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 Attivazione della superficie di coprioggetto di vetro <ol><li> Luogo coprioggetto di vetro circolare (diametro 25 mm) in una capsula di Petri e striscio 0,1 M di NaOH soluzione su di esso per 5 min (cappa consigliata). </li><li> Rimuovere la soluzione di NaOH e immergere completamente coprioggetto con DDH 2 O sterile per 20 minuti mentre delicatamente dondolo su un piatto oscillante in una cappa sterile cultura. </li><li> Scolare sterile DDH 2 O e ripetere il passaggio 1.2. </li><li> Togliere i coprioggetti con una pinzetta sterile e metterli in un nuovo piatto di Petri con la faccia attivata alto. </li><li> Coprioggetti secco sotto un flusso costante di gas azoto ad alta purezza. </li><li> In una cappa sterile cultura, spalmare uno strato sottile di 3-acrilato (trimetossisilil) propil (92%) sul lato attivo del vetrino per 1 ora. </li><li> Ampiamente lavare vetrini con 3 lavaggi di DDH 2 O sterile e immergerli in DDH 2 O sterile in un nuovo piatto di Petri. </li><li> Toccare la piastra di Petricon Parafilm e metterlo su un piatto rocker sotto agitazione per 10 min. </li><li> Togliere i coprioggetti dai DDH 2 O con una pinzetta sterile con punte sottili e metterli in un nuovo piatto di Petri con la faccia attivata alto. </li><li> Conservare a temperatura ambiente in un luogo asciutto con un foglio di alluminio per evitare che la polvere di attaccarsi i coprioggetti. </li></ol> 2 Preparazione di idrossi-PAAM idrogel <ol><li> Preparare un peso di 65 mg di N-idrossietil acrilammide (HEA) in una provetta Eppendorf da 1,5 ml. È importante preparare una soluzione HEA fresca. </li><li> Aggiungere 1 ml di tampone HEPES 50 mM a HEA e mescolare utilizzando un vortex fino a quando la dissoluzione HEA completo. </li><li> Aggiungere 400 ml di 40% w / w in HEPES soluzione di acrilammide e il volume del 2% richiesto w / w in HEPES soluzione di bis-acrilammide (vedi Tabella 1) per raggiungere la rigidità idrogel desiderata. Regolare con HEPES 50 mM ad un volume finale di 5 ml. </li><li> Mescolare la soluzione con un vortex e Degas itin una camera a vuoto per 20 minuti al fine di ridurre la concentrazione di ossigeno all&#39;interno della soluzione, che impedisce idrossi-Paam polimerizzazione. </li><li> Sotto una cappa sterile, filtrare la soluzione degased con un filtro di dimensione dei pori di 0,2 micron per sterilizzarlo. </li><li> Attivare vetrini circolari (diametro 22 mm) in un pulitore UV / ozono durante 7 min. </li><li> Preparare 100 ml di soluzione al 10% di ammonio persolfato (APS), che è di 10 APS mg su 100 ml DDH 2 O. È importante preparare una soluzione APS fresca. </li><li> Aggiungere 2,5 ml di tetramethylenediamine (TEMED) e 25 ml di soluzione di APS al idrossi-PAAM soluzione sterilizzata (punto 2.5) per avviare la polimerizzazione. Miscelare la soluzione da 3 pipettaggi successive senza introduzione di bolle, in condizioni di sterilità. </li><li> Sotto una cappa sterile, una goccia 25 ml della soluzione di idrossi-PAAM su un coprioggetto 25 mm (disponibile dal punto 1.9) e subito mettere un gl 22 millimetriass coprioggetto (preparato nel passaggio 2.5) sulla parte superiore della goccia per spremere la soluzione idrossi-PAAM. Centrare il coprioggetto di vetro 22 millimetri con una pinzetta sterile e appianare eventuali bolle. </li><li> Consenti idrogel-idrossi PAAM a polimerizzare a temperatura ambiente per 15 min. Invertire manualmente la restante soluzione idrossi-Paam nel tubo Eppendorf a seguire il completamento del processo di polimerizzazione. </li><li> Immergere completamente coprioggetto con sterile DDH 2 O e separare con attenzione le 22 coprioggetto mm di vetro, introducendo il bordo di una lama di rasoio tra i 22 coprioggetto mm di vetro e lo strato di idrogel idrossi-PAAM. </li><li> Lavare idrogel-idrossi PAAM con PBS sterile (3 scambi di PBS), e lasciare che i gel completamente immersi in PBS sterile per mantenere l&#39;idratazione. </li><li> Conservare idrogel-idrossi PAAM in PBS sterile a 4 ° C per un massimo di 3 giorni. </li></ol> 3. polidimetilsilossano (PDMS) Microstamp Fabrication NOTA: La realizzazione di un maestro di silicio è necessario prima di staRT Il PDMS fabbricazione microstamp. Questo microfabbricazione di un maestro di silicio può essere fatto con tecniche litografiche, che richiede attrezzature e formazione specializzata. Collaborazioni con un impianto di nanofabbricazione sono incoraggiati a realizzare il master di silicio. In alternativa, contattare una società che fabbrica i padroni di silicio microstrutturato personalizzati su richiesta. È importante notare che la realizzazione del master silicio deve solo essere fatto una volta. Infatti, maestri di silicio microstrutturate possono essere utilizzati a tempo indefinito per produrre francobolli elastomerici. <ol><li> Mix PDMS e il catalizzatore in un rapporto di 10: 1 in un bicchiere di plastica e mescolare accuratamente con una pipetta per 10 min. </li><li> Degassare la miscela PDMS sotto vuoto per rimuovere le bolle d&#39;aria che si sono formate durante la fase 3.1. </li><li> Posizionare il maestro silicio microstrutturata in una capsula di Petri e gettato uno strato di spessore di 10 mm di miscela degasato PDMS su di essa senza formare bolle. </li><li> Lasciate curare le PDMS per 2 ore a 60 ° C in unforno. </li><li> In un ambiente privo di polvere, peel-off lo strato PDMS e accise 1 cm 2 microstamps con un bisturi. </li><li> Utilizzando pinze, posto PDMS microstamps modello-up in una capsula di Petri. </li></ol> 4. micropatterning idrossi-PAAM idrogel <ol><li> Luogo PDMS microstamps in un etanolo / acqua e la soluzione sonicare (50/50) per 15 min. </li><li> Asciugare i francobolli con un flusso di flusso di azoto e di metterli modello-up in un pulitore UV / ozono (λ &lt;200 nm) per 7 min. </li><li> Sotto una cappa sterile, mettere una goccia di 150 ml di una soluzione di proteina desiderata (ad esempio, 100 mg / ml in PBS laminina o 25 mg / ml di fibronectina in PBS) sulla superficie microstrutturata di un 1 cm 2 PDMS bollo. </li><li> Opzionale: Modificare la concentrazione della soluzione proteica di modulare la densità cellulare-ligando. </li><li> Stendere la soluzione proteica sulla superficie timbro muovendo con una punta sterile di una pipetta verso l&#39;angolo diil timbro. </li><li> Lasciare la soluzione proteica per assorbire il timbro PDMS per 60 minuti sotto una cappa sterile. Spegnere le lampade per evitare danni proteine. </li><li> Sotto una cappa sterile, trasferire vetrini rivestiti idrossi-PAAM (disponibili dal punto 2.13) in una capsula di Petri. </li><li> Rimuovere l&#39;eccesso di PBS dalla superficie dei substrati idrossi-Paam con una bassa corrente di azoto in condizioni sterili. Interrompere la procedura appena alcuna prova di acqua in piedi sulla superficie del gel si osserva. Il gel non deve essere asciugata bene in questa fase. </li><li> Asciugare accuratamente la superficie strutturata di PDMS bollo con un flusso costante di gas azoto ad alta purezza. </li><li> Afferrare il timbro proteina rivestite con una pinza tessuto medicazione e posizionare la superficie strutturata in contatto con la superficie idrogel essiccato. Applicare punti di pressione brevi con la punta delle pinzette sulla parte superiore del timbro PDMS per assicurare un buon contatto tra microfeatures bollo e la superficie idrogel. </li><li> Lasciare il timbro PDMS su the superficie idrogel per 1 ora a temperatura ambiente. </li><li> Rimuovere delicatamente PDMS francobolli da idrogel idrossi-PAAM con una pinza di tessuto medicazione e seguire il passo 4.1 per pulire il timbro. </li><li> Lavare ampiamente idrogel-PAAM idrossi timbrato da 3 scambi di PBS (pH = 7,4) in condizioni di sterilità per 10 minuti per ogni cambio. </li><li> Optional: Ulteriori microdisegni di altre proteine ​​ECM possono essere aggiunti al-PAAM idrossi superficie seguendo i passaggi 4,5-4,11. </li><li> Zone non stampate passivare con una soluzione sterile di BSA a 5 mg / ml in PBS durante una notte a 4 ° C sotto agitazione delicata su una piastra oscillante. </li><li> Lavare estesamente da 3 scambi di PBS (pH = 7,4) in condizioni di sterilità per 10 minuti per ogni cambio. In questa fase, stampati idrogel-PAAM idrossi possono essere conservati a 4 ° C fino ad una settimana. </li></ol> 5. Deposizione cellulare su micropatterned idrossi-PAAM idrogel <ol><li> Incubare coprioggetto in mezzi cella per 30-45 minuti prima di placcatura cells. </li><li> Lavare le cellule aderenti coltivate in un pallone di 75 centimetri 2 coltura con PBS sterile a 37 ° C e staccarlo con 3 ml di tripsina-EDTA o accutase per 10 min. </li><li> Trasferire la quantità desiderata di pre-riscaldato completo terreno di coltura appropriato per la linea cellulare nel pallone contenente le cellule staccate e centrifugare la sospensione cellulare per 3 min a 650 x g. </li><li> Rimuovere il surnatante con una micropipetta e risospendere le cellule in terreno di coltura completo a 15-20.000 cellule / ml. </li><li> Aggiungere 4 ml della soluzione di cellule di un coprioggetto micropatterned (ottenuto dal punto 5.1) e posizionare la cella coprioggetto coperto in un incubatore a 37 ° C e 5% di umidità per 1-2 ore. </li><li> Aspirare delicatamente le cellule senza legami e sostituire il terreno di coltura. Riportare le cellule attaccate alla incubatore e farli diffondono completamente (3-6 ore, a seconda del tipo di cellula). </li></ol>

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No conflicts of interest declared.

Molte osservazioni in vitro nella moderna biologia cellulare sono state eseguite su vetrini rigidi, spesso rivestiti con un sottile strato di proteine ​​ECM o peptidi sintetici contenenti la sequenza RGD. Tuttavia, tali substrati di coltura di base non ricapitolano l&#39;intera complessità fisico-chimiche della ECM e quindi non forniscono un modello accurato per lo studio dei processi meccanotrasduzione cellulari. Per affrontare questo problema, vi proponiamo una semplice alternativa a funzionalizzare idro...

Molti aspetti del microambiente cellulare locale (ad esempio, la rigidità, dimensione dei pori, la natura delle proteine, o di densità delle cellule-ligando) forniscono una serie coordinata di spunti normativi che controllano processi cellulari come la motilità, proliferazione cellulare, differenziamento, e l&#39;espressione genica. Le modifiche delle proprietà fisico-chimiche dell&#39;ambiente extracellulare possono essere percepiti dalle cellule e causare diverse conseguenze fisiologiche, tra cui la deformazione della polarizzazione cellulare, la migrazione e la differenziazione. Non è chiaro, tuttavia, come le cellule traducono ECM modifiche in segnali biochimici cellulari. E &#39;quindi di grande importanza per Ingegnere controllata in microambienti in vitro in grado di riprodurre le interazioni tra cellule e il loro microambiente per studiare percorsi meccanotrasduzione. Per risolvere questo problema, abbiamo recentemente introdotto un nuovo metodo 1, chiamato idrogel-PAAM idrossi, di generare facilmente due dimematrici nsional morbidi che permettono di controllare in modo indipendente importanti spunti meccanotrasduzione: rigidezza, matrice di geometria cellulare e confinamento, la natura delle proteine ​​e densità cellulare-ligando. ECM dirige processi cellulari attraverso gradienti in morphogens (chemiotassi), proteine ​​adesive (haptotaxis) e rigidità (durotaxis). Nel corso degli ultimi decenni, avanzata in piattaforme vitro sono stati sviluppati per isolare questi segnali extracellulari, al fine di prendere in giro fuori come le cellule sono in grado di tradurre le caratteristiche biochimiche e biofisiche in processi fisiologici 2-5. Sono stati sviluppati a fascio elettronico 6, fotolitografia 7, 8 immobilizzazione fotochimico o tecniche plasma assistita 9 per dirigere la crescita delle cellule viventi su substrati micropatterned. Anche se queste tecniche hanno dato risultati importanti, la maggior parte di essi non consentono una discriminazione tra l&#39;influenza individuale di diversi spunti sul comportamento delle cellulee richiedono attrezzature tecniche che pochi laboratori possono permettersi. Tra queste tecniche, stampa microcontact (μCP), è emerso come un metodo affidabile e accessibile per creare cellule-adesivo micro-isole 10. Più di recente, grandi sforzi 11-14 sono stati fatti per sviluppare μCP su idrogel con rigidità accordabili in modo da riprodurre la vasta gamma di rigidità osservati nei tessuti viventi. Tra queste opere, poliacrilammide (PAAM) è diventata popolare 15 ed è già una delle matrici a base di polimeri più comunemente usati per la biomeccanica cellulari saggi. Superfici Paam sono comunemente funzionalizzati con il eterobifunzionale reticolante N-sulfosuccinimidyl-6-proteine ​​[4&#39;-azido-2&#39;-nitrophenylamido] (solfo-SANPAH) e ECM sono legati alla superficie mediante attivazione UV del nitrofenil solfo-SANPAH gruppi azide 16. Un&#39;altra tecnica consiste nel idrazina accoppiamento a proteine ​​che sono stati gravemente ossidaticon periodato 17. Hynd e collaboratori hanno introdotto una tecnica per patterning di superfici idrogel biomimetici con proteine ​​e peptidi che richiede fotopolimerizzazione in presenza di un monomero acroyl-streptavidina 18. Più recentemente, Tseng et al. Hanno segnalato un nuovo metodo micropatterning 19 sulla base dell&#39;esposizione ai raggi UV profondo di PAAM attraverso una maschera di quarzo ottico che richiede di incubare gel PA attivati ​​con 1-etil-3-[3-dimetilamminopropil] carbodiimide cloridrato (EDC) e N-idrossisuccinimmide (NHS) soluzioni di acqua prima di aggiungere la proteina. Nonostante la capacità di queste tecniche per creare omogenei e riproducibili proteine ​​microdisegni, la maggior parte di loro soffrono maggiori limitazioni: processi lunghi di sintesi (ad esempio, dialisi, liofilizzazione, ecc), composti chimici costosi (ad esempio, l&#39;acido ialuronico, solfo-SANPAH) o UV profondo irradiazione. Inoltre, queste tecniche non consentono la modulazione indipendente substrato rigidità, micropatterngeometria, ECM natura proteica, e la densità delle cellule-ligando. Prendendo queste limitazioni in considerazione, abbiamo sviluppato un nuovo approccio e semplice a base di acrilammide, che consente l&#39;immobilizzazione di una varietà di proteine ​​e biomolecole su idrogel morbido e permette la sintonizzazione indipendente di spunti meccanotrasduzione al fine di decifrare il loro ruolo sulle funzioni cellulari. Invece di trattare idrogel PAAM con composti chimici aggressivi, introduciamo un monomero acrilammide commerciale con gruppi idrossilici PAAM durante la polimerizzazione. Questa semplice operazione supera la proprietà anti-adesive intrinseca degli idrogel PAAM senza altri requisiti tecnici. La presenza di gruppi ossidrilici porta ad una elevata affinità di idrogel-idrossi Paam per proteine ​​e biomolecole che formano interazioni di idrogeno-bonding. In combinazione con μCP, idrogel-PAAM idrossi consentono una rapida generazione di piattaforma cultura bidimensionale con un controllo indipendentesulla rigidità matrice, tipo di proteine ​​ECM, densità cellulare-ligando e adesività limitata, che si immaginava di essere una potente piattaforma per lo studio meccanotrasduzione. Lo scopo di questo protocollo è quello di fornire le informazioni necessarie per rendere facilmente idrogel-PAAM idrossi senza alcuna competenza in scienze dei materiali. L&#39;obiettivo finale è quello di fornire un mezzo per i ricercatori di porre domande fisiologicamente rilevanti a livello cellulare e dei tessuti che possono portare ad una migliore comprensione delle vie meccanotrasduzione coinvolti nei meccanismi fisiopatologici.

<table border="0" cellpadding="0" cellspacing="0" width="573">
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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<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>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Rocking plate</td>
			<td style="width:71px;">IKAcWerke</td>
			<td style="width:119px;">Model KS 130 Basic</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Vortexer</td>
			<td style="width:71px;">Scientific Industries</td>
			<td style="width:119px;">Model Vortex Genie2</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:216px;">Vacuum degassing chamber</td>
			<td style="width:71px;">Applied Vacuum Engineering</td>
			<td style="width:119px;">DP- 8-KIT</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Parafilm</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">P7793-1EA</td>
			<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>
			<td style="width:167px;"></td>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
			<td style="width:119px;">GTTP Filter code</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Round glass coverslip (22 mm diameter)</td>
			<td style="width:71px;">Neuvitro</td>
			<td style="width:119px;">GG-22</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Round glass coverslip (25 mm diameter)</td>
			<td style="width:71px;">Neuvitro</td>
			<td style="width:119px;">GG-25</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Variable volume micropipette</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z114820</td>
			<td style="width:167px;"></td>
		</tr>
		<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>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Name of the Reagent</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalogue Number</td>
			<td style="width:167px;">Comments</td>
		</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>
		</tr>
		<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>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Human Plasma Fibronectin</td>
			<td style="width:71px;">Millipore</td>
			<td style="width:119px;">FC010</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Laminin from EHS</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">L2020</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Sodium hydroxyde</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">221465-25G</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:216px;">Double-distilled water (ddH2O)</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Endothelial cell growth medium</td>
			<td style="width:71px;">Cells Applications</td>
			<td style="width:119px;">211K-500</td>
			<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

E &#39;ormai ben noto che molte funzioni cellulari sono regolate da interazioni delle cellule con spunti fisico-chimiche e meccaniche della loro matrice extracellulare (ECM) ambiente. Le cellule eucariotiche sentono costantemente il loro microambiente locale attraverso meccanosensori superficie di trasdurre i cambiamenti fisici di ECM in segnali biochimici, e integrare questi segnali per raggiungere specifici cambiamenti nell&#39;espressione genica. È interessante notare che i parametri fisico-chimiche e meccaniche della coppia ECM lattina con l&#39;altro per regolare destino cellulare. Pertanto, una chiave per comprendere meccanotrasduzione è quello di disaccoppiare il contributo relativo di spunti ECM sulle funzioni cellulari. Qui vi presentiamo un protocollo dettagliato sperimentale per generare rapidamente e facilmente idrogel biologicamente rilevanti per la messa a punto indipendente di spunti meccanotrasduzione in vitro. Noi idrogel di poliacrilamide modificati chimicamente (PAAM) per superare i loro intrinsecamente non ADHESive proprietà incorporando monomeri di acrilammide idrossile-funzionalizzati durante la polimerizzazione. Abbiamo ottenuto un romanzo PAAM idrogel, chiamato idrossi-PAAM, che consente l&#39;immobilizzazione di qualsiasi natura desiderato di proteine ​​ECM. La combinazione di idrossi-Paam Idrogel con stampa microcontact permette di controllare indipendentemente la morfologia delle cellule singole, la rigidità della matrice, la natura e la densità delle proteine ​​ECM. Mettiamo a disposizione un metodo semplice e rapido che può essere impostato in ogni laboratorio di biologia per studiare nei processi meccanotrasduzione cellule in vitro. Abbiamo convalidare questo romanzo piattaforma bidimensionale conducendo esperimenti sulle cellule endoteliali che dimostrano un accoppiamento meccanico tra ECM rigidità e il nucleo.

Figura 1A presenta la co-polimerizzazione di acrilammide (AAm) e bisacrylamide (bis-AAm) con N-hydroxyethylacrylamide (HEA) monomeri contenenti un ossidrile primario formata da casuale polimerizzazione radicalica una rete idrofila di poliacrilammide con gruppi idrossilici incorporati (idrossi-Paam) . In questo protocollo, un peso di 65 mg di HEA deve essere diluita in un volume di 1 ml di HEPES. Sapendo che la densità di HEA è approssimativamente uguale a uno, assumiamo che si ottiene un volume di lav...

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

Vi presentiamo un nuovo idrogel di poliacrilamide, chiamato idrossi-PAAM, che permette un legame diretto di proteine ​​ECM con un costo minimo o esperienza. La combinazione di idrossi-PAAM Idrogel con stampa microcontact facilita il controllo indipendente di molti spunti del microambiente naturale delle cellule per studiare mechanostransduction cellulare.

Preparazione di idrossi-PAAM idrogel per dissociare gli effetti di meccanotrasduzione 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 Views.  Université de Mons.  Vi presentiamo un nuovo idrogel di poliacrilamide, chiamato idrossi-PAAM, che permette un legame diretto di proteine ​​ECM con un costo minimo o esperienza. La combinazione di idrossi-PAAM Idrogel con stampa microcontact facilita il controllo indipendente di molti spunti del microambiente naturale delle cellule per studiare mechanostransduction cellulare. 

Video: 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 crescita Stretch: Il meccanotrasduzione di crescita neuronale

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

Ricerca

Bioingegneria

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.

Idrogeli di alginato per tridimensionale Cultura Organo di ovaie e tube

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.

Basso Peso Molecolare proteina di arricchimento sul film sottili di silice mesoporosi per la scoperta di biomarcatori

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.

La sintesi di RGD-funzionalizzati idrogeli come strumento per applicazioni terapeutiche

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.

Formazione Light-mediata ed il modello di idrogel per colture cellulari

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.

Sintesi di idrogel con proprietà antivegetative come membrane per la purificazione dell&#39;acqua

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.

Produzione di elastina-come proteina idrogeli per incapsulamento e Immunostaining delle cellule 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.

Misurazione della proteinasi metallica della matrice cellulare globale e l'attività metabolica in idrogel 3D

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).

Una piattaforma Lab-On-A-Chip per stimolare la Meccanotrazione osteocite e analizzare i risultati funzionali della rimodellamento osseo

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.

Ceppo controllato di idrogel 3D in imaging a microscopia dal vivo

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