Briefmarken zu Acrylnitril-Butadien-Styrol (ABS) Träger oder einer anderen starren Materials unter Verwendung von Klebstoff. HINWEIS: Das Material der Wahl nicht transparent oder biokompatibel sein. Es muss jedoch eine flache Oberfläche für die Schnittstelle mit der Roboterwerkzeuge und höhere Steifigkeit als PDMS bereitzustellen. </li></ol> 2. Vorbereiten Coverslides <ol style="margin-left: 40px;"><li> Spülen Mikroskop coverslides (24 mm x 50 mm # 1) in Toluol, Methanol und beschallen in Aceton für 1 Minute vor dem Spülen mit Ethanol und Trocknen unter einem leichten Stickstoffstrom. Hinweis: Behandeln Toluol und Aceton nur in einer chemischen Abzugshaube. </li><li> Mantel coverslides mit ~ 3,5 nm Titan (Ti), gefolgt von 18,0 nm Gold (Au) mit einem fokussierten Elektronenstrahl-Verdampfers. Hinweis: Dieses Verfahren ist mit einer Vielzahl von mit Gold beschichteten Substraten, einschließlich Silicium und Polystyrol kompatibel. Während es möglich ist, diese vor Ort zu erzeugen, sind ebenfalls mit Gold beschichteten Substrate über kommerzielle Lieferanten erhältlich. HINWEIS: Ti-Schichten sollte mindestens 3 nm dick sein, während Au-Schichten auf Substraten sollte eint mindestens 5 nm dick und kann den ganzen Weg in der Dicke in Abhängigkeit von den idealen optischen Eigenschaften des fertigen Substrats 16,17 1mm liegen. Substrate sind nicht erforderlich für die Herstellung optisch klar zu sein; jedoch ermöglicht sie mikroskopische Analyse hergestellt Substraten. </li><li> Spülen Sie goldbeschichteten coverslides mit Ethanol und trocken unter leichtem Stickstoff unmittelbar vor der Verwendung. </li></ol> 3. R-mCP der Außenring <ol style="margin-left: 40px;"><li> Bevor R-mCP mit der selektiv kompatible Knickarmroboter (SCARA) System, kalibrieren Sie die Roboterwerkzeug Effektoren und begleitenden Dual-Kamera-Systeme mit der Software des Systems. ANMERKUNG: Diese Werkzeuge sind in Figur 1 dargestellt. </li></ol><img alt="Figur 1" src="/files/ftp_upload/52186/52186fig1highres.jpg" /> Abbildung 1. R- mCP System und Robotic Arm Tooling. </sTrong&gt; (A) Großansicht des R-mCP System mit allen Werkzeuge und Vorrichtungen (a) Vakuumspann, (b) Reagenz Bad, (c) nach unten gerichtete Kamera, (d) Stempel Verschachtelung Halterung, (e) Roboter Werkzeug. (B) Robotic arm tooling Darstellung (f) Diamant Ätzanlage und (g) pneumatische Saugwerkzeug hält eine (h) ABS-Backed PDMS-Stempel. <ol start="2" style="margin-left: 40px;"><li> Manuell legen Sie eine PDMS-Stempel mit 600 um ID und 900 &amp; mgr; m OD vorstehende Ringe nach unten im Stempel Verschachtelung Befestigung. Hand legen eine frisch gereinigte Gold beschichteten Deckglas auf der Oberseite der Vakuumspannvorrichtung, in Figur 1A dargestellt, und zu immobilisieren sie mit dem verbundenen Laborvakuum. </li><li> Mit den Robotersteuerungen, richten Sie den nach unten weisenden Kamera über dem Mittelpunkt des goldbeschichteten Deckglas, wie in 2A. HINWEIS: Dieser Punkt kann anfänglich visuell definiert werden und erfordert keine große Genauigkeit, wie der PDMS-Stempel ist viel largER als die goldbeschichteten Folie. </li><li> Weisen den Roboterarm zu vier Referenz ätzen &quot;X&quot; Markierungen an den Ecken eines Quadrats mit den Maßen 3,8 mm · 3,8 mm auf der Gold beschichteten Deckglas mit der Diamant Ätzanlage zum Roboterarm befestigt zentriert. (In 2B Dargestellt; automatisierten Prozess) HINWEIS: Dadurch wird sichergestellt, alle vier Referenzmarken sind innerhalb einer visuellen Rahmen der nach unten weisenden Kamera. </li><li> Mit den Robotern pneumatische Absaugung Tooling, Abholung und halten die PDMS-Stempel 1 mm über der Stempel Verschachtelung Halterung wie in 2C. (Automatisierten Prozess) </li><li> Mit dem nach oben zeigenden Kamera und LED-Beleuchtungsring in 2D und E abgebildet, visualisieren quadratischen Referenzmarken des Stempels und ihnen mit der Kamera-Software zu identifizieren 10 mal um durchschnittlich X des Stempels, Y zu bestimmen und von der Mitte der Winkelversatz Roboter Werkzeugachse. (Automatisierten Prozess) </li><li> Wie in <strong&gt; 2A, verwenden Sie den unten gerichtete Kamera zu visualisieren und zu erkennen geätzten Referenzmarken der Deckglas des 10-fachen der durchschnittlichen X Das Deckglas ist, Y-Center-Standorte und Winkelversatz von der Mitte des Roboters Werkzeugachse zu bestimmen. (Automatisierten Prozess). HINWEIS: Eine interne Berechnung wird von der Software des Robotersystems durchgeführt, um genau auszurichten der Stempel und Deckglas der X, Y-Center-Standorte und Winkelverschiebungen in künftige R-mCP Schritte. </li><li> 3.8) Den Stempel in ein Bad aus Alkanthiol ATRP Initiators, ω-mercaptoundecyl bromisobutyrat (2 mM in Ethanol), wie in Abbildung 2F dargestellt. (Automatisierten Prozess) </li><li> Entfernen Sie den Stempel aus der Alkanthiol-Lösung und legen Sie es über Stickstoffstrom auf 0,48 bar (5 psi) unter Druck gesetzt, um das Ethanol wie in 2G verdampfen. Nach 1 min erhöht der Stickstoffstrom Druck auf 1,03 bar (15 psi) zu einheitlichen Trockenheit zu gewährleisten. (Automatisierten Prozess) HINWEIS: Unvollständige Trocknung wird in vollständigen oder teilweisen Verlust der Mustertreue führen. </li><li> Nach dem Trocknen bewegen der Stempel über die berechnete Mittelstellung des Gold-beschichteten Objektträger und senken es bei 100 &amp; mgr; m-Schritten während der Überwachung Z-Achsen-Motorkraft, wie in Figur 2H dargestellt. ANMERKUNG: Diese wird als Drehmoment von der Z-Achsen-Motor erfahren und in die Roboterführung Software angezeigt kommuniziert. (Automatisierten Prozess) </li><li> Stop Absenken der Stempel einmal der vorbestimmte Druck von 79,2 kPa erreicht worden ist, und der Kontakt mit dem Gold-beschichteten Objektträger für 15 Sekunden zu halten. (Automatisierten Prozess) HINWEIS: Die Druckwerte werden hier für den aktuellen Stempel Design und Funktion Höhe optimiert. Wenn der Briefmarkenentwurf ist von einem Versuch und Irrtum-Prozess verändert, speziell für hohe Seitenverhältnis Briefmarken, tune diese entsprechend. </li><li> Entfernen Sie langsam den Stempel aus dem goldbeschichteten Objektträger und setzen Sie den Stempel wieder in die Stempel Verschachtelung Befestigung. (Automatisierten Prozess) </li></ol&gt; 4. SI-ATRP von PEGMEMA auf mikrostrukturierten Coverslides <ol style="margin-left: 40px;"><li> Lassen Unterdruck hält die mikroDeckGlas, und überträgt es auf einen 50-ml-Schlenk-Kolben. Seal und entgast Schlenkkolben mit einer Vakuumpumpe. </li><li> Hinzufügen von 5,5 ml ATRP Reaktionsmischung, die das Makromonomer PEGMEMA (208.75 mmol), Wasser (34,4 ml), Methanol (43,8 ml), Kupfer (II) -bromid (1 mmol) und 2 &#39;, 2-Bipyridin (3 mmol) an die Schlenk-Kolben. </li><li> 0,5 ml der L-Natriumascorbat (454,3 mM) in Wasser, um die Reaktion zu starten, und lassen Sie sie 16 Stunden lang bei RT unter Schutzgas fortzusetzen. HINWEIS: Nach der Zugabe von L-Natriumascorbat, wird Reaktionsfarbe von hellgrün bis dunkelbraun zu verschieben, wie in 3A-B gezeigt. HINWEIS: Die Reaktion kann über 16 Stunden weiter, aber es wird die Länge der PEG Bürsten nicht wesentlich erhöhen. </li></ol><imgalt = &quot;3&quot; src = &quot;/ files / ftp_upload / 52186 / 52186fig3highres.jpg&quot; /&gt; Abbildung 3. Einleitung SI-ATRP. (A) Einleitung der Reaktion und (B) anschließende Farbänderung nach Zugabe von L-Natriumascorbat. (C) Mikroskopische Aufnahme von mikroDeckGlas Oberfläche folgenden SI-ATRP Verfahren. Maßstabsleiste 1 mm. <ol start="4" style="margin-left: 40px;"><li> Entfernen Sie die mikroDeckGlas aus dem Schenk-Kolben und spülen mit Ethanol, Wasser und Ethanol und trocken unter einem leichten Stickstoffstrom. HINWEIS: Nach SI-ATRP, sollten Oberflächenmodifikationen für das Auge sichtbar sein und kann, wie in 3C abgebildet und unter einem Mikroskop untersucht werden. Anmerkung: Dies ist die einfachste Methode, um die Genauigkeit der Test testen, wie es entfällt die Notwendigkeit, die Substrate Immunfärbung. </li></ol> 5. Azid Funktionalisierung von mikrostrukturierten PEGMEMA Chains <ol style="margin-left: 40px;"><li> Legen Sie die mikroDeckGlas in einem 20 ml Glasreaktionsflasche. </li><li> 6 ml N, N-Dimethylformamid (DMF), enthaltend 100 mM Natriumazid in die Reaktionsflasche. Pflegen diese Reaktion bei 37 ° C für 24 Stunden. Hinweis: Behandeln DMF nur in einer chemischen Abzugshaube. Vorsicht beim Abwiegen Natriumazid. </li><li> Nach Abschluss entfernen Sie die mikroDeckGlas aus dem Reaktionsgefäß und spülen mit Ethanol und dann mit Wasser und trocken unter einem Stickstoffstrom. </li></ol> 6. Passivierung von Brom Funktionalisierte PEGMEMA Chains <ol style="margin-left: 40px;"><li> Zeigen mikrostrukturiert Deckglas in 20 ml Glasreaktionsgefäße. </li><li> 6 ml Dimethylsulfoxid (DMSO), die 100 mM Ethanolamin und 300 mM Triethylamin zu der Reaktionsflasche. Halten diese Reaktion bei 40 ° C für 24 Stunden. Hinweis: Behandeln DMSO, Ethanolamin und Triethylamin nur in einer chemischen Abzugshaube. </li><li> Nach Abschluss entfernen Sie die mikro coverslide aus dem Reaktionsgefäß gründlich mit Ethanol und dann mit Wasser und trocken unter einem Stickstoffstrom. </li></ol> 7. R-mCP der Innenring und SI-ATRP von PEGMEMA <ol style="margin-left: 40px;"><li> Durchführung R-mCP Schritte wie zuvor in den Schritten 3.2 und 3.6 - 3.12, Substitution eines PDMS-Stempel mit 300 um ID und 600 um Außendurchmesser vorstehenden Ringräume, so dass die Ringräume mit kleineren Merkmale sind in den zuvor mikrostrukturiert Ringräume angeordnet. HINWEIS: Die Druckschwelle für diese Briefmarke ist 132,0 kPa. Wie bereits erwähnt, werden diese Druckeinstellungen für wobei die Stempelfunktionen in diesem Experiment verwendet optimiert. </li><li> Führen Sie SI-ATRP Schritte wie zuvor in den Schritten 4.1 beschrieben - 4.4. </li></ol> 8. Acetylen Funktionalisierung von mikrostrukturierten PEGMEMA Chains <ol style="margin-left: 40px;"><li> Zeigen mikroDeckGlas in einem 20 ml Glasreaktionsflasche. </li><li> 6 ml DMSO, enthaltend 100 mMPropargylamin in die Reaktionsflasche. Halten Sie diese Reaktion bei RT für 24 h. Hinweis: Behandeln DMSO und Propargylamin nur in einer chemischen Abzugshaube. </li><li> Nach Abschluss entfernen Sie die mikroDeckGlas aus dem Reaktionsgefäß und spülen mit Ethanol und dann mit Wasser und trocken unter einem Stickstoffstrom. </li></ol> 9. Kupferkatalysierte &quot;Click&quot; Biotinylierung von Acetylen Abgebrochene PEGMEMA Chains <ol style="margin-left: 40px;"><li> Zeigen mikroDeckGlas in einem 20 ml Glasreaktionsflasche. </li><li> 6 ml Kupfersulfat (15 mM) / Tris [(1-Benzyl-1H-1,2,3-triazol-4-yl) methyl] amin (TBTA, 30 mM) (1: 1 v / v Wasser / DMF) enthalten 562 uM Azid-PEG 4 -Biotin Konjugat in die Reaktionsflasche. Mit 1,2 ml L-Ascorbinsäure (0,15 mM) in Wasser zu der Mischung, um die Reaktion zu initialisieren. </li><li> Bubble Stickstoff durch die Reaktionslösung für 10 sec, versiegeln die Phiole mit Parafilm und für 24 h bei RT reagieren. </li><li> Nach completion entfernen mikroDeckGlas aus dem Reaktionsgefäß, mit Wasser abspülen und legen Sie sie in einem 12-Well-Polystyrolschale. </li></ol> 10. Immunfluoreszenz-Nachweis von biotinylierter Acetylengruppen <ol style="margin-left: 40px;"><li> Block mikroDeckGlas in DPBS (3% Eselserum) 1 h bei RT. Färbung für biotinylierte Acetylengruppen mit Streptavidin-546-Konjugat (2 ug / ml) in DBP (3% Eselserum in PBS) für 2 h bei RT. </li><li> Spülen mit Mikrodeckglas 5 mal mit DPBS für 10 min unter leichtem Schütteln. ANMERKUNG: Abbildung 4B zeigt ein Bild der Folie, nach dieser Schritt abgeschlossen ist. </li></ol> 11. Kupferfreie &quot;Click&quot; Biotinylierung von Azid Abgebrochene PEGMEMA Chains HINWEIS: Wenn gewünscht, kann dieses Substrat Modifikationsschritt in situ während der Zellkultur durchgeführt werden. <ol style="margin-left: 40px;"><li> Lassen mikroDeckGlas in 12-Well-polystyrene Gericht folgenden DBPs spült. </li><li> 2 ml DBP (oder Zellkulturmedium), enthaltend 20 &amp; mgr; M-PEG DBCO 4 Biotin und lassen diese Reaktion für 24 h bei RT weiter (oder bei 37 ° C in einem Brutschrank). </li><li> Nach Abschluss gründlich mikroDeckGlas 5 mal mit DPBS (oder einfach führen Sie einen Medienwechsel). </li></ol> 12. Immunfluoreszenz-Nachweis von biotinylierter Azidgruppen <ol style="margin-left: 40px;"><li> Färbung für biotinylierte Azidgruppen mit Streptavidin-488-Konjugat (2 ug / ml) in 2 ml DPBS (3% Eselserum) für 2 h bei RT. </li><li> Spülen mit Mikrodeckglas 5 mal in DPBS für 10 min unter leichtem Schütteln. </li><li> Bildmikrodeckglas mit Hilfe der konfokalen Fluoreszenzmikroskops. 4A-C zeigt Bilder der Folie, nach diesem Schritt abgeschlossen. HINWEIS: Bei der Verwendung von Substraten für die Gewebekultur-Assays, überspringen Abschnitte 10 und 12 dieses Protokolls. Nach der gewünschten degree Funktionalisierungsgrad sterilisieren entworfen Deckglas durch Spülen beide Seiten mit 100% Ethanol und Trocknen unter einem Stickstoffstrom. Die Objektträger in einer sterilen Sicherheitswerkbank und spülen Sie die Objektträger mit PBS fünfmal, bevor mit Zellaussaat und Kultur voran. </li></ol>

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P., Chilkoti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface-Initiated+Atom+Transfer+Radical+Polymerization+of+Oligo(ethylene+glycol)+Methyl+Methacrylate+from+a+Mixed+Self-Assembled+Monolayer+on+Gold.">Surface-Initiated Atom Transfer Radical Polymerization of Oligo(ethylene glycol) Methyl Methacrylate from a Mixed Self-Assembled Monolayer on Gold.</a> Advanced Functional Materials. 16 (5), 640-648 (2006).</li><li>Scadden, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+stem-cell+niche+as+an+entity+of+action.">The stem-cell niche as an entity of action.</a> Nature. 441 (7097), (2006).</li><li>Codelli, J. A., Baskin, J. M., Agard, N. J., Bertozzi, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Second-Generation+Difluorinated+Cyclooctynes+for+Copper-Free+Click+Chemistry.">Second-Generation Difluorinated Cyclooctynes for Copper-Free Click Chemistry.</a> Journal of the American Chemical Society. 130 (34), 11486-11493 (2008).</li><li>Debets, M. F., van Berkel, S. S., Schoffelen, S., Rutjes, F. P. J. T., van Hest, J. C. M., van Delft, F. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aza-dibenzocyclooctynes+for+fast+and+efficient+enzyme+PEGylation+via+copper-free+(3+2)+cycloaddition.">Aza-dibenzocyclooctynes for fast and efficient enzyme PEGylation via copper-free (3+2) cycloaddition.</a> Chemical Communications. 46 (1), 97 (2010).</li><li>DeForest, C. A., Polizzotti, B. D., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+click+reactions+for+synthesizing+and+patterning+three-dimensional+cell+microenvironments.">Sequential click reactions for synthesizing and patterning three-dimensional cell microenvironments.</a> Nature Materials. 8 (8), 659-664 (2009).</li><li>Roth, E. A., Xu, T., Das, M., Gregory, C., Hickman, J. J., Boland, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inkjet+printing+for+high-throughput+cell+patterning.">Inkjet printing for high-throughput cell patterning.</a> Biomaterials. 25 (17), 3707-3715 (2004).</li><li>Xu, T., Zhao, W., Zhu, J. M., Albanna, M. Z., Yoo, J. J., Atala, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomaterials.">Biomaterials.</a> Biomaterials. 34 (1), 130-139 (2013).</li><li>Brouzes, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Droplet+microfluidic+technology+for+single-cell+high-throughput+screening.">Droplet microfluidic technology for single-cell high-throughput screening.</a> Proceedings of the National Academy of Sciences. 106 (34), 14195-14200 (2009).</li><li>Meitl, M. 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=Transfer+printing+by+kinetic+control+of+adhesion+to+an+elastomeric+stamp.">Transfer printing by kinetic control of adhesion to an elastomeric stamp.</a> Nature Materials. 5 (1), 33-38 (2005).</li></ol>

The authors declare that they have no competing financial interest.

Ideale Substrate für das Tissue Engineering Bioinspiriertes würde und dadurch die räumliche Verteilung der kritischen bioaktiven Liganden innerhalb der nativen Geweben zu rekapitulieren. Sie würden auch dynamische Eigenschaften besitzen, die zeitliche Änderungen der Liganden und die räumlichen Muster in der sie präsentiert gerichtet Morphogenese ermöglichen und räumlich begrenzte Induktion von Zellschicksal zu ermöglichen. Herstellung solcher Substrate erfordert die Immobilisierung von mehreren biochemischen S...

Die Fähigkeit von PEG-gepfropften Oberflächen kovalent gebunden biochemischen Liganden angezeigt während gleichzeitig inhärente Nicht-Fouling-Eigenschaften machen sie zu einer idealen Wahl für Engineering individuelle Mikroumgebungen auf Kultursubstraten 1,2,3. Die biospezifische Wechselwirkungen von Liganden konjugiert PEG vermittelte Bürsten ermöglicht reductionistic Analyse der Wirkungen von biochemischen Hinweisen im Komplex in vivo Gewebemikroumgebung über einzelne Zell Phänotypen gefunden. Weiterhin kann bio-orthogonal &quot;auf&quot; Chemie verwendet werden, um Richt Immobilisierung von Liganden zu erleichtern, so dass sie im nativen Konformationen 4-6 dargestellt werden. So mikroskaligen räumlichen Strukturierung von PEG Bürsten ist ein vielseitiges Werkzeug, um Designer in vitro Nischen schaffen, um Zellsignalisierung durch immobilisierte biochemischen Cues 6,7 induzierte untersuchen. Ein übliches Verfahren zur Erzeugung von räumlichen Mustern der biochemischen cues bringt Mikrokontakt-Drucken (mCP) gold-beschichteten Substrate mit Mustern von PEG konjugierten Alkanthiole. Dann werden die mikroselbstorganisierte Monoschichten (SAMs) von PEG-ylated Alkanthiole schränkt die physikalische Adsorption von biochemischen Molekülen, zB Proteine, nur für die nicht gemusterten Bereiche des Substrats 8,9. , Die durch diese Technik erzeugt SAMs sind jedoch empfindlich gegenüber Oxidation in der Langzeitzellkulturmedien. Somit μCP&#39;d Alkanthiol SAMs häufig weiter mit PEG Polymerbürsten mit oberflächeninitiierte Atom Transfer Radical Polymerisation (ATRP-SI), um die Region nicht-Fouling-Stabilität 10 zu erhöhen gepfropft. Insbesondere mCP der Alkanthiol Polymerisationsinitiator erzeugt ω-meraptoundecyl bromisobutyrat, auf Gold-beschichteten Oberflächen, gefolgt von SI-ATRP von Poly (ethylenglycol) methylethermethacrylat (PEGMEMA) Monomere Oberflächen mikro langfristig stabil und nicht Fouling PEG Bürsten. Darüber hinaus sind diese<html> sind in der Lage, die weiter modifiziert, um diverse chemische Einheiten 11 zu präsentieren. Unter Ausnutzung dieser Eigenschaft, Sha et. al. eine Methode entwickelt, um Kultursubstrate mit Mehrkomponenten PEGMEMA Bürsten präsentiert orthogonal &quot;auf&quot; Chemie-Ingenieur. Bei diesem Verfahren verwendet man eine Reihe von &amp; mgr; CP / SI-ATRP Schritten mit sequentieller Natriumazid, Ethanolamin vermischt und Propargylamin nucleophile Substitutionen Kultursubstrate präsentiert Mikromuster mehrere immobilisierte Liganden 6 zu erstellen. Während das Potential der Verwendung solcher Chemikalien in Verbindung mit manuellen mgr; CP neue Kultursubstrate Ingenieur ist immens, es wird von der Präzision und Genauigkeit, mit welcher mehrere &amp; mgr; CP Schritte können auf einem einzigen Substrat ausgerichtet werden, beschränkt. Ein hohes Maß an Präzision und Genauigkeit erforderlich wäre, um reproduzierbar herzustellen Komplex in vitro Nischen mit diesen vielseitigen Techniken werden. e_content &quot;&gt; Um diese Einschränkung zu begegnen, haben mehrere automatisierte und halbautomatisierte mCP Systemen erzeugt wurden. Chakra et. al. eine mCP System, in dem kundenspezifische Stempel sind auf einem Schienensystem platziert und mit Gold-beschichtete Objektträger mit in konformen Kontakt gebracht entwickelt ein computergesteuertes pneumatisches Stellglied. Allerdings erfordert dieses Verfahren die präzise Herstellung von kundenspezifischen Stempel entwickelt und berichtet über eine Präzisions 10 um ohne Bericht der erreicht wird, wenn die Durchführung mehrerer Schritte 12 &amp; mgr; CP Genauigkeit. Vor kurzem wurde ein Verfahren unter Verwendung eines integrierten kinematischen Kopplungssystem berichteten Genauigkeit unterhalb von 1 um unter Verwendung eines einzigen Musters, waren aber nicht in der Lage, mehrere Muster genau auszurichten aufgrund des Fehlens einer präzisen Steuerung der Stempelfunktionen von Form zu Form 13. Zusätzlich beiden vorhergehenden Verfahren erfordern das Substrat zwischen den Strukturierungsschritten fest bleiben und damit deutlich die Vielfalt der Oberflächenmodifizierung Chemie, die sein kann, begrenztverwendet. Hier beschreiben wir eine automatisierte R-mCP System in der Lage genau und präzise Ausrichtung mehrerer mCP Schritte und ermöglicht maximale Flexibilität bei der Briefmarkenentwurf und Fertigung. Weiterhin können die gemusterten Substraten wiederholt aus dem System zwischen Stanz entfernt werden, wodurch die Verwendung von verschiedenen Substratmodifikation Chemien, einschließlich sequentieller nucleophile Substitutionen erlaubt. Substrate entwickelt unter Verwendung solcher Chemikalien wurden für Zellkultur zuvor sowohl von uns 6,14 und andere 7 verwendet. So haben wir R-mgr; CP und sequenzielle nukleophile Substitutionsreaktionen zusammengeführt, um ein Verfahren zur Herstellung von skalierbaren Kultursubstrate mit komplexen biochemischen und mikro Signale zu entwickeln.

<table border="0" cellpadding="0" cellspacing="0" width="740">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:185px;">Name</td>
			<td style="width:185px;">Company</td>
			<td style="width:185px;">Catalog Number</td>
			<td style="width:185px;">Comments</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:185px;">SCARA&#160;</td>
			<td style="width:185px;">Epson</td>
			<td style="width:185px;">LS3-401ST</td>
			<td style="width:185px;">Higher end models with increased precision are available if desired.&#160;</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:185px;">(TRIDECAFLUORO-1,1,2,2-TETRAHYDROOCTYL)TRICHLOROSILANE</td>
			<td style="width:185px;">Gelest</td>
			<td style="width:185px;">SIT8174.0</td>
			<td style="width:185px;">CAUTION, Should only be handled in a chemical fume hood. When silanizing wafers no one should enter the hood until all silane has been evaporated.</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:185px;">Sylgard 184 Silicone Elastomer Kit</td>
			<td style="width:185px;">Ellsworth Adhesive Co</td>
			<td style="width:185px;">NC9020938</td>
			<td style="width:185px;">Thouroughly degass solutions via vacuum exposure before use. Alternative kits such as Kit 182 are acceptable.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:185px;">24mm X 50 mm #1 Cover Glass Slides</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:185px;">48393106</td>
			<td style="width:185px;">These can be purchased from a number of suppliers with varying dimensions to suit need.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">CHA-600 Telemark Electron Beam Evaporator</td>
			<td style="width:185px;">Telemark</td>
			<td style="width:185px;">SEC-600-RAP</td>
			<td style="width:185px;">Requries specialized training.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:185px;">EPSON LS3 SCARA</td>
			<td style="width:185px;">EPSON</td>
			<td style="width:185px;">LS3-401ST</td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:185px;">&#969;-mertcaptoundecyl bromoisobutyrate</td>
			<td style="width:185px;">Prochimia</td>
			<td style="width:185px;">FT 015-m11-0.2</td>
			<td style="width:185px;">Store at -20&#176;C. Other ATRP initiators may be used as this R-&#956;CP platform is applicable to all micropatterning modalities.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">Schlenk Tube Flask 50 mL</td>
			<td style="width:185px;">Synthware</td>
			<td style="width:185px;">60003-078</td>
			<td style="width:185px;">Requires rubber stoppers with diaphram.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:185px;">Poly(ethylene glycol) methyl ether methacrylate</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">447943</td>
			<td style="width:185px;">Shipped containing MEHQ and BHT free readical inhibitors.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">Methanol (Certified ACS)</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:185px;">A412-4</td>
			<td style="width:185px;">CAUTION, only handle in chemical fume hood.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">Copper(II) Bromide</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">437867</td>
			<td style="width:185px;">CAUTION, limit exposure with surgical mask.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">2&#39;,2-Bipyridine</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">D216305</td>
			<td style="width:185px;">CAUTION, limit exposure with surgical mask.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:185px;">Sodium L-Ascorbate</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">A4034</td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">20mL Borosilicate Glass Scintillation Vials</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:185px;">03-340-4E</td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">Sodium Azide</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">S2002</td>
			<td style="width:185px;">CAUTION, limit exposure with surgical mask.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">N,N-dimethyformamide</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">227056</td>
			<td style="width:185px;">CAUTION, only handle in chemical fume hood.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">Ethanolamine</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">398136</td>
			<td style="width:185px;">CAUTION, only handle in chemical fume hood.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">Triethylamine</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">T0886</td>
			<td style="width:185px;">CAUTION, only handle in chemical fume hood.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">Dimethylsulfoxide</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">276855</td>
			<td style="width:185px;">CAUTION, only handle in chemical fume hood.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">Propargylamine</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">P50900</td>
			<td style="width:185px;">CAUTION, only handle in chemical fume hood.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:185px;">200 Proof Ethanol</td>
			<td style="width:185px;">University of Wisconsin Material Distribution Services</td>
			<td style="width:185px;">2292</td>
			<td style="width:185px;">CAUTION, only handle in chemical fume hood.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:185px;">Azide-PEG3-Biotin</td>
			<td style="width:185px;">ClickChemistryTools</td>
			<td style="width:185px;">AZ104-100</td>
			<td style="width:185px;">Solubilized in DMF</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">Copper(II) Sulfate</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">C1297</td>
			<td style="width:185px;">CAUTION, limit exposure with surgical mask.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:185px;">Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA)</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">678937</td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:185px;">L-Ascorbic Acid</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">A7506</td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:185px;">Phosphate Buffer Saline</td>
			<td style="width:185px;">Invitrogen</td>
			<td style="width:185px;">14190144</td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="168">
			<td height="168" style="height:168px;width:185px;">Donkey Serum</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">D9663</td>
			<td style="width:185px;">Donkey serum contaminated items are considered bio-hazardous material and should be disposed of accordingly. Various other compounds (e.g. BSA) are available and serve this purpose.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:185px;">12-Well Polystyrene Plate</td>
			<td style="width:185px;">Thermo Scientifit - NUNC</td>
			<td style="width:185px;">07-200-81</td>
			<td style="width:185px;">Plates can be purchased form a number of suppliers with varying dimensions.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:185px;">DBCO-PEG4-Biotin</td>
			<td style="width:185px;">Clickchemistytools</td>
			<td style="width:185px;">A105P4-10</td>
			<td style="width:185px;">Solubilized in DMF</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">Streptavidin, Alexa Fluor 488 Conjugate</td>
			<td style="width:185px;">Life Technologies</td>
			<td style="width:185px;">S-11223</td>
			<td style="width:185px;">Solubilized in PBS</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">Streptavidin, Alexa Fluor 546 conjugate</td>
			<td style="width:185px;">Life Technologies</td>
			<td style="width:185px;">S-11225</td>
			<td style="width:185px;">Solubilized in PBS</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:185px;">Nikon A1-R Confocal Microscope</td>
			<td style="width:185px;">Nikon</td>
			<td style="width:185px;">Nikon Eclipse Ti, A1R</td>
			<td style="width:185px;">An epifluorescent microscope is sufficient to image functionalized micropatterned substrates.</td>
		</tr>
	</tbody>
</table>

fabricating complex culture substrates using robotic microcontact printing (r-&#181;cp) and sequential nucleophilic substitution

In tissue engineering, it is desirable to exhibit spatial control of tissue morphology and cell fate in culture on the micron scale. Culture substrates presenting grafted poly(ethylene glycol) (PEG) brushes can be used to achieve this task by creating microscale, non-fouling and cell adhesion resistant regions as well as regions where cells participate in biospecific interactions with covalently tethered ligands. To engineer complex tissues using such substrates, it will be necessary to sequentially pattern multiple PEG brushes functionalized to confer differential bioactivities and aligned in microscale orientations that mimic in vivo niches. Microcontact printing (&#956;CP) is a versatile technique to pattern such grafted PEG brushes, but manual &#956;CP cannot be performed with microscale precision. Thus, we combined advanced robotics with soft-lithography techniques and emerging surface chemistry reactions to develop a robotic microcontact printing (R-&#956;CP)-assisted method for fabricating culture substrates with complex, microscale, and highly ordered patterns of PEG brushes presenting orthogonal &#8216;click&#8217; chemistries. Here, we describe in detail the workflow to manufacture such substrates.

Der Einsatz von manuellen Ausrichtung mCP Techniken, um Kultursubstrate mit Arrays von PEG-gepfropft Bürsten mit orthogonaler funktionalisiert Engineer &quot;klicken Sie auf&quot; Chemie hat in früheren Arbeiten 6 nachgewiesen. Allerdings bietet diese minimale Steuerung der Musterausrichtung und führt oft zu einer Überlappung von funktionalisierten Bereichen. Hier wird eine neue R-mgr; CP-System verwendet, um diese Beschränkung zu überwinden, und ihre Fähigkeit, genau Muster eine Anordnung von PEG Bür...

Watch this Scientific Journal Video about Fabricating Complex Culture Substrates Using Robotic Microcontact Printing R-Ã‚ÂµCP and Sequential Nucleophilic Substitution at JoVE.com

Fabricating Complex Culture Substrates Using Robotic Microcontact Printing R- &#181;CP and Sequential Nucleophilic Substitution

Cell culture substrates functionalized with microscale patterns of biological ligands have immense utility in the field of tissue engineering. Here, we demonstrate the versatile and automated manufacture of tissue culture substrates with multiple, micropatterned poly(ethylene glycol) brushes presenting orthogonal chemistries that enable spatially precise and site-specific immobilization of biological ligands.

Herstellung von komplexen Kultursubstrate verwenden Robotic Mikrokontaktdrucken (R-mCP) und Sequential Nucleophile Substitution

In tissue engineering, it is desirable to exhibit spatial control of tissue morphology and cell fate in culture on the micron scale. Culture substrates ...

fabricating-complex-culture-substrates-using-robotic-microcontact

Research

JoVE Journal

Bioengineering

Fabricating Complex Culture Substrates Using Robotic Microcontact Printing (R-&#181;CP) and Sequential Nucleophilic Substitution

10.4K Aufrufe.  University of Wisconsin, Madison. Cell culture substrates functionalized with microscale patterns of biological ligands have immense utility in the field of tissue engineering. Here, we demonstrate the versatile and automated manufacture of tissue culture substrates with multiple, micropatterned poly(ethylene glycol) brushes presenting orthogonal chemistries that enable spatially precise and site-specific immobilization of biological ligands. 

Serie: Fabricating Complex Culture Substrates Using Robotic Microcontact Printing R- µCP and Sequential Nucleophilic Substitution

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the need for 3D environments to represent in vitro experiments which better mimic in vivo qualities 1-3. Studies in fields such as cancer research 4, neural engineering 5, cardiac physiology 6, and cell-matrix interaction7,8have shown cell behavior differs substantially between traditional monolayer cultures and 3D constructs.
Hydrogels are used as 3D environments because of their variety, versatility and ability to tailor molecular composition through functionalization 9-12. Numerous techniques exist for creation of constructs as cell-supportive matrices, including electrospinning13, elastomer stamps14, inkjet printing15, additive photopatterning16, static photomask projection-lithography17, and dynamic mask microstereolithography18. Unfortunately, these methods involve multiple production steps and/or equipment not readily adaptable to conventional cell and tissue culture methods. The technique employed in this protocol adapts the latter two methods, using a digital micromirror device (DMD) to create dynamic photomasks for crosslinking geometrically specific poly-(ethylene glycol) (PEG) hydrogels, induced through UV initiated free radical polymerization. The resulting "2.5D" structures provide a constrained 3D environment for neural growth. We employ a dual-hydrogel approach, where PEG serves as a cell-restrictive region supplying structure to an otherwise shapeless but cell-permissive self-assembling gel made from either Puramatrix or agarose. The process is a quick simple one step fabrication which is highly reproducible and easily adapted for use with conventional cell culture methods and substrates.
Whole tissue explants, such as embryonic dorsal root ganglia (DRG), can be incorporated into the dual hydrogel constructs for experimental assays such as neurite outgrowth. Additionally, dissociated cells can be encapsulated in the photocrosslinkable or self polymerizing hydrogel, or selectively adhered to the permeable support membrane using cell-restrictive photopatterning. Using the DMD, we created hydrogel constructs up to ~1mm thick, but thin film (&lt;200 &mu;m) PEG structures were limited by oxygen quenching of the free radical polymerization reaction. We subsequently developed a technique utilizing a layer of oil above the polymerization liquid which allowed thin PEG structure polymerization.
In this protocol, we describe the expeditious creation of 3D hydrogel systems for production of microfabricated neural cell and tissue cultures. The dual hydrogel constructs demonstrated herein represent versatile in vitro models that may prove useful for studies in neuroscience involving cell survival, migration, and/or neurite growth and guidance. Moreover, as the protocol can work for many types of hydrogels and cells, the potential applications are both varied and vast.

Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.

Herstellung von mikrostrukturierten Hydrogele für Neural Kultur-Systeme mit Dynamic Mask Projektion Photolithographie

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the ...

Forschung

Biotechnik

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

Erstellen Zweidimensionale Patterned Substrate für Protein-und Zell-Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Soft Lithographic Functionalization and Patterning Oxide-free Silicon and Germanium

The development of hybrid electronic devices relies in large part on the integration of (bio)organic materials and inorganic semiconductors through a stable interface that permits efficient electron transport and protects underlying substrates from oxidative degradation. Group IV semiconductors can be effectively protected with highly-ordered self-assembled monolayers (SAMs) composed of simple alkyl chains that act as impervious barriers to both organic and aqueous solutions. Simple alkyl SAMs, however, are inert and not amenable to traditional patterning techniques. The motivation for immobilizing organic molecular systems on semiconductors is to impart new functionality to the surface that can provide optical, electronic, and mechanical function, as well as chemical and biological activity. 
Microcontact printing (&mu;CP) is a soft-lithographic technique for patterning SAMs on myriad surfaces.1-9 Despite its simplicity and versatility, the approach has been largely limited to noble metal surfaces and has not been well developed for pattern transfer to technologically important substrates such as oxide-free silicon and germanium. Furthermore, because this technique relies on the ink diffusion to transfer pattern from the elastomer to substrate, the resolution of such traditional printing is essentially limited to near 1 &mu;m.10-16
In contrast to traditional printing, inkless &mu;CP patterning relies on a specific reaction between a surface-immobilized substrate and a stamp-bound catalyst. Because the technique does not rely on diffusive SAM formation, it significantly expands the diversity of patternable surfaces. In addition, the inkless technique obviates the feature size limitations imposed by molecular diffusion, facilitating replication of very small (&lt;200 nm) features.17-23 However, up till now, inkless &mu;CP has been mainly used for patterning relatively disordered molecular systems, which do not protect underlying surfaces from degradation.
Here, we report a simple, reliable high-throughput method for patterning passivated silicon and germanium with reactive organic monolayers and demonstrate selective functionalization of the patterned substrates with both small molecules and proteins. The technique utilizes a preformed NHS-reactive bilayered system on oxide-free silicon and germanium. The NHS moiety is hydrolyzed in a pattern-specific manner with a sulfonic acid-modified acrylate stamp to produce chemically distinct patterns of NHS-activated and free carboxylic acids. A significant limitation to the resolution of many &mu;CP techniques is the use of PDMS material which lacks the mechanical rigidity necessary for high fidelity transfer. To alleviate this limitation we utilized a polyurethane acrylate polymer, a relatively rigid material that can be easily functionalized with different organic moieties. Our patterning approach completely protects both silicon and germanium from chemical oxidation, provides precise control over the shape and size of the patterned features, and gives ready access to chemically discriminated patterns that can be further functionalized with both organic and biological molecules. The approach is general and applicable to other technologically-relevant surfaces.

Here we describe a simple method for patterning oxide-free silicon and germanium with reactive organic monolayers and demonstrate functionalization of the patterned substrates with small molecules and proteins. The approach completely protects surfaces from chemical oxidation, provides precise control over feature morphology, and provides ready access to chemically discriminated patterns.

Soft-Lithographie Funktionalisierung und Strukturierung Oxydfreie Silizium und Germanium

The development of hybrid electronic devices relies in large part on the integration of (bio)organic materials and inorganic semiconductors through a ...

Microcontact Printing of Proteins for Cell Biology

The ability to pattern proteins and other biomolecules onto substrates is important for capturing the spatial complexity of the extracellular environment. Development of microcontact printing by the Whitesides group (<a href="http://gmwgroup.harvard.edu/">http://gmwgroup.harvard.edu/</a>) in the mid-1990s revolutionalized this field by making microelectronics/microfabrication techniques accessible to laboratories focused on the life sciences. Initial implementations of this method used polydimethylsiloxane (PDMS) stamps to create patterns of functionalized chemicals on material surfaces1. Since then, a range of innovative approaches have been developed to pattern other molecules, including proteins2. This video demonstrates the basic process of creating PDMS stamps and uses them to pattern proteins, as these steps are difficult to accurately express in words. We focus on patterning the extracellular matrix protein fibronectin onto glass coverslips as a specific example of patterning.
An important component of the microcontact printing process is a topological master, from which the stamps are cast; the raised and lowered regions of the master are mirrored into the stamp and define the final pattern. Typically, a master consists of a silicon wafer coated with photoresist and then patterned by photolithography, as is done here. Creation of masters containing a specific pattern requires specialized equipment, and is best approached in consultation with a fabrication center or facility. However, almost any substrate with topology can be used as a master, such as plastic diffraction gratings (see Reagents for one example), and such serendipitous masters provide readily available, simple patterns. This protocol begins at the point of having a master in hand.

Microcontact printing is used extensively to pattern proteins and other molecules on material surfaces. We demonstrate the basic steps of this process, stamping patterns of fibronectin onto glass.

Mikrokontaktdrucken von Proteinen für Zellbiologie

The ability to pattern proteins and other biomolecules onto substrates is important for capturing the spatial complexity of the extracellular environment. ...

Biologie

Automated Robotic Dispensing Technique for Surface Guidance and Bioprinting of Cells

This manuscript describes the introduction of cell guidance features followed by the direct delivery of cells to these features in a hydrogel bioink using an automated robotic dispensing system. The particular bioink was selected as it allows cells to sediment towards and sense the features. The dispensing system bioprints viable cells in hydrogel bioinks using a backpressure assisted print head. However, by replacing the print head with a sharpened stylus or scalpel, the dispensing system can also be employed to create topographical cues through surface etching. The stylus movement can be programmed in steps of 10 &#181;m in the X, Y and Z directions. The patterned grooves were able to orientate mesenchymal stem cells, influencing them to adopt an elongated morphology in alignment with the grooves&#39; direction. The patterning could be designed using plotting software in straight lines, concentric circles, and sinusoidal waves. In a subsequent procedure, fibroblasts and mesenchymal stem cells were suspended in a 2% gelatin bioink, for bioprinting in a backpressure driven extrusion printhead. The cell bearing bioink was then printed using the same programmed coordinates used for the etching. The bioprinted cells were able to sense and react to the etched features as demonstrated by their elongated orientation along the direction of the etched grooves.

This protocol describes a bioprinting methodology using an automated robotic depositing system that incorporates etched topographical guidance cues with the precision deposition of a cell bearing hydrogel bioink. The printed cells are directly delivered to the etched features and are able to sense and orientate with them.

Automatische Robotic Dosiertechnik für Oberflächen Ausrichtungs- und Bioprinting von Zellen

This manuscript describes the introduction of cell guidance features followed by the direct delivery of cells to these features in a hydrogel bioink using ...

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

Automatisierte Roboter Liquid Handling Versammlung Modular DNA-Geräte

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

Functional Surface-immobilization of Genes Using Multistep Strand Displacement Lithography

Immobilization of genes on lithographically structured surfaces allows the study of compartmentalized gene expression processes in an open microfluidic bioreactor system. In contrast to other approaches towards artificial cellular systems, such a setup allows for a continuous supply with gene expression reagents and simultaneous draining of waste products. This facilitates the implementation of cell-free gene expression processes over extended periods of time, which is important for the realization of dynamic gene regulatory feedback systems. Here we provide a detailed protocol for the fabrication of genetic biochips using a simple-to-use lithographic technique based on DNA strand displacement reactions, which exclusively uses commercially available components. We also provide a protocol on the integration of compartmentalized genes with a polydimethylsiloxane (PDMS)-based microfluidic system. Furthermore, we show that the system is compatible with total internal reflection fluorescence (TIRF) microscopy, which can be used for the direct observation of molecular interactions between DNA and molecules contained in the expression mix.

We describe a simple lithographic procedure for the immobilization of gene-length DNA molecules on a surface, which can be used to perform cell-free gene expression experiments on biochips.

Funktionale Oberfläche-Immobilisierung von Genen mit mehrstufigen Strang Verschiebung Lithographie

Immobilization of genes on lithographically structured surfaces allows the study of compartmentalized gene expression processes in an open microfluidic ...

Biomimetic Replication of Root Surface Microstructure using Alteration of Soft Lithography

Biomimetics is the use of chemistry and material sciences to mimic biological systems, specifically biological structures, to better humankind. Recently, biomimetic surfaces mimicking the microstructure of leaf surface, were used to study the effects of leaf microstructure on leaf-environment interactions. However, no such tool exists for roots. We developed a tool allowing the synthetic mimicry of the root surface microstructure into an artificial surface. We relied on the soft lithography method, known for leaf surface microstructure replication, using a two-step process. The first step is the more challenging one as it involves the biological tissue. Here, we used a different polymer and curing strategy, relying on the strong, rigid, polyurethane, cured by UV for the root molding. This allowed us to achieve a reliable negative image of the root surface microstructure including the delicate, challenging features such as root hairs. We then used this negative image as a template to achieve the root surface microstructure replication using both the well-established polydimethyl siloxane (PDMS) as well as a cellulose derivative, ethyl cellulose, which represents a closer mimic of the root and which can also be degraded by cellulase enzymes secreted by microorganisms. This newly formed platform can be used to study the microstructural effects of the surface in root-microorganism interactions in a similar manner to what has previously been shown in leaves. Additionally, the system enables us to track the microorganism&#8217;s locations, relative to surface features, and in the future its activity, in the form of cellulase secretion.

Biomimetics has been previously used as a tool to study leaf-microorganism interactions. However, no such tool exists for roots. Here, we develop a protocol to form synthetic surfaces mimicking root surface microstructure for the study of root-environment interactions.

Biomimetische Replikation der Wurzeloberflächen-Mikrostruktur mit Änderung der weichen Lithographie

Biomimetics is the use of chemistry and material sciences to mimic biological systems, specifically biological structures, to better humankind. Recently, ...

Generation of Multicue Cellular Microenvironments by UV-Photopatterning of Three-Dimensional Cell Culture Substrates

The extracellular matrix is an important regulator of cell function. Environmental cues existing in the cellular microenvironment, such as ligand distribution and tissue geometry, have been increasingly shown to play critical roles in governing cell phenotype and behavior. However, these environmental cues and their effects on cells are often studied separately using in vitro platforms that isolate individual cues, a strategy that heavily oversimplifies the complex in vivo situation of multiple cues. Engineering approaches can be particularly useful to bridge this gap, by developing experimental setups that capture the complexity of the in vivo microenvironment, yet retain the degree of precision and manipulability of in vitro systems.
This study highlights an approach combining ultraviolet (UV)-based protein patterning and lithography-based substrate microfabrication, which together enable high-throughput investigation into cell behaviors in multicue environments. By means of maskless UV-photopatterning, it is possible to create complex, adhesive protein distributions on three-dimensional (3D) cell culture substrates on chips that contain a variety of well-defined geometrical cues. The proposed technique can be employed for culture substrates made from different polymeric materials and combined with adhesive patterned areas of a broad range of proteins. With this approach, single cells, as well as monolayers, can be subjected to combinations of geometrical cues and contact guidance cues presented by the patterned substrates. Systematic research using combinations of chip materials, protein patterns, and cell types can thus provide fundamental insights into cellular responses to multicue environments.

Traditionally, cell culture is performed on planar substrates that poorly mimic the natural environment of cells in vivo. Here we describe a method to produce cell culture substrates with physiologically relevant curved geometries and micropatterned extracellular proteins, allowing systematic investigations into cellular sensing of these extracellular cues.

Erzeugung von Multicue-Zellmikroumgebungen durch UV-Photostrukturierung von dreidimensionalen Zellkultursubstraten

The extracellular matrix is an important regulator of cell function. Environmental cues existing in the cellular microenvironment, such as ligand ...

Anwendungen des 3D-Drucks für mikroskalige neuronale Zellkulturgeräte

Two-Photon Polymerization 3D-Printing of Micro-scale Neuronal Cell Culture Devices

Neuronal cultures have been a reference experimental model for several decades. However, 3D cell arrangement, spatial constraints on neurite outgrowth, and realistic synaptic connectivity are missing. The latter limits the study of structure and function in the context of compartmentalization and diminishes the significance of cultures in neuroscience. Approximating ex vivo the structured anatomical arrangement of synaptic connectivity is not trivial, despite being key for the emergence of rhythms, synaptic plasticity, and ultimately, brain pathophysiology. Here, two-photon polymerization (2PP) is employed as a 3D printing technique, enabling the rapid fabrication of polymeric cell culture devices using polydimethyl-siloxane (PDMS) at the micrometer scale. Compared to conventional replica molding techniques based on microphotolitography, 2PP micro-scale printing enables rapid and affordable turnaround of prototypes. This protocol illustrates the design and fabrication of PDMS-based microfluidic devices aimed at culturing modular neuronal networks. As a proof-of-principle, a two-chamber device is presented to physically constrain connectivity. Specifically, an asymmetric axonal outgrowth during ex vivo development is favored and allowed to be directed from one chamber to the other. In order to probe the functional consequences of unidirectional synaptic interactions, commercial microelectrode arrays are chosen to monitor the bioelectrical activity of interconnected neuronal modules. Here, methods to 1) fabricate molds with micrometer precision and 2) perform in vitro multisite extracellular recordings in rat cortical neuronal cultures are illustrated. By decreasing costs and future widespread accessibility of 2PP 3D-printing, this method will become more and more relevant across research labs worldwide. Especially in neurotechnology and high-throughput neural data recording, the ease and rapidity of prototyping simplified in vitro models will improve experimental control and theoretical understanding of in vivo large-scale neural systems.

Autor im Rampenlicht: Modulare neuronale Netze zur Analyse von Gehirnfunktionen

3D printing at the micrometer scale enables rapid prototyping of polymeric devices for neuronal cell cultures. As a proof-of-principle, structural connections between neurons were constrained by creating barriers and channels influencing neurite outgrowth, while the functional consequences of such manipulation were observed by extracellular electrophysiology.

Zwei-Photonen-Polymerisation 3D-Druck von neuronalen Zellkulturgeräten im Mikromaßstab

Neuronal cultures have been a reference experimental model for several decades. However, 3D cell arrangement, spatial constraints on neurite outgrowth, ...