This work was supported by the Belgian National Foundation for Scientific Research (F.R.S.-FNRS) through &#8220;MIS Confocal Microscopy&#8221;, &#8220;Cr&#233;dit aux Chercheurs&#8221; grants and the &#8220;Nanomotility FRFC project&#8221; (no. 2.4622.11). T.G. doctoral fellowship is supported by the Foundation for Training in Industrial and Agricultural Research (FRIA). The authors gratefully acknowledge Sylvain Desprez for mechanical characterization and G&#233;raldine Circelli for confocal imaging.

1. Aktivieren der Oberfläche des Deckgläser <ol><li> Ort Glaskreisdeckgläser (25 mm Durchmesser) in einer Petrischale und Abstrich 0,1 M NaOH-Lösung auf sie für 5 min (chemischen Abzugshaube empfohlen). </li><li> Entfernen Sie die NaOH-Lösung und vollständig eintauchen Deckgläser mit sterilem ddH2O für 20 min unter leichtem Schaukeln auf einem Schaukelplatte in einem sterilen Kultur Kapuze. </li><li> Abtropfen sterilen ddH2O und wiederholen Sie den Schritt 1.2. </li><li> Entfernen Sie die Deckgläser mit einer sterilen Pinzette und legen Sie sie in eine neue Petrischale mit der Aktivseite nach oben. </li><li> Trockendeckgläser unter einem ständigen Strom von hochreinem Stickstoffgas. </li><li> In einem sterilen Kultur Haube, schmieren eine dünne Schicht von 3 (trimethoxysilyl) propylacrylat (92%) auf der Aktivseite der Deckglas für 1 Stunde. </li><li> Ausgiebig waschen Deckgläschen mit 3 Waschungen von sterilen ddH2O und tauchen sie in sterilen ddH2O in einem neuen Petrischale. </li><li> Tippen Sie auf die Petrischalemit Parafilm und legen Sie es auf einer Wippe Platte unter leichtem Schütteln für 10 min. </li><li> Deckgläser abnehmen ddH2O mit einer sterilen Pinzette mit feinen Spitzen und legen Sie sie in eine neue Petrischale mit der Aktivseite nach oben. </li><li> Speichern bei RT in einem trockenen Ort mit Aluminiumfolie, um Staub vom Festhalten an den Deckgläsern zu vermeiden. </li></ol> 2. Herstellung von Hydroxy-PAAm Hydrogele <ol><li> Vorbereitung ein Gewicht von 65 mg N-Hydroxyethyl-acrylamid (HEA) in einem 1,5 ml Eppendorf-Röhrchen. Es ist wichtig, um eine frische HEA-Lösung herzustellen. </li><li> 1 ml 50 mM HEPES-Puffer, um HEA und mischen mit einem Vortexer bis zur vollständigen Auflösung HEA. </li><li> Werden 400 ul 40% w / w in HEPES Acrylamid-Lösung und der gewünschten Menge von 2% w / w in HEPES-bis-acrylamid-Lösung (siehe Tabelle 1), um die gewünschte Steifigkeit zu erreichen Hydrogel. Einstellen mit 50 mM HEPES auf ein Endvolumen von 5 ml. </li><li> Mischen Sie die Lösung mit einem Vortexer und entgasenin einer Vakuumkammer für 20 Minuten, um die Sauerstoffkonzentration in der Lösung, die Hydroxy-PAAm Polymerisation verhindert reduzieren. </li><li> Unter einer sterilen Haube, filtern das entgaste Lösung mit einer 0,2 um Porengröße Filter, um sie zu sterilisieren. </li><li> Aktivieren runden Glasdeckgläschen (22 mm Durchmesser) in einem UV / Ozon-Reiniger während 7 min. </li><li> Planen 100 ul 10% Ammoniumpersulfat (APS)-Lösung, das heißt 10 mg APS in 100 ul ddH 2 O. Es ist wichtig, um eine frische APS-Lösung herzustellen. </li><li> Fügen 2,5 ul Tetramethylendiamin (TEMED) und 25 ul APS Lösung des sterilisierten Hydroxy-PAAm-Lösung (Schritt 2.5), um die Polymerisation zu initiieren. Mischen Sie die Lösung von 3 aufeinander folgenden Pipettierungen ohne Einführung von Blasen, unter sterilen Bedingungen. </li><li> Unter einer sterilen Haube, legen Sie einen 25 ul Tropfen der Hydroxy-PAAm Lösung im 25 mm Deckglas (ab Schritt 1.9) und sofort legen Sie eine 22 mm glArsch Deckglas (auf Stufe 2.5 vorbereitet) auf der Oberseite des Tropfens, um das Hydroxy-PAAm Lösung zu quetschen. Zentrieren Sie die 22 mm Deckglas mit einer sterilen Pinzette und glätten keine Blasen. </li><li> Ermöglichen Hydroxy-PAAm Hydrogele bei RT für 15 min polymerisiert. Kehren Sie manuell die restlichen Hydroxy-PAAm Lösung in der Eppendorf-Röhrchen, um die Beendigung der Polymerisation Prozess zu folgen. </li><li> Voll eintauchen Deckgläser mit sterilem ddH2O und sorgfältig trennen die 22 mm Deckgläschen durch die Einführung der Kante einer Rasierklinge zwischen den 22 mm Deckgläschen und dem Hydroxy-PAAm Hydrogel-Schicht. </li><li> Mit sterilem PBS waschen Hydroxy-PAAm Hydrogele (3 Austausch von PBS) und lassen Sie die Gele vollständig in sterilem PBS eingetaucht, um Hydratation beizubehalten. </li><li> Speichern Hydroxy-PAAm Hydrogele in sterilem PBS bei 4 ° C für bis zu 3 Tagen. </li></ol> 3. Polydimethylsiloxan (PDMS) Microstamp Fabrication HINWEIS: Die Herstellung eines Silizium-Master ist vor der staRT Das PDMS microstamp Fertigung. Diese Mikro eines Silizium-Master kann durch lithographische Techniken, die spezielle Ausrüstung und Ausbildung erfordert geführt werden. Kooperationen mit einer Nanofabrikation Anlage werden ermutigt, den Silizium-Master herzustellen. Alternativ wenden Sie ein Unternehmen, das maßgeschneiderte mikrostrukturierte Silizium-Meister fertigt auf Anfrage. Es ist wichtig zu beachten, daß die Herstellung des Silizium-Master muss nur einmal durchgeführt werden. In der Tat können mikrostrukturierte Silizium-Meister auf unbestimmte Zeit zu Elastomer Briefmarken verwendet werden. <ol><li> Mix PDMS und Härter in einem 10: 1-Verhältnis in einem Kunststoffbecher und mischen mit einer Pipette für 10 min. </li><li> Entgast PDMS-Mischung unter Vakuum, um Luftblasen, die während der Stufe 3.1 gebildet wurden zu entfernen. </li><li> Legen Sie die mikrostrukturierte Silizium-Master in einer Petrischale und warf einen 10-mm-dicke Schicht von entgastem PDMS-Mischung auf, ohne Bildung von Blasen. </li><li> Lassen Sie die PDMS für 2 Stunden bei 60 ° C in einer heilenOfen. </li><li> In einer staubfreien Umgebung, Peel-off der PDMS-Schicht und der Verbrauch 1 cm 2 microstamps mit einem Skalpell. </li><li> Mit einer Pinzette, microstamps Ort PDMS Muster-up in einer Petrischale. </li></ol> 4. Mikrostrukturierung Hydroxy-PAAm Hydrogele <ol><li> Ort PDMS microstamps in einem Ethanol / Wasser (50:50)-Lösung und beschallen für 15 min. </li><li> Trocknen der Stempel mit einem Strom von Stickstofffluss und sie muster in einem UV / Ozon-Reinigungsmittel (λ &lt;200 nm) für 7 min. </li><li> Unter einer sterilen Haube, legen eine 150 ul-Tropfen einer gewünschten Proteinlösung (beispielsweise 100 ug / ml Laminin in PBS oder 25 ug / ml Fibronektin in PBS) auf die mikrostrukturierte Oberfläche eine 1-cm 2 PDMS-Stempel. </li><li> Optional: Ändern der Konzentration der Proteinlösung in die Zelle-Ligandendichte zu modulieren. </li><li> Ausbreitung der Proteinlösung über die Stempeloberfläche durch Verschieben mit einer sterilen Spitze einer Pipette zu jeder Eckeder Stempel. </li><li> Verlassen Sie die Proteinlösung auf der PDMS-Stempel für 60 Minuten unter einer sterilen Haube zu adsorbieren. Schalten Sie Lampen, um die Proteinschäden zu vermeiden. </li><li> Unter einer sterilen Haube, übertragen Hydroxy-PAAm beschichtete Deckgläser (erhältlich bei Schritt 2.13) in eine Petrischale. </li><li> Entfernen überschüssigen PBS von der Oberfläche Hydroxy-PAAm Substrate mit einem geringen Stickstoffstrom unter sterilen Bedingungen. Stoppen des Verfahrens, sobald keine Hinweise stehendem Wasser auf der Gel-Oberfläche beobachtet. Das Gel sollte nicht gründlich in diesem Stadium getrocknet werden. </li><li> Trocken sorgfältig die strukturierte Oberfläche der PDMS-Stempel mit einem stetigen Strom von hochreinem Stickstoffgas. </li><li> Fassen Sie die Protein-beschichteten Stempel mit Dressing Gewebe Pinzette und legen Sie die strukturierte Oberfläche in Kontakt mit der Oberfläche getrocknete Hydrogel. Gelten kurze Druckpunkte mit der Spitze der Pinzette auf der Oberseite der PDMS-Stempel, um einen guten Kontakt zwischen Stempel und Mikroformen des Hydrogels Oberfläche zu gewährleisten. </li><li> Verlassen Sie die PDMS-Stempel auf the Hydrogel Oberfläche für 1 h bei RT. </li><li> Entfernen Sie vorsichtig PDMS-Stempel aus Hydroxy-PAAm Hydrogele mit Dressing Gewebe Pinzette und folgen Sie den Schritt 4.1, um den Stempel zu reinigen. </li><li> Waschen umfassend die Stanz Hydroxy-PAAm Hydrogele von 3 Austausch von PBS (pH = 7,4) unter sterilen Bedingungen 10 min pro Austausch. </li><li> Optional: Zusätzliche Mikro anderer ECM-Proteine ​​können durch folgende Schritte 4,5-4,11 auf die Hydroxy-PAAm Oberfläche hinzugefügt werden. </li><li> Passivierung nicht bedruckten Bereiche mit einer sterilen Lösung von BSA bei 5 mg / ml in PBS während einer Nacht bei 4 ° C unter leichtem Schütteln auf einer Schwingplatte. </li><li> Umfassend Waschen von 3 Austausch von PBS (pH = 7,4) unter sterilen Bedingungen 10 min pro Austausch. In diesem Stadium kann gestanzt Hydroxy-PAAm Hydrogele bei 4 ° C bis zu einer Woche gelagert werden. </li></ol> 5. Zellablage auf mikrostrukturierten Hydroxy-PAAm Hydrogele <ol><li> Deckgläschen in Zellmedium inkubieren 30-45 min vor dem Beschichten cEllen. </li><li> Waschen adhärenten Zellen in einer 75 cm 2 Kulturflasche mit sterilem PBS bei 37 ° C, und nehmen Sie mit 3 ml Trypsin-EDTA oder Accutase für 10 min. </li><li> Übertragen Sie die gewünschte Menge des vorgewärmten komplette Wachstumsmedium entsprechend Ihrer Zelllinie in den Kolben mit den abgelösten Zellen und Zentrifugieren der Zellsuspension für 3 min bei 650 x g. </li><li> Den Überstand mit einer Mikropipette und Die Zellen in komplettem Kulturmedium bei 15-20.000 Zellen / ml. </li><li> 4 ml der Zelllösung zu einem mikroDeckGlas (aus Stufe 5.1) erhalten und Küvette bedeckten Deckglas in einem Brutschrank bei 37 ° C und 5% Feuchtigkeit für 1-2 Stunden. </li><li> Saugen Sie sanft ungebunden Zellen und ersetzen Kulturmedium. Bringen Sie die anhaftenden Zellen in den Inkubator und lassen Sie sie vollständig verteilt (3-6 Stunden, je nach Zelltyp). </li></ol>

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No conflicts of interest declared.

Viele in vitro Beobachtungen in modernen Zellbiologie haben auf starren Glasplättchen, die oft mit einer dünnen Schicht aus ECM-Proteine ​​oder synthetische Peptide, die die RGD-Sequenz beschichtet geführt. Allerdings sind solche grundlegenden Kultursubstraten nicht rekapitulieren die gesamte physikalisch-chemische Komplexität des ECM und somit nicht ein genaues Modell für die Untersuchung zellulärer Mechanotransduktion Prozesse. Um dieses Problem anzugehen, schlagen wir eine einfache Alternative zu z...

Viele Aspekte der zellulären Mikroumgebung lokalen (zB Steifigkeit, Porengröße, Art der Proteine ​​oder Zell-Ligandendichte) eine regulatorische Hinweise, die zelluläre Prozesse wie Motilität, Zellproliferation, Differenzierung und Genexpression steuern koordinieren. Modifikationen der physikalisch-chemischen Eigenschaften der extrazellulären Umgebung durch Zellen wahrgenommen werden und zu unterschiedlichen physiologischen Auswirkungen, einschließlich Verformung der Zellpolarisation, Migration und Differenzierung. Es bleibt jedoch unklar, wie die Zellen zu übersetzen ECM Änderungen in zelluläre biochemische Signale. Es ist daher von großer Bedeutung, um in vitro Mikroumgebungen, die die Interaktionen zwischen Zellen und ihrer Mikroumgebung für die Untersuchung Mechanotransduktion Wege reproduzieren gesteuert Ingenieur. Um dieses Problem anzugehen, haben wir vor kurzem eine neuartige Methode 1, genannt Hydroxy-PAAm Hydrogele, auf einfache Weise zwei Centnsional weichen Matrizen, die unabhängig voneinander zu steuern wichtige Hinweise Mechanotransduktion erlauben: Matrix Steifigkeit, Zellgeometrie und Haft, Natur des Proteins und Zell-Ligandendichte. ECM lenkt zelluläre Prozesse über Gradienten in Morphogenen (Chemotaxis), Klebeproteine ​​(Haptotaxis) und Steifigkeit (durotaxis). Im Laufe der letzten Jahrzehnte, fortschrittliche In-vitro-Plattformen wurden entwickelt, um diese extrazelluläre Signale, um zu isolieren, um herauszuarbeiten, wie die Zellen in der Lage, biochemische und biophysikalische Eigenschaften in physiologische Prozesse 2-5 zu übersetzen. Elektronenstrahl 6, Photolithographie 7, photochemische Immobilisierung 8 oder plasmaunterstützte Techniken 9 entwickelt worden, um das Wachstum der lebenden Zellen auf mikro Substrate zu lenken. Obwohl diese Techniken haben wichtige Ergebnisse gebracht, die meisten von ihnen Diskriminierung zwischen den einzelnen Einfluss verschiedener Hinweise auf das Zellverhalten nicht zulassen,und sie erfordern technische Einrichtungen, die nur wenige Labors leisten können. Unter diesen Techniken, Mikrokontakt-Drucken (mCP), wurde als robust und leicht zugängliche Verfahren zur Zell-anhaftenden Mikroinseln 10 zu schaffen taucht. In jüngerer Zeit umfangreiche Bemühungen 11-14 wurden unternommen, um mCP auf Hydrogelen mit abstimmbaren Rigiditäten, um das breite Spektrum der in lebenden Geweben beobachtet Rigiditäten reproduzieren zu entwickeln. Unter diesen Werken, Polyacrylamid (PAAm) populär geworden 15 und ist schon jetzt einer der am häufigsten verwendeten Polymerbasis Matrizen für die Zell Biomechanik-Assays. PAAm Oberflächen sind häufig mit dem heterobifunktionellen Vernetzungsmittel N-Sulfosuccinimidyl-6-[4&#39;-Azido-2&#39;-nitrophenylamido] (Sulfo-SANPAH) und ECM-Proteine ​​auf der Oberfläche durch UV-Aktivierung des Sulfo-SANPAH nitrophenyl verbunden funktionalisierten Azid-Gruppen 16. Eine weitere Technik besteht in Kupplungs Hydrazin, Proteine, die stark oxidiert worden sindmit Perjodat 17. Hynd und Mitarbeiter wurde eine Technik zur Strukturierung biomimetische Oberflächen Hydrogel mit Protein und Peptide, die Photopolymerisation in Gegenwart eines acroyl-Streptavidin-Monomer 18 erfordert. In jüngerer Zeit, Tseng et al. Einen neuen Mikrostrukturierungsverfahren 19 basierend auf tiefen UV-Belichtung durch einen optischen PAAm Quarzmaske, die aktiviert PA-Gelen mit 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimidhydrochlorid inkubieren erfordert (EDC) berichtet und N-Hydroxysuccinimid (NHS) wässrigen Lösungen vor, die das Protein hinzuzufügen. Trotz der Fähigkeit dieser Techniken, um homogene und reproduzierbare Mikroproteine ​​zu erzeugen, die meisten von ihnen leiden wesentliche Einschränkungen: lange Syntheseverfahren (zum Beispiel Dialyse, Lyophilisierung usw.), teure chemische Verbindungen (zB Hyaluronsäure, Sulfo-SANPAH) oder tiefen UV- Bestrahlung. Hinzu kommt, dass diese Techniken nicht zulassen, dass unabhängige Modulation der Steifigkeit Substrat, MikroGeometrie, ECM-Protein der Natur, und Zell-Ligandendichte. Unter Berücksichtigung dieser Einschränkungen zu berücksichtigen, wurde ein neues und einfach auf Acrylamid basierenden Ansatz, der Immobilisierung einer Vielzahl von Proteinen und Biomolekülen auf weichen Hydrogele ermöglicht und erlaubt unabhängige Abstimmung der Mechanotransduktion Signale, um ihre Rolle bei Zellfunktionen entschlüsseln entwickelt. Statt der Behandlung PAAm Hydrogele mit aggressiven chemischen Verbindungen führen wir eine kommerzielle Acrylamid-Monomer mit Hydroxylgruppen während PAAm Polymerisation. Dieser einfache Vorgang überwindet den inneren Antihafteigenschaft PAAm Hydrogele ohne weitere technische Anforderungen. Die Gegenwart von Hydroxyl-Gruppen führt zu einer hohen Affinität Hydroxy-PAAm Hydrogele Proteine ​​und Biomoleküle, die Wasserstoffbrücken bilden. In Kombination mit mCP, Hydroxy-PAAm Hydrogele ermöglichen eine schnelle Erzeugung von zweidimensionalen Kulturplattform mit einem unabhängigen Steuerauf Matrix Steifigkeit, Art der ECM-Proteine, Zelldichte und-Liganden beschränkt Haft, die geplant, um eine leistungsstarke Plattform für die Untersuchung Mechanotransduktion sein werden. Der Zweck dieses Protokolls ist es, die notwendigen Informationen für leicht machen Hydroxy-PAAm Hydrogele ohne Know-how in den Materialwissenschaften liefern. Das ultimative Ziel ist es, ein Mittel bereitzustellen, für Forscher, die physiologisch relevanten Fragen an den zellulären und Gewebespiegel, die zu einem besseren Verständnis der Mechanotransduktion Wege in pathophysiologische Mechanismen führen kann fragen.

<table border="0" cellpadding="0" cellspacing="0" width="573">
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		<tr height="64">
			<td height="64" style="height:64px;width:216px;">UV/Ozone Photoreactor</td>
			<td style="width:71px;">Ultra-Violet Products</td>
			<td style="width:119px;">Model PR-100</td>
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		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Rocking plate</td>
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		<tr height="85">
			<td height="85" style="height:85px;width:216px;">Vacuum degassing chamber</td>
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			<td style="width:167px;"></td>
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		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Parafilm</td>
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			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Stainless steel forceps with fine tip</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z225304-1EA</td>
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		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Dressing tissue forceps</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">F4392-1EA</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Petri dishes in polystyrene</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">P5731-500EA</td>
			<td style="width:167px;"></td>
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		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Aluminium foil, thickness 0.5&#160;mm</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">266574-3.4G</td>
			<td style="width:167px;"></td>
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		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Isopore membrane filter (0,2 &#181;m pore size)</td>
			<td style="width:71px;">Millipore</td>
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			<td height="43" style="height:43px;width:216px;">Round glass coverslip (22 mm diameter)</td>
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		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Variable volume micropipette</td>
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			<td style="width:119px;">Z114820</td>
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		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Protein microcentrifuge tubes</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z666505-100EA</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Scalpel handles</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">S2896-1EA</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Scalpel blades</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">S2771-100EA</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Cell culture flasks (75 cm2)</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">CLS430641</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Ultrasonic bath tray, solid (stainless steel)</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z613983-1EA</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;"></td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Name of the Reagent</td>
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		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Polydimethylsiloxane&#160;</td>
			<td style="width:71px;">Dow Corning</td>
			<td style="width:119px;">Sylgard 184 silicone elastomer kit</td>
			<td style="width:167px;"></td>
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		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Acrylamide (powder)</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">A3553</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">N,N&#8217;-Methylenebis(acrylamide)</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">146072</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">N-Hydroxyethylacrylamide</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">697931</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">N,N,N&#8217;,N&#8217;-Tetramethylethylenediamine</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">T9281</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Amonium PerSulfate (APS)</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">A3678</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">3-(Trimetoxysilyl)propyle acrylate</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">1805</td>
			<td style="width:167px;"></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Human Plasma Fibronectin</td>
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		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Laminin from EHS</td>
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			<td style="width:119px;">L2020</td>
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		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Sodium hydroxyde</td>
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		<tr height="26">
			<td height="26" style="height:26px;width:216px;">Double-distilled water (ddH2O)</td>
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			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Human Umbilical Vein Endothelial Cells (HUVEC)</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">C-003-5C</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Accutase</td>
			<td style="width:71px;">PAA laboratories</td>
			<td style="width:119px;">L11-007</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">HEPES buffer solution 1M in H20</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">83264-500ML-F</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Antibiotics-antimycotics</td>
			<td style="width:71px;">PAA laboratories</td>
			<td style="width:119px;">P11-002</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Phosphate Buffer Saline solution</td>
			<td style="width:71px;">PAA laboratories</td>
			<td style="width:119px;">H15-002</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Alexa Fluor 488 Phaloidin</td>
			<td style="width:71px;">Molecular Probes</td>
			<td style="width:119px;">A12379</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-vinculin antibody produced in mouse</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">V9131</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Goat anti-mouse antibody-tetramethylrhodamine</td>
			<td style="width:71px;">Molecular Probes</td>
			<td style="width:119px;">T-2762</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-Fibronectin</td>
			<td rowspan="2" style="width:71px;">Sigma-Aldrich</td>
			<td rowspan="2" style="width:119px;">F3648</td>
			<td rowspan="2" style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">(rabbit)</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Streptavidin&#160;</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">41469</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-Laminin antibody</td>
			<td rowspan="2" style="width:71px;">Sigma-Aldrich</td>
			<td rowspan="2" style="width:119px;">L9393</td>
			<td rowspan="2" style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">(rabbit)</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-rabbit IgG-FITC</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">F7512</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Trypsin-EDTA solution</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">T3924-100ML</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Absolute ethanol</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">459844-2.5L</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

preparation of hydroxy-paam hydrogels for decoupling the effects of mechanotransduction cues

Es ist nun gut bekannt, daß viele zelluläre Funktionen durch Wechselwirkungen von Zellen mit physikalisch-chemischen und mechanischen Signale der extrazellulären Matrix (ECM)-Umgebung reguliert. Eukaryotischen Zellen spüren ständig ihre lokalen Mikroumgebung durch Oberflächen Mechanosensoren zu körperlichen Veränderungen von ECM in biochemische Signale transduzieren, und integrieren diese Signale an bestimmten Veränderungen in der Genexpression zu erzielen. Interessant ist, physikalisch-chemischen und mechanischen Parameter des ECM Paar miteinander, um das Schicksal der Zelle regulieren. Daher ist ein Schlüssel zum Verständnis Mechanotransduktion um den relativen Beitrag der ECM Hinweise auf zelluläre Funktionen zu entkoppeln. Hier präsentieren wir Ihnen ein detailliertes Versuchsprotokoll, um schnell und einfach erzeugen biologisch relevanten Hydrogele für die unabhängige Abstimmung der Mechanotransduktion Cues in vitro. Wir chemisch modifiziertes Polyacrylamid-Hydrogele (PAAm), um ihre nicht-eigen ADHES überwindenive Eigenschaften durch Einbau Hydroxyl-funktionalisierten Acrylamid-Monomeren während der Polymerisation. Wir erhalten eine neue PAAm Hydrogel genannt Hydroxy-PAAm, die Immobilisierung von jeder gewünschten Art von ECM-Proteinen erlaubt. Die Kombination von Hydroxy-PAAm Hydrogele mit Mikrokontaktdrucken ermöglicht, unabhängig die Morphologie der einzelnen Zellen, die Matrix Steifigkeit, die Art und die Dichte der extrazellulären Matrixproteinen. Wir bieten eine einfache und schnelle Methode, die in jedem Biologielabor eingestellt werden können, in-vitro-Zell Mechanotransduktion Prozesse zu untersuchen. Wir validieren diesem Roman zweidimensionale Plattform durch die Durchführung von Experimenten auf Endothelzellen, die eine mechanische Kopplung zwischen ECM Steifigkeit und den Zellkern zu demonstrieren.

Figur 1A zeigt die Co-Polymerisation von Acrylamid (AAM) und Bisacrylamid (bis-AAM) mit N-Hydroxyethylacrylamid (HEA) Monomeren, die eine primäre Hydroxylgruppe durch statistische radikalische Polymerisation mit eingelagerten Hydroxygruppen gebildet ein hydrophiles Netzwerk von Polyacrylamid (hydroxy-PAAm) . In diesem Protokoll muss ein Gewicht 65 mg HEA in einem Volumen von 1 ml HEPES verdünnt werden. Wissend, dass die Dichte der HEA ist ungefähr gleich eins ist, gehen wir davon aus, dass wir erhalt...

Watch this Scientific Journal Video about Preparation of Hydroxy-PAAm Hydrogels for Decoupling the Effects of Mechanotransduction Cues at JoVE.com

Preparation of Hydroxy-PAAm Hydrogels for Decoupling the Effects of Mechanotransduction Cues

Wir präsentieren ein neues Polyacrylamid-Hydrogel, genannt Hydroxy-PAAm, die mit minimalen Kosten oder Know-how eine direkte Bindung von ECM-Proteine ​​ermöglicht. Die Kombination von Hydroxy-PAAm Hydrogele mit Mikrokontaktdruck erleichtert eine unabhängige Steuerung der vielen Hinweise des natürlichen Zellmikroumgebung für die Untersuchung zellulärer mechanostransduction.

Herstellung von Hydroxy-PAAm Hydrogele zur Entkopplung der Auswirkungen von Mechanotransduction Cues

It is now well established that many cellular functions are regulated by interactions of cells with physicochemical and mechanical cues of their ...

preparation-hydroxy-paam-hydrogels-for-decoupling-effects

Research

JoVE Journal

Bioengineering

13.2K Aufrufe.  Université de Mons.  Wir präsentieren ein neues Polyacrylamid-Hydrogel, genannt Hydroxy-PAAm, die mit minimalen Kosten oder Know-how eine direkte Bindung von ECM-Proteine ​​ermöglicht. Die Kombination von Hydroxy-PAAm Hydrogele mit Mikrokontaktdruck erleichtert eine unabhängige Steuerung der vielen Hinweise des natürlichen Zellmikroumgebung für die Untersuchung zellulärer mechanostransduction. 

Serie: Preparation of Hydroxy-PAAm Hydrogels for Decoupling the Effects of Mechanotransduction Cues

Axon Stretch Growth: The Mechanotransduction of Neuronal Growth

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

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

Axon Stretch Growth: The Mechanotransduktion des neuronalen Wachstums

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

Forschung

Biotechnik

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

Alginat Hydrogele für Three-Dimensional Organ Culture der Eierstöcke und Eileitern

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the monitoring of therapeutic efficiency.1-3 The circulating low molecular weight proteome (LMWP) composed of small proteins shed from tissues and cells or peptide fragments derived from the proteolytic degradation of larger proteins, has been associated with the pathological condition in patients and likely reflects the state of disease.4,5 Despite these potential clinical applications, the use of Mass Spectrometry (MS) to profile the LMWP from biological fluids has proven to be very challenging due to the large dynamic range of protein and peptide concentrations in serum.6 Without sample pre-treatment, some of the more highly abundant proteins obscure the detection of low-abundance species in serum/plasma. Current proteomic-based approaches, such as two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE) and shotgun proteomics methods are labor-intensive, low throughput and offer limited suitability for clinical applications.7-9 Therefore, a more effective strategy is needed to isolate LMWP from blood and allow the high throughput screening of clinical samples. 
Here, we present a fast, efficient and reliable multi-fractionation system based on mesoporous silica chips to specifically target and enrich LMWP.10,11 Mesoporous silica (MPS) thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway. Using different polymer templates and polymer concentrations in the precursor solution, various pore size distributions, pore structures, connectivity and surface properties were determined and applied for selective recovery of low mass proteins. The selective parsing of the enriched peptides into different subclasses according to their physicochemical properties will enhance the efficiency of recovery and detection of low abundance species. In combination with mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin films and the specificity and efficacy of low mass proteome harvesting. The results presented herein reveal the potential of the nanotechnology-based technology to provide a powerful alternative to conventional methods for LMWP harvesting from complex biological fluids. Because of the ability to tune the material properties, the capability for low-cost production, the simplicity and rapidity of sample collection, and the greatly reduced sample requirements for analysis, this novel nanotechnology will substantially impact the field of proteomic biomarker research and clinical proteomic assessment.

We developed a technology based on mesoporous silica thin film for the selective recovery of low molecular weight proteins and peptides from human serum. The physico-chemical properties of our mesoporous chips were finely tuned to provide substantial control in peptide enrichment and consequently profile the serum proteome for diagnostic purposes.

Niedermolekulare Protein Enrichment auf mesoporösem Siliciumdioxid Dünnschichten für Biomarker Discovery

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the ...

The Synthesis of RGD-functionalized Hydrogels as a Tool for Therapeutic Applications

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and functionalize polymer chains with compounds that are able to improve biocompatibility, mechanical properties, or cell viability allows the design of novel materials to meet new challenges in the biomedical field. With the polymer functionalization strategies, click chemistry is a powerful tool to improve cell-compatibility and drug delivery properties of polymeric devices. Similarly, the fundamental need of biomedicine to use sterile tools to avoid potential adverse-side effects, such as toxicity or contamination of the biological environment, gives rise to increasing interest in the microwave-assisted strategy.
The combination of click chemistry and the microwave-assisted method is suitable to produce biocompatible hydrogels with desired functionalities and improved performances in biomedical applications. This work aims to synthesize RGD-functionalized hydrogels. RGD (arginylglycylaspartic acid) is a tripeptide that can mimic cell adhesion proteins and bind to cell-surface receptors, creating a hospitable microenvironment for cells within the 3D polymeric network of the hydrogels. RGD functionalization occurs through Huisgen 1,3-dipolar cycloaddition. Some PAA carboxyl groups are modified with an alkyne moiety, whereas RGD is functionalized with azido acid as the terminal residue of the peptide sequence. Finally, both products are used in a copper catalyzed click reaction to permanently link the peptide to PAA. This modified polymer is used with carbomer, agarose and polyethylene glycol (PEG) to synthesize a hydrogel matrix. The 3D structure is formed due to an esterification reaction involving carboxyl groups from PAA and carbomer and hydroxyl groups from agarose and PEG through microwave-assisted polycondensation. The efficiency of the gelation mechanism ensures a high degree of RGD functionalization. In addition, the procedure to load therapeutic compounds or biological tools within this functionalized network is very simple and reproducible.

We present a protocol for the synthesis of RGD-functionalized hydrogels as devices for cell and drug delivery. The procedure involves copper catalyzed alkyne-azide cycloaddition (CuAAC) between alkyne-modified polyacrylic acid (PAA) and a RGD-azide derivative. The hydrogels are formed using microwave-assisted polycondensation and their physicochemical properties are investigated.

Die Synthese von RGD-funktionalisierten Hydrogele als Werkzeug für therapeutische Anwendungen

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

Licht-vermittelte Bildung und Musterung von Hydrogelen für Zellkulturanwendungen

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Synthesis of Hydrogels with Antifouling Properties As Membranes for Water Purification

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and thus achieving stable water permeability through membranes over time. Here, we report a facile method to prepare hydrogels based on zwitterions for membrane applications. Freestanding films can be prepared from sulfobetaine methacrylate (SBMA) with a crosslinker of poly(ethylene glycol) diacrylate (PEGDA) via photopolymerization. The hydrogels can also be prepared by impregnation into hydrophobic porous supports to enhance the mechanical strength. These films can be characterized by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) to determine the degree of conversion of the (meth)acrylate groups, using goniometers for hydrophilicity and differential scanning calorimetry (DSC) for polymer chain dynamics. We also report protocols to determine the water permeability in dead-end filtration systems and the effect of foulants (bovine serum albumin, BSA) on membrane performance.

This paper reports practical methods to prepare hydrogels in freestanding films and impregnated membranes and to characterize their physical properties, including water transport properties.

Synthese von Hydrogelen mit Antifouling-Eigenschaften als Membranen für die Wasserreinigung

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and ...

Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue culture systems lack a three-dimensional (3D) matrix, resulting in a significant disconnect between results collected in vitro and in vivo. To address this limitation, researchers have engineered 3D hydrogel tissue culture platforms that can mimic the biochemical and biophysical properties of the in vivo cell microenvironment. This research has motivated the need to develop material platforms that support 3D cell encapsulation and downstream biochemical assays. Recombinant protein engineering offers a unique toolset for 3D hydrogel material design and development by allowing for the specific control of protein sequence and therefore, by extension, the potential mechanical and biochemical properties of the resultant matrix. Here, we present a protocol for the expression of recombinantly-derived elastin-like protein (ELP), which can be used to form hydrogels with independently tunable mechanical properties and cell-adhesive ligand concentration. We further present a methodology for cell encapsulation within ELP hydrogels and subsequent immunofluorescent staining of embedded cells for downstream analysis and quantification.

Recombinant protein-engineered hydrogels are advantageous for 3D cell culture as they allow for complete tunability of the polymer backbone and therefore, the cell microenvironment. Here, we describe the process of recombinant elastin-like protein purification and its application in 3D hydrogel cell encapsulation.

Produktion von Elastin-Like Protein Hydrogele für Kapselung und Immunostaining Zellen in 3D

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue ...

Measuring Global Cellular Matrix Metalloproteinase and Metabolic Activity in 3D Hydrogels

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) culture systems. However, measurement of cell function in 3D culture is often more challenging. Many biological assays require retrieval of cellular material which can be difficult in 3D cultures. One way to address this challenge is to develop new materials that enable measurement of cell function within the material. Here, a method is presented for measurement of cellular matrix metalloproteinase (MMP) activity in 3D hydrogels in a 96-well format. In this system, a poly(ethylene glycol) (PEG) hydrogel is functionalized with a fluorogenic MMP cleavable sensor. Cellular MMP activity is proportional to fluorescence intensity and can be measured with a standard microplate reader. Miniaturization of this assay to a 96-well format reduced the time required for experimental set up by 50% and reagent usage by 80% per condition as compared to the previous 24-well version of the assay. This assay is also compatible with other measurements of cellular function. For example, a metabolic activity assay is demonstrated here, which can be conducted simultaneously with MMP activity measurements within the same hydrogel. The assay is demonstrated with human melanoma cells encapsulated across a range of cell seeding densities to determine the appropriate encapsulation density for the working range of the assay. After 24 h of cell encapsulation, MMP and metabolic activity readouts were proportional to cell seeding density. While the assay is demonstrated here with one fluorogenic degradable substrate, the assay and methodology could be adapted for a wide variety of hydrogel systems and other fluorescent sensors. Such an assay provides a practical, efficient and easily accessible 3D culturing platform for a wide variety of applications.

Here, a protocol is presented for encapsulating and culturing cells in poly(ethylene glycol) (PEG) hydrogels functionalized with a fluorogenic matrix metalloproteinase (MMP)-degradable peptide. Cellular MMP and metabolic activity are measured directly from the hydrogel cultures using a standard microplate reader.

Messung der globalen zellulären Matrix Metalloproteinase und metabolische Aktivität in 3D Hydrogele

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) ...

A Lab-On-A-Chip Platform for Stimulating Osteocyte Mechanotransduction and Analyzing Functional Outcomes of Bone Remodeling

Bone remodeling is a tightly regulated process that is required for skeletal growth and repair as well as adapting to changes in the mechanical environment. During this process, mechanosensitive osteocytes regulate the opposing responses between the catabolic osteoclasts and anabolic osteoblasts. To better understand the highly intricate signaling pathways that regulate this process, our lab has developed a foundationary lab-on-a-chip (LOC) platform for analyzing functional outcomes (formation and resorption) of bone remodeling within a small scale system. As bone remodeling is a lengthy process that occurs on the order of weeks to months, we developed long-term cell culturing protocols within the system. Osteoblasts and osteoclasts were grown on functional activity substrates within the LOC and maintained for up to seven weeks. Afterward, chips were disassembled to allow for the quantification of bone formation and resorption. Additionally, we have designed a 3D printed mechanical loading device that pairs with the LOC platform and can be used to induce osteocyte mechanotransduction by deforming the cellular matrix. We have optimized cell culturing protocols for osteocytes, osteoblasts, and osteoclasts within the LOC platform and have addressed concerns of sterility and cytotoxicity. Here, we present the protocols for fabricating and sterilizing the LOC, seeding cells on functional substrates, inducing mechanical load, and disassembling the LOC to quantify endpoint results. We believe that these techniques lay the groundwork for developing a true organ-on-a-chip for bone remodeling.

Here, we present protocols for analyzing bone remodeling within a lab-on-a-chip platform. A 3D printed mechanical loading device can be paired with the platform to induce osteocyte mechanostransduction by deforming the cellular matrix. The platform can also be used to quantify bone remodeling functional outcomes from osteoclasts and osteoblasts (resorption/formation).

Eine Lab-On-A-Chip-Plattform zur Stimulierung der Osteozyten-Mechanotransduktion und zur Analyse der funktionellen Ergebnisse des Knochenumbaus

Bone remodeling is a tightly regulated process that is required for skeletal growth and repair as well as adapting to changes in the mechanical ...

Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

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

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

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

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