Questo lavoro è stato sostenuto dai programmi Delaware Cobre in Drug Discovery e in Biomateriali avanzata finanziati dallo sviluppo Premi Istituzionali presso l&#39;Istituto Nazionale di generali scienze mediche presso il National Institutes of Health (P20GM104316 e P30 GM110758-01, rispettivamente), la Pew Charitable Trusts (00026178), un Science Foundation Premio nazionale Carriera (DMR-1.253.906), il Fondo di Burroughs Wellcome (1.006.787), e il programma National Science Foundation IGERT SBE2 presso l&#39;Università di Delaware (borsa di studio di L. Sawicki). Gli autori ringraziano il Centro Bioimmagini del Delaware Biotechnology Institute presso l&#39;Università di Delaware per la formazione e l&#39;accesso alla microscopia confocale, la signora Katherine Wiley per l&#39;assistenza durante le riprese video, il signor Matthew Rehmann per fornire generosamente hMSCs isolate dal midollo osseo, il Prof. Christopher J . Kloxin e il signor Stephen Ma per fornire generosamente fotomaschere, e il Prof. Wilfred Chen per l&#39;utilizzo del lettore di piastre automatizzato. </p&gt;

<ol>
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N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thiol-ene+click+chemistry.">Thiol-ene click chemistry.</a> Angew Chemie - Int Ed. 49 (9), 1540-1573 (2010).</li><li>Fan, Y., Deng, C., Cheng, R., Meng, F., Zhong, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+forming+hydrogels+via+catalyst-free+and+bioorthogonal+&amp;#34;tetrazole-Alkene&amp;#34;+photo-click+chemistry.">In situ forming hydrogels via catalyst-free and bioorthogonal &amp;#34;tetrazole-Alkene&amp;#34; photo-click chemistry.</a> Biomacromolecules. 14 (8), 2814-2821 (2013).</li><li>Burdick, J. A., Murphy, W. 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The authors have nothing to disclose.

La procedura qui presentata illustra le tecniche per photoencapsulate cellule all&#39;interno idrogel formate da tiolo-ene clic chimica e successivamente photopattern i gel con segnali biochimici. L&#39;uso della luce per formare inizialmente idrogel permette una miscelazione omogenea sospensione delle cellule all&#39;interno della soluzione di polimero prima della polimerizzazione. polimerizzazione &#39;blocca&#39; Rapid gel nella forma dello stampo definita e incapsula cellule all&#39;interno della rete idrogel sospesi. I gel possono anche essere stampati in numerose forme diverse (ad esempio, vetrini o siringhe) a seconda dell&#39;applicazione finale desiderato. Ad esempio, cellule incapsulate cultura 3D in idrogel allegati al vetrini sono particolarmente utili per applicazioni di imaging come attenuazione della luce è limitata all&#39;interno di un campione sottile. stampi siringa può essere utilizzato per una rapida incapsulamento delle cellule, consentendo un maggior numero di campioni da preparati in breve tempo (rispetto al vetrinos) che può essere utilizzato per esperimenti che richiedono un gran numero di cellule, come la citometria a flusso o qPCR. Successivamente, questi gel quindi possono essere modellati con segnali biochimici per sollecitare risposte cellulari desiderati quali la differenziazione o l&#39;invasione. 30,31 Saggi di redditività e attività metabolica indicano la sopravvivenza delle cellule del sistema materiale e condizioni patterning presentati. Si noti che l&#39;attività metabolica è stata monitorata fino al giorno 3 in gel non degradabili (RGKGRK peptide reticolazione) per valutare gli effetti iniziali di condizioni di polimerizzazione e patterning sulla funzione cellulare. Inoltre, un test di integrità della membrana (Live / Dead vitalità / citotossicità colorazione) di hMSCs a 1 e 6 giorni post-incapsulamento in gel formulati con un peptide reticolazione degradabile (GPQGIWGQ) supporta che le cellule rimangono vitali e diffondere a 1 settimana in cultura. La vitalità di linee cellulari aggiuntive stato segnalato per le condizioni di photopatterning 32 simili a quelleusato qui e può essere valutato per il sistema idrogel descritto utilizzando vivo colorazione / morti e saggi di attività metabolica. Anche se non abbiamo osservato problemi con la vitalità delle cellule di utilizzare questo sistema di materiali e relative procedure (4.2 e 4.3), alcuni tipi di cellule possono essere sensibili a radicali liberi e / o esposizione alla luce. In questo caso, gli utenti potrebbero considerare l&#39;utilizzo di sistemi di materiali non photoinitiated come l&#39;azoturo-alchino, 12 FXIII, 31 o chimiche di formazione idrogel Diels Alder-based. 33 Facile tecniche per rilevare e quantificare patterning di segnali biochimici all&#39;interno di gel sono anche presentati (3.1 e 3.2). Dosaggio di Ellman è di particolare interesse perché i reagenti sono commercialmente disponibili e non sono richieste fasi di lavorazione sintetica extra o reagenti più costosi (per esempio, peptide fluorescenza marcata). dosaggio di Ellman può essere utilizzato per determinare con precisione la modifica di tioli liberi con segnali biochimici sotto different photopatterning condizioni, nonché di visualizzare rapidamente modelli. Per quantificare incorporazione peptide, la concentrazione gruppo funzionale tiolo prima e dopo patterning, come misura quantitativa di peptide incorporazione, è direttamente valutata con test di Ellman. Mentre questo tipo di quantificazione può essere fatto con peptidi fluorescenza marcata, 34 quantificazione di imaging basata richiede più fasi di manipolazione e analisi in termini di tempo (ad esempio, la sintesi di un peptide fluorescenza marcata e la generazione di una curva di calibrazione per correlare fluorescenza concentrazione peptide utilizzando l&#39;analisi delle immagini). Per l&#39;imaging peptide incorporazione, il reagente di Ellman può essere applicato direttamente ai campioni ed immediatamente visualizzato. Mentre limitato alla xey piani per la visualizzazione del modello, la tecnica può essere usato come un semplice metodo di routine per determinare se sono state modellato matrici contenenti gruppi tiolici liberi. È importante notare che dosaggio di Ellman non è ccytocompatible onsidered, così mentre può essere usata per osservare e quantificare photopatterns, non può essere eseguita in presenza di cellule. Per l&#39;imaging patterning in tre dimensioni e in presenza di cellule, la coniugazione di peptidi fluorescenti all&#39;interno matrici di idrogel rimane un approccio potente e ampiamente utilizzato. Risoluzione dei modelli può essere valutata nelle direzioni x, ye z-aerei usando la microscopia confocale, e questo metodo è cytocompatible modo che le cellule all&#39;interno delle regioni fantasia o regioni non-fantasia possono essere identificati. Nel loro insieme, le tecniche di analisi e di imaging-based di Ellman sono strumenti complementari per i ricercatori di valutare quantitativamente e qualitativamente la photopatterning di segnali biochimici all&#39;interno del sistema materiali. Photoclick, o più in generale photoinitiated, chimiche per la formazione e la modifica di idrogeli in presenza di cellule sono numerosi. Lo stampaggio, l&#39;incapsulamento, e patterning tecniche presentate qui non sono limmitata al sistema materiale descritto e può essere applicata a chimiche a base di luce alternativi, come tiolo-alchino, 35 azide-alchino, 4 e altre chimiche tiolo-ene (ad esempio, tiolo-norbornene), 10,13 nonché con diversi fotoiniziatori, come Irgacure 2959, eosina Y, e canforochinone. Nota, gli utenti possono avere bisogno di regolare i parametri di procedura (ad esempio, tempi di incubazione, i tempi di polimerizzazione, densità cellulare) per garantire che le condizioni rimangano cytocompatible per questi altri sistemi. Poiché il processo di patterning richiede diffusione del peptide (s) alloc-modificato in idrogel (2.2.3-2.2.5), questo processo può rivelarsi più utile per l&#39;aggiunta di porzioni integrine vincolante (ad esempio, peptidi o proteine della matrice extracellulare frammenti) al idrogel, dove è richiesta attacco del ligando alla rete per la generazione di forze di trazione dall&#39;attivazione cellulare e completa integrina. 36 Note, per biomolecole che possonoessere altrettanto attivi in soluzione o su immobilizzazione (ad esempio, fattori di crescita o citochine), la fase di incubazione per porzione diffusione (~ 1 ora) nel idrogel potrebbe portare ad eventi che Convolute risultati patterning segnalazione. Altri metodi sono stati stabiliti per il fattore di crescita immobilizzazione o il sequestro del locale per il loro patterning. 37-39 Inoltre, la risoluzione modello è dettata dal controllo sulla esposizione alla luce. Qui, fotomaschere consentire la creazione di modelli attraverso la profondità di gel e in x e y aerei; tuttavia, una maggiore controllo spaziale sopra patterning di segnali biochimici all&#39;interno gel può essere ottenuto con metodi alternativi di irradiazione come l&#39;uso di due fotoni microscopio confocale per generare modelli nel assi X, Y e aerei z-. 34,40 Infine, è importante notare che, mentre il sistema materiale utilizzato in questa procedura è solo inizialmente modificato con segnali biochimici, chimiche Photoclick ortogonali potrebbero essere utilizzati per consentire alterazioni di proprietà della matrice nel tempo. 12 La procedura e le tecniche qui presentate aggiungono diversità delle attuali approcci per la creazione di matrici sintetiche con caratteristiche ben definite e spatiotemporally controllati. In particolare, la disponibilità commerciale dei reagenti e materiali utilizzati in questo procedimento sarà utile per una vasta gamma di ricercatori interessati all&#39;uso di biomateriali idrogel a base per applicazioni in coltura cellulare controllata.

Istruzioni chimiche sono sempre più utilizzati nella progettazione di materiali per numerose applicazioni biomediche, tra somministrazione di farmaci, ingegneria dei tessuti, e coltura cellulare controllata, a causa dei loro selettive, reattività cytocompatible efficienti, e spesso. 1-3 chimiche Photoclick che utilizzano la luce per attivare o avviare reazioni (ad esempio, azide-alchino, 4 tiolo-ene, 5 e tetrazolo-alchene 6) sono di particolare interesse per la formazione o la modifica di biomateriali. Tassi rapidi in condizioni miti e il controllo di quando e dove si svolgono con la luce rendono queste reazioni particolarmente adatto per il controllo user-diretto di proprietà biomateriali in presenza di cellule. 7,8 In particolare, sono stati utilizzati chimiche tiolo-ene Photoclick per generare biomateriali a base di idrogel con robuste caratteristiche meccaniche 5,9 e per l&#39;incapsulamento di una grande varietà di tipi di cellule, tra cui, ma nont limitato a, le cellule umane staminali mesenchimali (hMSCs), i fibroblasti, condrociti e cellule pancreatiche, con la promessa per coltura cellulare e la consegna. 10,11 Inoltre, questi chimiche sono state utilizzate per il patterning spaziale di segnali biochimici per imitare gli aspetti chiave della microambienti cellulari nativi e agevola gli opportuni interazioni cellula-matrice, tra cui l&#39;adesione, la differenziazione, e l&#39;invasione. 3,12 Per la costruzione di idrogel tiolo-ene con luce, peptidi contenenti residui di cisteina (tiolo) comunemente vengono fatte reagire con polimeri funzionalizzati con acrilati o norborneni ( &#39;ene&#39;) per una rapida, la polimerizzazione photoinitiated in condizioni cytocompatible. 13 Espansione questa cassetta degli attrezzi, abbiamo cercato di stabilire metodi per la formazione di idrogel con nuovi blocchi di costruzione versatile e accessibile che ha richiesto l&#39;elaborazione di sintesi minimo o erano disponibili in commercio verso la loro ampio utilizzo come matrici extracellulari sintetici.Uno per il ciondolo, gruppi integrine vincolante per promuovere l&#39;adesione delle cellule [K (alloc) GWGRGDS = Pep1Alloc] o due per legami crociati non degradabili o cellule-degradabili [K (14: In particolare, i peptidi sono stati modificati con allilossicarbonil (Alloc) lisine -protected alloc) RGKGRKGK (alloc) G o KK (alloc) GGPQGIWGQGK (alloc) K = Pep2Alloc, rispettivamente]. Con queste sequenze, le condizioni sono state stabilite per la reazione rapida (1-5 min), con quattro bracci poli tiolo-modificato (glicole etilenico) (PEG4SH) utilizzando dosi cytocompatible della lunghezza d&#39;onda della luce UV (10 mW / cm 2 a 365 nm) e litio fotoiniziatore fenil-2,4,6-trimethylbenzoylphosphinate (LAP). Gli idrogel risultanti erano stabile in condizioni di coltura cellulare per settimane. Per abilitare la degradazione delle cellule-driven e rimodellamento, un peptide enzimaticamente-cleavable è stata costituita entro i legami crociati gel (cioè, GPQGIWGQ), 15 e un modello di cellula primaria, le cellule staminali mesenchimali umane (hMSCs), è rimasto altamente praticabile dopo l&#39;incapsulamento e during cultura all&#39;interno di queste matrici. Inoltre, i peptidi sono stati spazialmente fantasia all&#39;interno di questi materiali, e hMSCs rimanere vitali e metabolicamente attiva in condizioni photopatterning. Alternative sequenze peptidiche pendente, non è riportato (es IKVAV, YIGSR, GFOGER, ecc), anche può essere incorporato all&#39;interno di matrici per sondare interazioni cellulari aggiuntivi con il microambiente circostante. Questi risultati sono promettenti per l&#39;applicazione di questi materiali idrogel a base per tridimensionale coltura cellulare (3D) e la consegna di studiare e interazioni dirette cellula-matrice per una varietà di tipi cellulari. Qui, metodi per photoencapsulate cellule e segnali biochimici successivamente photopattern all&#39;interno del sistema idrogel proposto sono presentati (Figura 1). Tecniche per osservare e quantificare questi photopatterns anche sono dimostrati: in particolare, i) l&#39;utilizzo quantitativo e qualitativo dei test di Ellman per determinare la Modificazione dei tioli liberi all&#39;interno substrati a motivi geometrici e ii) l&#39;uso qualitativa complementare di peptidi fluorescenti (AF488Pep1Alloc) per osservare questi modelli in tre dimensioni. Inoltre, metodi per determinare la vitalità (Live / Dead vitalità / citotossicità colorazione) e l&#39;attività metabolica (MTS; 3- (4,5-dimetiltiazol-2-il) -5- (3-carboxymethoxyphenyl) -2- (4-solfofenil) -2H-tetrazolium) sono presentati in modo tale che gli utenti possono determinare il citocompatibilità delle condizioni photoencapsulation e photopatterning per le diverse linee di cellule all&#39;interno di matrici di idrogel. Mentre il protocollo è dimostrata per un sistema Photoclick idrogel basato sulla luce facile, le tecniche possono essere applicate a numerosi altri sistemi idrogel radicalmente avviate per photoencapsulation e photopatterning in presenza di cellule.

<table border="0" cellpadding="0" cellspacing="0" width="823">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:208px;">Custom Peptides</td>
			<td style="width:161px;">Various Vendors</td>
			<td style="width:123px;">----</td>
			<td style="width:331px;">Peptides may also be synthesized via standard SPPS techniques with materials from vendors including ChemImpex and ChemPep.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:208px;">4arm PEG Thiol, MW 20k</td>
			<td style="width:161px;">JenKem USA</td>
			<td style="width:123px;">4ARM-SH</td>
			<td style="width:331px;">Listed under Multi-arm Homofunctional PEGs. PEG4SH may also be synthesized as previously referenced.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Lithium Phenyl-2,4,6-trimethylbenzoylphosphinate</td>
			<td style="width:161px;">Colorado Photopolymer Solutions</td>
			<td style="width:123px;">Li-TPO</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:208px;">Dimethyl Phenylphosphonite</td>
			<td style="width:161px;">Sigma Aldrich</td>
			<td style="width:123px;">149470</td>
			<td style="width:331px;">Caution, causes severe skin burns and eye damage. Wear protective gloves, clothing, and eye protection.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:208px;">2.4.6-Trimethylbenzoyl Chloride</td>
			<td style="width:161px;">Sigma Aldrich</td>
			<td style="width:123px;">682519</td>
			<td style="width:331px;">Caution, causes severe skin burns and eye damage. Wear protective gloves, clothing, and eye protection.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">Lithium Bromide</td>
			<td style="width:161px;">Sigma Aldrich</td>
			<td style="width:123px;">213225</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">2-Butanone</td>
			<td style="width:161px;">Sigma Aldrich</td>
			<td style="width:123px;">360473</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">Flask, Round Bottom, 100 mL</td>
			<td style="width:161px;">Chemglass</td>
			<td style="width:123px;">CG-1506-05</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">80 mL Filter Funnel, Buchner, Medium Frit</td>
			<td style="width:161px;">Chemglass</td>
			<td style="width:123px;">CG-1402-L-02</td>
			<td style="width:331px;">Filter paper inside a regular glass funnel may be used if desired.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">Magnetic Stir Bars</td>
			<td style="width:161px;">Various Vendors</td>
			<td style="width:123px;">----</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Magnetic Stirring and Hot Plate</td>
			<td style="width:161px;">Various Vendors</td>
			<td style="width:123px;">----</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">Vacuum Dessicator</td>
			<td style="width:161px;">Various Vendors</td>
			<td style="width:123px;">----</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">Deuterium Oxide</td>
			<td style="width:161px;">Acros Organics</td>
			<td style="width:123px;">16630</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td style="width:161px;">ThermoFisher Scientific</td>
			<td style="width:123px;">14190-250</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">Penicillin Streptomycin</td>
			<td style="width:161px;">ThermoFisher Scientific</td>
			<td style="width:123px;">15070-063</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">Fungizone</td>
			<td style="width:161px;">ThermoFisher Scientific</td>
			<td style="width:123px;">15290-018</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Trypsin-EDTA (0.5%), no phenol red</td>
			<td style="width:161px;">ThermoFisher Scientific</td>
			<td style="width:123px;">15400-054</td>
			<td style="width:331px;">Select appropriate medium and trypsin depending on cell type.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">Microcentrifuge tubes</td>
			<td style="width:161px;">Various Vendors</td>
			<td style="width:123px;">----</td>
			<td style="width:331px;">1.5 or 2 mL sizes, sterile.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">BD Syringe with Slip (Luer) Tips (Without Needle)</td>
			<td style="width:161px;">Fisher Scientific</td>
			<td style="width:123px;">14-823-16H</td>
			<td style="width:331px;">Product number listed here is for a 1 mL syringe (16H), Various sizes are available (14-823-XX).</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Fisherfinest Premium Plain Glass Microscope Slides</td>
			<td style="width:161px;">Fisher Scientific</td>
			<td style="width:123px;">12-544-1</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:208px;">High-Purity Silicone Rubber, 0.010&#34; Thick, 6&#34; x 8&#34; Sheet, 55A Durometer (Gasket)</td>
			<td style="width:161px;">McMaster Carr</td>
			<td style="width:123px;">87315K62</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">Ethanol, 200 Proof</td>
			<td style="width:161px;">Decon Labs</td>
			<td style="width:123px;">2701</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">RainX, Original</td>
			<td style="width:161px;">Amazon</td>
			<td style="width:123px;">800002243</td>
			<td style="width:331px;">May be purchased from other vendors.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">Sigmacote</td>
			<td style="width:161px;">Sigma Aldrich</td>
			<td style="width:123px;">SL2</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Photomasks</td>
			<td style="width:161px;">Advance Reproductions Corporation</td>
			<td style="width:123px;">----</td>
			<td style="width:331px;">Photomask Division, different designs may be printed as desired.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">DTNB; Ellman&#39;s Reagent; 5,5-dithio-bis(2-nitrobenzoic acid)</td>
			<td style="width:161px;">ThermoFisher Scientific</td>
			<td style="width:123px;">PI-22582</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">Sodium Phosphate Dibasic</td>
			<td style="width:161px;">Sigma Aldrich</td>
			<td style="width:123px;">S5136</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Ethylenediaminetetraacetic acid</td>
			<td style="width:161px;">Sigma Aldrich</td>
			<td style="width:123px;">E6758</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Sodium Hydroxide, Pellets/Certified ACS</td>
			<td style="width:161px;">Fisher Scientific</td>
			<td style="width:123px;">S318</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">Orthophosphoric Acid</td>
			<td style="width:161px;">Alfa Aesar</td>
			<td style="width:123px;">33266</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Cysteine Hydrochloride Monohydrate</td>
			<td style="width:161px;">Sigma Aldrich</td>
			<td style="width:123px;">C7880</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:208px;">LIVE/DEAD Viability/Cytotoxicity Kit, for mammalian cells</td>
			<td style="width:161px;">ThermoFisher Scientific</td>
			<td style="width:123px;">L-3224</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">CellTiter 96 Aqueous One Solution Assay</td>
			<td style="width:161px;">Promega</td>
			<td style="width:123px;">G3582</td>
			<td style="width:331px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">Centrifuge</td>
			<td style="width:161px;">Various Vendors</td>
			<td style="width:123px;">----</td>
			<td style="width:331px;">Capable of speeds at 90-110 x g.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Multiwell Plate Reader</td>
			<td style="width:161px;">Various Vendors</td>
			<td style="width:123px;">----</td>
			<td style="width:331px;">Capable of reading absorbance at 405 nm in a 96-well plate.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Epifluorescent or Confocal Microscope</td>
			<td style="width:161px;">Various Vendors</td>
			<td style="width:123px;">----</td>
			<td style="width:331px;">To visualize peptide patterns and cells within hydrogels.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Omnicure Exfo Series 2000</td>
			<td style="width:161px;">Excelitas Technologies</td>
			<td style="width:123px;">----</td>
			<td style="width:331px;">Alternate light systems may be used to polymerize hydrogels.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Zeiss Zen Lite Software</td>
			<td style="width:161px;">Zeiss</td>
			<td style="width:123px;">----</td>
			<td style="width:331px;">Available at zeiss.com; compatible with images taken on Zeiss microscopes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">ImageJ</td>
			<td style="width:161px;">NIH</td>
			<td style="width:123px;">----</td>
			<td style="width:331px;">Available at imagej.nih.gov; applicable for general image analysis</td>
		</tr>
	</tbody>
</table>

light-mediated formation and patterning of hydrogels for cell culture applications

Clicca chimiche sono stati studiati per l&#39;impiego in numerose applicazioni biomateriali, tra cui la somministrazione di farmaci, ingegneria dei tessuti, e la coltura cellulare. In particolare, reazioni click luce-mediata, quali reazioni tiolo-ene e tiolo-ino photoinitiated, garantire un controllo spaziotemporale sopra proprietà del materiale e consentire la progettazione di sistemi con un alto grado di controllo di proprietà dall&#39;utente diretto. Fabbricazione e modifica di biomateriali a base di idrogel che utilizzano la precisione offerta dalla luce e la versatilità offerta da questi chimiche tiolo-X Photoclick sono di crescente interesse, in particolare per la coltura di cellule all&#39;interno di ben definiti, microambienti biomimetici. Qui, descriviamo i metodi per la photoencapsulation delle cellule e la successiva photopatterning di segnali biochimici all&#39;interno di matrici di idrogel che utilizzano blocchi di costruzione versatili e modulari polimerizzati da una reazione Photoclick tiolo-ene. In particolare, un approccio è presentato per constructing idrogel da allilossicarbonil (Alloc) legami crociati del peptide -functionalized e frazioni peptidiche ciondolo e poli tiolo-funzionalizzati (glicole etilenico) (PEG), che rapidamente polimerizzare in presenza di litio acylphosphinate fotoiniziatore e dosi cytocompatible di lunghezza d&#39;onda ultravioletta (UV). Facile tecniche di visualizzare photopatterning e quantificare la concentrazione di peptidi aggiunti sono descritti. Inoltre, i metodi sono stabiliti per le cellule incapsulamento, in particolare le cellule staminali mesenchimali umane, e determinare la loro vitalità e l&#39;attività. Mentre la formazione ed il modello iniziale di idrogel tiolo-alloc sono mostrati qui, queste tecniche ampiamente possono essere applicati ad un certo numero di sistemi materiali altra luce e radicale-iniziati (ad esempio, tiolo-norbornene, tiolo-acrilato) per generare substrati fantasia.

La messa a punto e la procedura per photoencapsulate cellule e gel successivamente photopattern contenenti cellule incapsulate è rappresentato in figura 1, e un esempio per la preparazione di soluzioni madre per formare una% gel 10 in peso è fornita in tabella 1. Utilizzando la tabella 1, la quantità di monomeri (PEG4SH , Pep2Alloc, ± Pep1Alloc) e fotoiniziatore (LAP) necessario per polimerizzare idrogel viene calcolato. In base a questi calcoli, soluzioni stock di PEG4SH, Pep1Alloc, Pep2Alloc, e LAP sono preparati e mescolati con e senza Pep1Alloc per la formazione e photopatterning gel, rispettivamente (Figura 1A). Successivamente, le cellule vengono raccolte dalle piastre, contati e centrifugati in quantità appropriate per incapsulamento (Figura 1B). Il pellet cellulare viene risospeso in soluzione gelificante (peptide / polimero / LAP in PBS), e miscele di cellule / monomero vengono trasferiti al vetro o una siringa molds. Le cellule sono incapsulati all&#39;interno idrogel all&#39;atto dell&#39;applicazione della luce (1-5 minuti e 10 mW / cm 2 a 365 nm) (Figura 1C). Per photopatterning (Figura 1D), i gel sono impregnati con Pep1Alloc e LAP per 30 minuti a 1 ora e 30 min, permettendo la diffusione di peptide e iniziatore nella matrice polimerizzato. Questi gel peptide-carico sono coperti di fotomaschere con motivi desiderati ed esposto ad una seconda dose di luce (1 min) per coniugare i peptidi a tioli liberi all&#39;interno della matrice. Cavezze pendente solo sono covalentemente legati al gel in regioni esposte alla luce, facilitando appropriate interazioni cellula-matrice e mimando principali proprietà meccaniche e biochimiche del microambiente cellule nativa verso tastatura funzione cellulare e destino in vitro. dosaggio di Ellman fornisce un metodo facile e poco costoso da quantificare modifica gel e peptide incorporazione all&#39;interno photopattesubstrati rned. Fantasia e gel non-fantasia (ad esempio, gel con o senza modifiche peptide) sono immersi in tampone di reazione post-polimerizzazione di Ellman. Successivamente, standard cisteina ei gel imbevuti in tampone sono poste in piastre da 48 pozzetti e incubate con il reagente di Ellman (Figura 2A, sinistra). Dopo 1 ora e 30 minuti, aliquote di campioni vengono posti in singoli pozzetti di una piastra a 96 pozzetti e l&#39;assorbanza è registrata a 405 nm. Una curva di calibrazione (Figura 2A, destra) dagli standard cisteina è tracciata (assorbanza vs concentrazione, adattamento lineare), e la quantità di tioli liberi in gel può essere determinato in base al fattore di diluizione. Queste concentrazioni tioli liberi per vari polimerizzazione e photopatterning condizioni di un gel 10% in peso sono mostrati nella Figura 2B. Gel polimerizzati per 1 o 5 minuti senza Pep1Alloc (blu bar, I = 1 min e II = 5 min) hanno statisticamente simili concentrazioni tioli liberi, indicando tcappello una reazione rapida verifica e gelificazione è completata entro 1 minuto (a due code t-test, p&gt; 0.05). Così, esposizione aggiuntiva alla luce (2-5 minuti) non provoca ulteriore conversione dei gruppi funzionali. Gel polimerizzati per 1 min senza Pep1Alloc erano intrisi di Pep1Alloc (3 mg / ml) e LAP (2,2 mm) per 30 e 90 min (barre verdi, II = 30 min e IV = 90 min) ed esposti a una seconda dose di luce per 1 min. La diminuzione tioli liberi (60-80% rispetto alla condizione 1 min) indica modifica efficiente di gel con stecche pendente in queste condizioni. Se modifica superiore è desiderato, aumento delle concentrazioni di soluzione Pep1Alloc possono essere usati come accessibilità Pep1Alloc di tioli liberi in soluzioni diluite peptidici possono limitare conversione; per esempio, abbiamo trovato che le concentrazioni fino a 20 mg / ml Pep1Alloc producono&gt; conversione del 90% di tioli liberi. Unicamente, modelli di peptidi aggiunti al idrogel puòessere rapidamente ripreso con il reagente di Ellman (Figura 3A, in 5 min). Tuttavia, la visualizzazione del modello è perso nel tempo (maggiore di 5 min) a causa della diffusione giallo TNB 2- dianione attraverso il gel. Per migliorare la risoluzione delle immagini e osservare i modelli in tre dimensioni (X, Y, e Z piani) e in presenza di cellule, peptide fluorescente addizione (AF488Pep1Alloc) può essere utilizzata. Nella Figura 3B X, Y e Z-proiezioni di pile di immagini scattate con un microscopio confocale sono mostrati, a dimostrazione risoluzione modello (scala micron). Circa (94 ± 2)% e (94 ± 1)% di hMSCs incapsulati all&#39;interno di gel degradabili (Pep2Alloc = GPQG ↓ WGQ) rimangono (corpi cellulari verdi) vitali 1 e 6 giorni dopo l&#39;incapsulamento, rispettivamente, con poche cellule morte (nuclei rossi ) osservato (Figura 4A). Inoltre, hMSC diffusione è osservata a 6 giorni post-incapsulamento ( <st<html> rong&gt; Figura 4A, nel riquadro), che indica che le cellule possono rimodellare e interagire con queste matrici MMP-degradabili modificati con RGDS integrine vincolante. Saggi di attività metaboliche effettuati su cellule incapsulate in gel non degradabili (Pep2Alloc = RGKGRK) 1 e 3 giorni dopo l&#39;incapsulamento (Figura 4B) di fornire una seconda misura della vitalità cellulare e dimostrano che le cellule rimangono attivi per le varie condizioni photoencapsulation e photopatterning testati con dosaggio di Ellman (Figura 2B). In particolare, non vi è alcuna differenza significativa dell&#39;attività metabolica tra i 30 min e 90 min incubazione con Pep1Alloc + LAP (condizioni III e IV) e l&#39;incapsulamento iniziale (condizioni di I e II), che indica che la procedura è appropriata per le applicazioni di photopatterning a la presenza di cellule incapsulate (due code t-test, p&gt; 0.05). 2fig1.jpg &quot;/&gt; Figura 1:. Installazione per incapsulare le celle all&#39;interno di idrogel e successivamente photopatterning con l&#39;indicazione biochimica (A) Le soluzioni madre di macromeri e fotoiniziatore sono preparati e misto (PEG4SH = spina dorsale, Pep2Alloc = reticolazione, Pep1Alloc = ciondolo parte adesiva (RGDS), LAP = fotoiniziatore ). (B) Le cellule sono raccolti per l&#39;incapsulamento. (C) Le cellule sono mescolate con le soluzioni macromero senza o con peptidi integrine vincolante (PEG4SH / Pep2Alloc / LAP o PEG4SH / Pep2Alloc / Pep1Alloc / LAP, rispettivamente) e sono incapsulati in seguito all&#39;esposizione alla luce. (D) gel contenenti gruppi tiolici in eccesso durante la formazione del gel (qui, PEG4SH / Pep2Alloc / LAP, formata con 2 mm in eccesso tioli) possono essere modellati con segnali biochimici dalla successiva aggiunta di peptidi funzionalizzati con un singolo gruppo alloc (Pep1Alloc, ad esempio, RGDS, IKVAV, etc.) per promuovere l&#39;adesione delle celluleall&#39;interno di specifiche regioni del gel. Qui, viene mostrato patterning di gel con RGDS fluorescenza marcata. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54462/54462fig1large.jpg" target="_blank">Fai clic qui per vedere una versione più grande di questa figura.</a> <img alt="figura 2" src="/files/ftp_upload/54462/54462fig2.jpg" /> Figura 2:. Impostazione del test e risultati quantitativi di Ellman per valutare la modifica degli idrogel fantasia Gel (A) e gli standard di cisteina sono incubati in piastre a 48 pozzetti con il reagente di Ellman. Una curva standard lineare viene tracciata per determinare la concentrazione di cisteina nei campioni di gel. (B) tioli liberi in eccesso sono incorporati all&#39;interno di idrogel durante formazione di gel e consumati su patterning con un ciondolo alloc-peptide (I = 1 min polimerizzazione; II = 5 min polimerizzazione; III = 30 min di incubazione con Pep1Alloc; IV = 90 min di incubazione wi<html> esimo Pep1Alloc; sia III e IV polimerizzati e modellati con la luce per 1 min). I dati riportati illustrano la media (n = 3) con barre di errore che mostra l&#39;errore standard. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54462/54462fig2large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 3" src="/files/ftp_upload/54462/54462fig3.jpg" /> Figura 3:. Assay di Ellman e immagini fluorescenti di visualizzare idrogel photopatterned Reattivo (A) di Ellman può essere utilizzato per individuare rapidamente i modelli (linee) in X e Y-piani (giallo = regione non nanostrutturata, Barra di scala = 1 mm). (B) peptidi fluorescenti possono essere utilizzati per osservare i modelli nelle direzioni x, ye z-aerei (verde = regione fantasia; 200 bar scala micron; 10X obiettivo acqua-immersione; Ex / Em 488/525 nm).large.jpg &quot;target =&quot; _ blank &quot;&gt; Clicca qui per vedere una versione più grande di questa figura. <img alt="Figura 4" src="/files/ftp_upload/54462/54462fig4.jpg" /> . Figura 4: La vitalità e l&#39;attività metabolica delle cellule all&#39;interno non degradabili idrogel photopatterned (A) Esempio confocale Z-stack (Z-proiezione; 10X obiettivo acqua-immersione) di vitali (verde; Ex / Em 488/525 nm) e morti hMSCs (rosso; Ex / Em 543/580 nm) incapsulato all&#39;interno di idrogel 24 ore post-incapsulamento (Scala bar = 200 micron). Le cellule sparse all&#39;interno di questi idrogel 6 giorni dopo l&#39;incapsulamento (immagine inserto, Scala bar = 50 micron). (B) Le cellule sono metabolicamente attivo 1 e 3 giorni (barre chiare e scure, rispettivamente) post-incapsulamento per i vari polimerizzazione (I = 1 min polimerizzazione; II = 5 min di polimerizzazione) e le condizioni di patterning (III = 30 min di incubazione con Pep1Alloc ; IV = 90 min incubation con Pep1Alloc; sia polimerizzati e modellato con la luce per 1 min). I dati riportati illustrano la media (n = 3) con barre di errore che mostra l&#39;errore standard. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54462/54462fig4large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> <img alt="Tabella 1" src="/files/ftp_upload/54462/54462tbl1.jpg" /> Tabella 1: configurazione di esempio per calcolare i volumi di soluzioni madre per fare idrogel cellule all&#39;interno del foglio di calcolo che sono evidenziati blu indicano parametri definiti dall&#39;utente;. le altre quantità sono calcolate sulla base di queste impostazioni. I volumi finali per ciascuna soluzione madre per fare un gel sono evidenziati arancione. I globuli bianchi contengono formule utilizzate per calcolare i volumi finali in base ai parametri definiti dall&#39;utente. Si noti che una concentrazione di 2,2 mM LAP è equivalente a 0,067% in peso.

Watch this Scientific Journal Video about Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications at JoVE.com

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

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 ...

light-mediated-formation-patterning-hydrogels-for-cell-culture

Nanyang Technological University

Research

JoVE Journal

Bioengineering

12.9K Views.  University of Delaware. 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. 

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

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 ...

Ricerca

Bioingegneria

Plasma Lithography Surface Patterning for Creation of Cell Networks

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular phenomena. To address this requirement, we have developed a plasma lithography technique to manipulate the cellular microenvironment by creating a patterned surface with feature sizes ranging from 100 nm to millimeters. The goal of this technique is to be able to study, in a controlled way, the behaviors of individual cells as well as groups of cells and their interactions.
This plasma lithography method is based on selective modification of the surface chemistry on a substrate by means of shielding the contact of low-temperature plasma with a physical mold. This selective shielding leaves a chemical pattern which can guide cell attachment and movement. This pattern, or surface template, can then be used to create networks of cells whose structure can mimic that found in nature and produces a controllable environment for experimental investigations. The technique is well suited to studying biological phenomenon as it produces stable surface patterns on transparent polymeric substrates in a biocompatible manner. The surface patterns last for weeks to months and can thus guide interaction with cells for long time periods which facilitates the study of long-term cellular processes, such as differentiation and adaption. The modification to the surface is primarily chemical in nature and thus does not introduce topographical or physical interference for interpretation of results. It also does not involve any harsh or toxic substances to achieve patterning and is compatible for tissue culture. Furthermore, it can be applied to modify various types of polymeric substrates, which due to the ability to tune their properties are ideal for and are widely used in biological applications. The resolution achievable is also beneficial, as isolation of specific processes such as migration, adhesion, or binding allows for discrete, clear observations at the single to multicell level.
This method has been employed to form diverse networks of different cell types for investigations involving migration, signaling, tissue formation, and the behavior and interactions of neurons arraigned in a network.

A versatile plasma lithography technique has been developed to generate stable surface patterns for guiding cellular attachment. This technique can be applied to create cell networks including those that mimic natural tissues and has been used for studying several, distinct cell types.

Plasma Surface Patterning litografia per la creazione di reti di cella

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular ...

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

Preparazione del 3D fibrina ponteggi per le applicazioni delle cellule staminali Cultura

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy

TIRF microscopy has emerged as a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules in vitro and in living cells1. The optical phenomenon of total internal reflection occurs when light passes from a medium with high refractive index into a medium with low refractive index at an angle larger than a characteristic critical angle (i.e. closer to being parallel with the boundary). Although all light is reflected back under such conditions, an evanescent wave is created that propagates across and along the boundary, which decays exponentially with distance, and only penetrates sample areas that are 100-200 nm near the interface2. In addition to providing superior axial resolution, the reduced excitation of out of focus fluorophores creates a very high signal to noise ratios and minimizes damaging effects of photobleaching2,3. Being a widefield technique, TIRF also allows faster image acquisition than most scanning based confocal setups.
At first glance, the low penetration depth of TIRF seems to be incompatible with imaging of bacterial and fungal cells, which are often surrounded by thick cell walls. On the contrary, we have found that the cell walls of yeast and bacterial cells actually improve the usability of TIRF and increase the range of observable structures4-8. Many cellular processes can therefore be directly accessed by TIRF in small, single-cell microorganisms, which often offer powerful genetic manipulation techniques. This allows us to perform in vivo biochemistry experiments, where kinetics of protein interactions and activities can be directly assessed in living cells.
We describe here the individual steps required to obtain high quality TIRF images for Saccharomyces cerevisiae or Bacillus subtilis cells. We point out various problems that can affect TIRF visualization of fluorescent probes in cells and illustrate the procedure with several application examples. Finally, we demonstrate how TIRF images can be further improved using established image restoration techniques.

Total Internal Reflection Fluorescence (TIRF) microscopy is a powerful approach to observe structures close to the cell surface at high contrast and temporal resolution. We demonstrate how TIRF can be employed to study protein dynamics at the cortex of cell wall-enclosed bacterial and fungal cells.

Visualizzazione di Cortex Organizzazione e Dynamics in microrganismi, utilizzando Total Internal Reflection Fluorescence Microscopy

TIRF microscopy has emerged as  a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules  in vitro and in living cells1. ...

Cell Co-culture Patterning Using Aqueous Two-phase Systems

Cell patterning technologies that are fast, easy to use and affordable will be required for the future development of high throughput cell assays, platforms for studying cell-cell interactions and tissue engineered systems. This detailed protocol describes a method for generating co-cultures of cells using biocompatible solutions of dextran (DEX) and polyethylene glycol (PEG) that phase-separate when combined above threshold concentrations. Cells can be patterned in a variety of configurations using this method. Cell exclusion patterning can be performed by printing droplets of DEX on a substrate and covering them with a solution of PEG containing cells. The interfacial tension formed between the two polymer solutions causes cells to fall around the outside of the DEX droplet and form a circular clearing that can be used for migration assays. Cell islands can be patterned by dispensing a cell-rich DEX phase into a PEG solution or by covering the DEX droplet with a solution of PEG. Co-cultures can be formed directly by combining cell exclusion with DEX island patterning. These methods are compatible with a variety of liquid handling approaches, including manual micropipetting, and can be used with virtually any adherent cell type.

Aqueous two-phase systems were used to simultaneously pattern multiple populations of cells. This fast and easy method for cell patterning takes advantage of the phase separation of aqueous solutions of dextran and polyethylene glycol and the interfacial tension that exists between the two polymer solutions.

Cella Co-cultura Patterning Uso acquose due sistemi monofase

Cell patterning  technologies that are fast, easy to use and affordable will be required for the  future development of high throughput cell assays, ...

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based &#8216;lab on a chip&#8217; technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO2&#160;wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO2 regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

This protocol describes a microfabrication-compatible method for cell patterning on SiO2. A predefined parylene-C design is photolithographically printed on SiO2 wafers. Following incubation with serum (or other activation solution) cells adhere specifically to (and grow according to the conformity of) underlying parylene-C, whilst being repulsed by SiO2 regions.

Patterning cellulare su fotolitograficamente Definito parylene-C: SiO<sub&gt; 2</sub&gt; Substrati

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain ...

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 ...

Mesenchymal Stromal Cell Culture and Delivery in Autologous Conditions: A Smart Approach for Orthopedic Applications

Human Mesenchymal Stromal Cells (hMSCs) are cultured in vitro with different media. Limits on their use in clinical settings, however, mainly depend on potential biohazard and inflammation risks exerted by xenogeneic nutrients for their culture. Human derivatives or recombinant materials are the first choice candidates to reduce these reactions. Therefore, culture supplements and materials of autologous origin represent the best nutrients and the safest products.
Here, we describe a new protocol for the isolation and culture of bone marrow hMSCs in autologous conditions &#8212;&#160;namely,&#160;patient-derived serum as a supplement for the culture medium and fibrin as a scaffold for hMSC administration. Indeed, hMSC/fibrin clot constructs could be extremely useful for several clinical applications. In particular, we focus on their use in orthopedic surgery, where the fibrin clot derived from the donor&#39;s own blood allowed effective cell delivery and nutrient/waste exchanges. To ensure optimal safety conditions, it is of the utmost importance to avoid the risks of hMSC transformation and tissue overgrowth. For these reasons, the approach described in this paper also indicates a minimally ex vivo hMSC expansion, to reduce cell senescence and morphologic changes, and short-term osteo-differentiation before implantation, to induce osteogenic lineage specification, thus decreasing the risk of subsequent uncontrolled proliferation.

Culturing human Mesenchymal Stromal Cells (hMSCs) with autologous serum, reduces the risks of rejection by xenogeneic material and other negative effects. It also allows for the recovery of a subset of mesodermal progenitors, which can deliver fresh hMSCs. Embedding hMSCs in an autologous fibrin clot enables easy handling and effective surgical implantation.

Mesenchimali stromali coltura cellulare e la consegna in autologhi Condizioni: un approccio intelligente per applicazioni ortopediche

Human Mesenchymal Stromal Cells (hMSCs) are cultured in vitro with different media. Limits on their use in clinical settings, however, mainly depend on ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

Sintonizzabile idrogel da polmonare matrice extracellulare per colture cellulari 3D

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Patterning Bioactive Proteins or Peptides on Hydrogel Using Photochemistry for Biological Applications

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors within the medium to control cell behavior. However, soluble additions cannot mimic certain signaling motifs, such as matrix-bound growth factors, cell-cell signaling, and spatial biochemical cues, which are common influences on cells. Furthermore, biophysical properties of the matrix, such as substrate stiffness, play important roles in cell fate, which is not easily manipulated using conventional cell culturing practices. In this method, we describe a straightforward protocol to provide patterned bioactive proteins on synthetic polyethylene glycol (PEG) hydrogels using photochemistry. This platform allows for the independent control of substrate stiffness and spatial biochemical cues. These hydrogels can achieve a large range of physiologically relevant stiffness values. Additionally, the surfaces of these hydrogels can be photopatterned with bioactive peptides or proteins via thiol-ene click chemistry reactions. These methods have been optimized to retain protein function after surface immobilization. This is a versatile protocol that can be applied to any protein or peptide of interest to create a variety of patterns. Finally, cells seeded onto the surfaces of these bioactive hydrogels can be monitored over time as they respond to spatially specific signals.

In this method, we use photopolymerization and click chemistry techniques to create protein or peptide patterns on the surface of polyethylene glycol (PEG) hydrogels, providing immobilized bioactive signals for the study of cellular responses in vitro.

Patterning bioattivi proteine o peptidi su idrogel utilizzando fotochimica per applicazioni biologiche

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors ...