I finanziamenti del BBSRC è gentilmente riconosciuto (codice di autorizzazione BB H011293 / 1).

Panoramica Sezione 1 descrive la preparazione di pH nanosensori reattivo e la caratterizzazione della risposta nanosensori a pH utilizzando la spettrometria di fluorescenza e la loro dimensione al SEM. Sezione 2 descrive la preparazione di elettrofilate scaffold polimerici e caratterizzazione della loro morfologia e dimensioni utilizzando SEM. Sezione 2 descrive anche la preparazione di scaffolds self-reporting, che sono ponteggi elettrofilate con l&#39;inclusione dei nanosensori reattive pH. I risultanti ponteggi self-report sono caratterizzati da SEM per valutare se si è verificato eventuali modifiche morfologiche delle fibre elettrofilate. La fluorescenza dalle impalcature auto-segnalazione risultanti dai nanosensori incorporati è valutata mediante microscopia confocale. Sezione 3 descrive la coltura di cellule sul patibolo electrospun e il self-report patibolo. I ponteggi vengono prima sterilizzati in preparatione per la coltura cellulare a seguito della quale i ponteggi sono valutati per determinare se la sterilizzazione e la cultura di cellule influenzano la capacità di fluorescenza dei ponteggi auto-segnalazione. Il protocollo descrive inoltre la consegna di nanosensori alle cellule che vengono coltivate su scaffold elettrofilate. Lysotracker colorante viene utilizzato per garantire che nanosensori non sono state prese in compartimenti cellulari acide. 1. Preparazione e analisi di pH Responsive nanosensori sol-gel 1.1 Preparazione del pH Responsive nanosensori sol-gel Nota: Questa preparazione deve essere eseguita in una cappa aspirante. <ol><li> Preparare i fluorofori per l&#39;uso entro il nanosensori pH sciogliendo 5 - (e-6)-carbossifluoresceina, estere succinimidyl (FAM-SE) (1,5 mg) in dimetilformammide (DMF) (1 ml) in un pallone a fondo rotondo e 6-carboxytetramethylrhodamine , estere succinimidyl (TAMRA-SE) (1,5 mg) in DMF (1 ml) in un altro round pallone a fondo.Aggiungere 3-amminopropiltrietossisilano (APTES) (1,5 mL) per ogni beuta e agitare sotto atmosfera di azoto secco (21 ° C) per 24 ore al buio cioè avvolgere i campioni in stagnola. </li><li> Aggiungere entrambi i coloranti di cui sopra (250 mL) ad una miscela di etanolo (6 ml) e idrossido di ammonio (30% in peso, 4 ml) contenuta in un pallone a fondo tondo e agitare per 1 ora (21 ° C). </li><li> Aggiungere lentamente tetraetilortosilicato (TEOS) (0,5 ml) alla miscela, continuare a mescolare per altri 2 hr. La miscela si accende nuvoloso. I nanosensori possono essere raccolti mediante evaporazione rotante. Nanosensori possono essere memorizzati in una fiala di vetro a 4 ° C per un uso futuro. ATTENZIONE: ulteriore manipolazione di nanosensori nella loro forma secca deve essere eseguita in una cappa aspirante o in caso di pesatura loro l&#39;equilibrio dovrebbe essere chiuso, ad esempio in un Weighsafe. </li></ol> 1.2 nanosensori risposta a pH <ol><li> Preparare soluzioni tampone fosfato di Sorensen che vanno dal pH 5,5-8,0 per Mixing rapporti specifici di sodio fosfato monobasico (0,2 M) e sodio fosfato bibasico (0,2 M) soluzioni stock. Controllare il pH finale utilizzando un pHmetro. Apportare le modifiche minori al pH con idrossido di sodio (NaOH) (4 M) o acido cloridrico (HCl) (2 M). </li><li> Sospendere i nanosensori pH nei tamponi di pH (5 mg / ml) da un lato tenendo il recipiente contenente la soluzione di nanosensori in un vortice per 3 minuti poi immergere il vaso in un ultrasonicatore fino a quando la soluzione diventa torbida (circa 5 min). Ripetere la procedura per sospendere pienamente i nanosensori in soluzione. </li><li> Posizionare la prima soluzione in una cuvetta ottica e utilizzare lunghezze d&#39;onda di eccitazione di 488 nm per 5 - (e-6)-carbossifluoresceina (FAM) e 568 nm per 6-carboxytetramethylrhodamine (TAMRA). Raccogliere le lunghezze d&#39;onda di emissione a 500-530 nm per FAM e 558-580 nm per TAMRA con uno spettrometro a fluorescenza. </li><li> Modificare le soluzioni nella cuvetta ottico in modo che il pH può essere osservata a diversi pH e continuare collecting spettri di emissione. </li><li> Calcolare il rapporto dei valori massimi di emissione prodotta a ciascun valore di pH per produrre una curva di calibrazione. </li></ol> 1.3 SEM di nanosensori <ol><li> Porre un campione dei nanosensori su un carbonio rivestito microscopio elettronico stub e farfugliare mantello d&#39;oro per 5 min in atmosfera di argon. </li><li> Osservare da microscopia elettronica a scansione (SEM). Durante l&#39;imaging regolare la distanza di lavoro, tensione e ingrandimento di minimizzare elettrone di carica (Figura 1). </li></ol> 2. Preparazione e analisi di PLGA Ponteggi 2.1 Electrospinning PLGA Ponteggi <ol><li> In una cappa aspirante sciogliere acido poli (lattico-co-glicolico) (PLGA) in diclorometano (DCM) (20% (w / w)) con piridinio formiato (PF) (1% (w / w)). Porre la soluzione in una siringa da 10 ml con un ago smussato riempimento 18 G e sicuro idonei a una pompa a siringa. </li><li> Dist. la punta dell&#39;ago 20 centimetri di distanza da un 20cm x 15 cm piano di raccolta alluminio. </li><li> Fissare l&#39;elettrodo di un alimentatore ad alta tensione alla punta della siringa e la terra alla piastra di raccolta alluminio. ATTENZIONE: Si deve prestare attenzione in quanto l&#39;alta tensione è usato cioè di spegnere sempre l&#39;alimentazione elettrica prima di gestire qualsiasi apparecchiatura elettrofilatura. </li><li> Consegnare la soluzione con un flusso costante di 3,5 m / ora a 12 kV per 2 ore. Elettrospinning utilizzando queste condizioni darà una profondità un&#39;impalcatura di circa 60 micron. </li><li> Lasciare il patibolo in una cappa per 24 ore per consentire i residui di solvente di evaporare. </li></ol> 2.2 Electrospinning pH Responsive PLGA Ponteggi <ol><li> Preparare ponteggi incorporano pH nanosensori reattivi come descritto nella sezione 2.1, ma con la modifica di aggiungere nanosensori per la soluzione di polimero (5 mg / ml). Sospendere il nanosensori nella soluzione PLGA con l&#39;assistenza di ultrasuoni (circa 5 min) prima<html> per la messa in una siringa da 10 ml. </li></ol> 2.3 SEM di PLGA impalcatura e pH Impalcatura Responsive <ol><li> Porre un campione del patibolo PLGA o pH patibolo reattivo su un microscopio elettronico rivestito stub carbonio e procedere come descritto per SEM di nanosensori (Figura 1). </li></ol> 2.4 Calibrazione del pH impalcatura Responsive <ol><li> Porre un campione di pH scaffold reattivo in una piastra di coltura a 35 mm e immergersi in apposite soluzioni tampone. </li><li> In un microscopio confocale utilizzare un laser argon 488 nm per eccitare la FAM e un laser krypton 568 nm per eccitare il TAMRA entro i nanosensori. Osservare l&#39;emissione di fluorescenza di pH ponteggi rispondenti a 500-530 nm per FAM e 558-580 nm per TAMRA. (Figura 2). Utilizzare la scansione sequenziale per evitare la raccolta di lunghezze d&#39;onda di eccitazione. </li></ol> 3. Coltura cellulare su PLGA Ponteggi e pH Responsive PLGA Ponteggi S copi &quot;&gt; Nota: Le seguenti deve essere eseguita in un armadio coltura cellulare. 3.1 Preparazione di PLGA Ponteggi per coltura cellulare <ol><li> Tagliare le PLGA o pH reattivo ponteggi PLGA in due centimetri 2 pezzi. </li><li> Sterilizzare scaffold mediante irradiazione con raggi ultravioletti (UV) ad una distanza di 8 cm per 15 min per lato. </li><li> Trasferire i ponteggi ad una piastra di coltura 12 pozzetti e mettere un anello in acciaio sulla parte superiore. Aggiungere una soluzione di penicillina / streptomicina (5% v / v) in tampone fosfato salino (PBS) all&#39;interno e all&#39;esterno dell&#39;anello dell&#39;acciaio, incubare una notte (37 ° C, 5% CO 2). Nota: l&#39;anello di acciaio è stato realizzato presso l&#39;Università di Nottingham e ha le seguenti dimensioni: diametro esterno 2 cm, 1 cm di diametro interno, 1 cm di profondità. </li><li> Rimuovere la soluzione sterilizzante e lavare con PBS. </li><li> Aggiungere cella di coltura verso l&#39;interno (500 microlitri) e all&#39;esterno (500 microlitri) dell&#39;anello acciaio, posto in un incubatore (37 ° C, 5% CO 2 </sub&gt;) mentre preparare una sospensione cellulare. </li><li> Preparare una sospensione cellulare ed eseguire una conta cellulare utilizzando un saggio di esclusione Trypan Blue. </li><li> Creare una sospensione di cellule per ottenere un numero di cellule di 1 x 10 6 cellule / ml. </li><li> Rimuovere i supporti da dentro e fuori dal ring in acciaio. </li><li> Sostituire con preriscaldata mezzi di coltura delle cellule sulla parte esterna dell&#39;anello dell&#39;acciaio (1 m). </li><li> Aggiungere 300 microlitri della sospensione cellulare all&#39;interno dell&#39;anello acciaio - piastra roccia avanti e indietro delicatamente per distribuire le cellule. </li><li> Posizionare la piastra di coltura cellulare in un incubatore (37 ° C, 5% CO 2) - l&#39;adesione cellulare avrebbe dovuto aver luogo entro 24 ore, ma continuare cultura per tutto il tempo dell&#39;esperimento richiede - rinfrescante media ogni 2-3 giorni. </li></ol> 3.2 nanosensori Consegna in cellule coltivate su PLGA impalcatura <ol><li> Cellule Seed Onto PLGA patibolo come descritto nella sezione 3.1. Per questo studio un vuoto PLGA patibolo (non contenanosensori Ning) è stato utilizzato a causa della sovrapposizione di emissione di fluorescenza durante l&#39;imaging. </li><li> Posizionare la cella scaffold seminato in un incubatore (37 ° C, 5% CO 2) per 72 ore per permettere alle cellule di crescere e migrano in tutto il ponteggio prima nanosensori consegna. </li><li> Prima di nanosensori consegna lavare le cellule con PBS e cambiare il supporto da terreni di coltura standard per i media liberi antibiotici e siero, incubare (37 ° C, 5% CO 2) per 1 ora. Nota: mezzi di coltura standard per 3T3 compone di mezzo di Eagle modificato da Dulbecco supplementato con siero fetale di vitello, soluzione ((v / v) 10%) L-glutammina (2 mM), penicillina / streptomicina (1% (v / v)) . </li><li> Durante il periodo di incubazione preparare il complesso nanosensori-liposomi da sonicating brevemente nanosensori (5 mg) con OptiMEM (50 pl) per creare una soluzione A. </li><li> Crea soluzione B aggiungendo Lipofectamine 2000 (5 ml) a OptiMEM (45 microlitri), incubare a temperatura ambiente per 5 min. </li><li> Aggiungere la soluzioneA alla soluzione B, incubare a temperatura ambiente per 20 minuti per formare la soluzione C, che è il complesso nanosensori-liposoma. </li><li> Aggiungere la soluzione C (100 microlitri) di cellule e incubare (37 ° C, 5% CO 2) per 3 ore. </li><li> Rimuovere le cellule scaffold seminati dal termostato, supporto Aspirare e lavare due volte con PBS prima di aggiungere PBS (1 ml) per consentire la visualizzazione di cellule in vivo mediante microscopia confocale. Nota: PBS è stato utilizzato in questo protocollo in modo che l&#39;indicatore rosso fenolo presente nella cella di coltura non ha causato autofluorescenza e anche perché veniva eseguita una calibrazione a differenti pH. Tuttavia fenolo media liberi rosse potrebbero essere utilizzati per l&#39;analisi a lungo termine delle colture cellulari. </li><li> Immergere la cella scaffold seminato nei buffer di diverso pH e monitorare la risposta dei nanosensori associati con le cellule. </li><li> Monitorare Phi determinando il rapporto tra le intensità di fluorescenza da FAM e TAMRA osservando fluorescenza utilizzando la microscopia confocale (Figure 3). </li></ol> 3.3 nanosensori posizione all&#39;interno di cellule di mammifero <ol><li> Incubare le cellule con nanosensors contenenti solo il colorante FAM come descritto nella sezione 3.2 (questo perché l&#39;emissione di fluorescenza di TAMRA è nella stessa regione spettrale come LysoTracker Rosso). Preparare questi nanosensori come descritto nella sezione 1.1, ma con l&#39;omissione di preparazione e aggiungendo TAMRA. </li><li> Dopo il periodo di consegna nanosensori diluire la soluzione LysoTracker Rosso a 50 nm. </li><li> Aggiungere una aliquota della soluzione diluita (2 ml) per cellule in coltura su scaffold PLGA. </li><li> Incubare le cellule con LysoTracker rosso per 1 ora (37 ° C, 5% CO 2). </li><li> Dopo il periodo di incubazione rimuovere il supporto e lavare le cellule due volte con PBS (pH 7,4) prima di cambiare il supporto di PBS (pH 7,4) (2 ml) in preparazione per la visualizzazione mediante microscopia confocale. Nota: fenolo media liberi rosse potrebbero essere utilizzati al posto di PBS. </li><li> Aggiungere la macchia nucleare DraQ5 al PBS così una concentrazione finale di 5 mM e incubare le cellule per 3 min (37 ° C 5% CO 2). Non è necessario cambiare il supporto successivo al periodo di incubazione con DRAQ5. </li><li> Utilizzare un laser a 488 nm argon per eccitare il FAM solo nanosensori, una krypton laser 568 nm per eccitare il LysoTracker Rossa e 633 nm elio / neon laser per eccitare il cromoforo DRAQ5 e osservare la fluorescenza a 500-530 nm, 580-600 nm, 650 -750 nm rispettivamente (Figura 3). Utilizzare la scansione sequenziale per evitare la raccolta di lunghezze d&#39;onda di eccitazione. </li></ol>

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Gli autori dichiarano di non avere interessi finanziari in competizione.

L&#39;ingegneria tissutale aspira a creare sostituti biologici che può essere utilizzato sia come in vivo come modelli in vitro e in tessuto in terapia sostitutiva tessuto di riparare, sostituire, mantenere o migliorare la funzione di un particolare tessuto o organo. Sostituti sintetici sono stati utilizzati per molti anni a sostituire o riparare ausilio di tessuti, ma questi spesso falliscono a causa della scarsa integrazione con il tessuto ospite e / o infezione, che alla fine porta al rifi...

Una strategia chiave nell&#39;ingegneria dei tessuti è l&#39;uso di materiali biocompatibili per fabbricare scaffold cui morfologia assomiglia al tessuto che sta per sostituire ed è anche in grado di sostenere la crescita e la funzione delle cellule 1,2. L&#39;impalcatura fornisce supporto meccanico consentendo attaccamento cellulare e la proliferazione tuttavia permette la migrazione cellulare durante gli interstizi di un costrutto cellulare 3D. Il ponteggio deve permettere il trasporto di massa di nutrienti cellulari e non inibire la rimozione dei rifiuti metabolici 3. Electrospinning è emerso come un metodo promettente per la realizzazione di supporti polimerici in grado di supportare la crescita cellulare 4-6. Le fibre elettrofilate non tessuti prodotti sono adatti per la crescita cellulare in quanto sono spesso porosi e permettono l&#39;interazione cellula-cellula e migrazione cellulare durante gli interstizi di un costrutto cellulare 3D 7. E &#39;importante monitorare la vitalità cellulare durante tl periodo di coltura e per garantire che la vitalità cellulare è mantenuta per tutto l&#39;arco del costrutto 3D. Ad esempio, condizioni di coltura come l&#39;ossigeno e pH richiedono un attento controllo, come gradienti di concentrazione di analiti possono esistere all&#39;interno del costrutto 3D. Bioreattori o sistemi di perfusione possono essere impiegati per simulare le condizioni in vivo di flusso interstiziale e come risultato un trasferimento incremento dei nutrienti e la rimozione metabolica rifiuti 8. La questione se tali sistemi sono garantire condizioni microambientali costanti può essere affrontato valutando il microambiente cellulare in tempo reale. Metriche chiave microambiente che possono essere monitorati in tempo reale le seguenti: temperatura, composizione chimica di supporti cellulari, concentrazione di ossigeno disciolto e anidride carbonica, pH e umidità. Di questi parametri, la temperatura può essere più facilmente monitorata utilizzando sonde in situ. Metodi per monitorare i restanti parametri di cui comunemente inrimozione volve di una aliquota di campionamento e quindi disturbare la coltura cellulare e aumentano il rischio di contaminazione. Si cercano continui, metodi in tempo reale. Metodi di controllo attuali di solito contare su strumenti che analizzano fisicamente il costrutto cellulare come un monitor pH o sonda ossigeno. Tuttavia, questi metodi intrusivi possono danneggiare il costrutto cellulare e disturbare l&#39;esperimento in corso. Monitoraggio non invasivo di concentrazioni dell&#39;analita all&#39;interno del costrutto 3D potrebbe consentire il monitoraggio in tempo reale di vari aspetti ambientali quali la riduzione dei nutrienti 9. Ciò consentirebbe di valutare apporto di sostanze nutritive alle regioni più profonde all&#39;interno della struttura e determinare se scorie metaboliche veniva eliminato in modo efficace 10,11. Sistemi che tentano di risolvere il problema di invasività genere comportano l&#39;uso di una camera di perfusione che passa mezzo di coltura sia attraverso il recipiente di coltura e ai sensori esterni per monitorare pH, ossigeno e glucosio 12. Ilre è crescente interesse nello sviluppo di sensori che possono essere integrati direttamente nel recipiente di coltura che non richiede la rimozione di una aliquota di campionamento e come tale sarebbe fornire monitoraggio in situ. Per affrontare tali carenze per in situ e monitoraggio non invasivo delle condizioni microambientali abbiamo incorporato analiti nanosensori reattivo in scaffolds elettrofilate per la produzione di ponteggi auto-segnalazione 13. Ponteggi che agiscono come dispositivi di rilevamento per il monitoraggio dell&#39;attività di fluorescenza sono stati preparati in precedenza, in cui il dispositivo di rilevamento era o scaffold polimerico effettivo creato da elettrospinning o attraverso l&#39;uso di un colorante reattivo analita che è incorporato nel polimero prima della formazione del ponteggio 14,15 . Tuttavia, questi dispositivi di rilevamento hanno il potenziale per dare uscite ottiche erronee causate da possibili interferenze di altri analiti. L&#39;uso di un dispositivo di rilevamento su raziometricoch come quelli preparati nel protocollo descritto ha il potenziale per eliminare questi possibili effetti negativi e fornire una risposta specifica per l&#39;analita in questione. I ponteggi elettrofilate qui presentati sono stati preparati dal sintetico co-polimero di poli (acido lattico-co-glicolico) (PLGA), selezionato a causa di avere Food and Drug Administration (FDA), grazie alle sue proprietà biodegradabili e biocompatibili ed una pista record di sostenere la crescita e la funzione dei vari tipi di cellule 16-18. Gli analiti raziometrico nanosensori reattivo pronti sono sensibili al pH. I nanosensori incorporano due coloranti fluorescenti in una matrice sol-gel biocompatibile in cui un colorante, FAM è sensibile al pH e l&#39;altra, TAMRA agisce come standard interno in quanto non è sensibile al pH. Inoltre la fluorescenza sia FAM e TAMRA può essere analizzata separatamente in quanto non si sovrappongono in modo significativo. Determinare il rapporto tra la emi fluorescenzaSsion di entrambi i coloranti a specifiche lunghezze d&#39;onda dà una risposta pH indipendente da altre condizioni ambientali. Le impalcature self-reporting potrebbero consentire la valutazione ripetizione del pH in situ e in tempo reale senza interrompere il modello 3D sviluppato. Abbiamo dimostrato che questi scaffold sono in grado di supportare attaccamento e la proliferazione cellulare e rimangono reattivo al analita in questione. La cinetica di acidi sottoprodotti di costrutti ingegnerizzati rimane poco studiato e come tale utilizzando i ponteggi reattive pH possono notevolmente facilitare tali studi 19. Inoltre, l&#39;uso dei ponteggi auto-segnalazione per applicazioni di ingegneria tissutale presenta l&#39;opportunità di comprendere appieno, monitorare e ottimizzare la crescita del modello 3d costrutti di tessuti in vitro, non invasivo e in tempo reale. I nanosensori reattiva di pH sono stati consegnati per l&#39;ambiente intracellulare di fibroblasti in coltura su electrospun PLGA ponteggiods. Il rapporto tra l&#39;emissione di fluorescenza dei coloranti sono stati utilizzati per monitorare pHi e rispetto ad un auto-segnalazione ponteggio Incorporando nanosensori pH. La consegna di nanosensori a cellule coltivate in un ambiente 3D potrebbe consentire il monitoraggio della concentrazione di analita in profondità all&#39;interno del costrutto in modo non distruttivo. Pertanto nanosensori possono essere uno strumento di imaging valida per valutare in modo non distruttivo il comportamento delle cellule in tutto 3D costruisce consentendo analisi a lungo termine. Screening della concentrazione dell&#39;analita di singole celle all&#39;interno di un costrutto 3D potrebbe assicurare che stanno ricevendo concentrazioni di nutrienti e di ossigeno sufficienti. Parametri di processo di monitoraggio potrebbe contribuire allo sviluppo di tecniche standardizzate per il trasporto di massa effettiva di ossigeno e nutrienti. La consegna dei nanosensori per l&#39;ambiente intracellulare e l&#39;incorporazione di nanosensori in scaffold polimerici potrebbe essere combinati per consentire la valutazione della vitalità cellulare così come la prestazione impalcatura all&#39;interno const 3Dructs durante il processo di crescita dei tessuti. Questo può portare ad un aumento della conoscenza di questi costrutti e progredire la realizzazione di sostituti del tessuto biologico pertinenti.

<table border="0" cellpadding="0" cellspacing="0" width="481">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">Ethanol</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:137px;">32221</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">Anhydrous dimethylformamide (DMF)</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">270547</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:195px;">Ammonium hydroxide 50% (v/v) aqueous solution</td>
			<td style="width:71px;">Alfa Aesar</td>
			<td style="width:137px;">35574</td>
			<td style="width:79px;">diluted to 30% (v/v) with pure water</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">TEOS</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">13190-3</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">3-Aminopropyltriethoxysilane (APTES)</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">A3648</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">5-(and-6)-carboxyfluorescein, succinimidyl ester (FAM-SE)</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:137px;">C1311</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:195px;">6-carboxytetramethylrhodamine, succinimidyl ester (TAMRA-SE)</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:137px;">C1171</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">Sodium phosphate monobasic (0.2 M)</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:137px;">S-9638</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">Sodium phosphate dibasic (0.2 M)</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:137px;">S-0876</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">NaOH</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:137px;">S8045</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">Trypsin/EDTA</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:137px;">T4174</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">Penicillin/Streptomycin</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:137px;">P0781</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">PBS</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:137px;">D8537</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">DMEM</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:137px;">D6046</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:195px;">FBS</td>
			<td style="width:71px;">Source Bioscience</td>
			<td style="width:137px;">Batch-213-101992</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">L-Glutamine</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:137px;">G7513</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">Lipofectamine 2000</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:137px;">11668-019</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">Optimem</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:137px;">11058-021</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">LysoTracker Red</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:137px;">L-7528</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">Draq5</td>
			<td style="width:71px;">Biostatus Ltd</td>
			<td style="width:137px;">DR50050</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">Nitrogen gas</td>
			<td style="width:71px;">BOC</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">DCM</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:137px;">320269</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">TFA</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:137px;">T6508</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="211">
			<td height="211" style="height:211px;width:195px;">Confocal microscope</td>
			<td style="width:71px;">Leica TCS-SP</td>
			<td style="width:137px;"></td>
			<td style="width:79px;">equipped with argon and krypton lasers and a 63X 0.9NA water immersion lens</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:195px;">UV light</td>
			<td style="width:71px;">UVLS28 UVP, USA</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:195px;">Stirrer plate</td>
			<td style="width:71px;">SB161-3 Jencons-PLS</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:195px;">pH meter</td>
			<td style="width:71px;">Jenway model 3510</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:195px;">Rotary Evaporator</td>
			<td style="width:71px;">Buchi Rotary Evaporator R200</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">Centrifuge (nanosensors)</td>
			<td style="width:71px;">Hermle Z300</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:195px;">Centrifuge (cell culture)</td>
			<td style="width:71px;">Thermo Scientific Heraeus Biofuge Primo</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:195px;">Vortex</td>
			<td style="width:71px;">Whirlimixer Fisherbrand</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:195px;">Ultrasonicator</td>
			<td style="width:71px;">FB11021 Fisherbrand</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:195px;">Aluminum sheet</td>
			<td style="width:71px;">Nottingham University</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:195px;">35mm cell culture plate</td>
			<td style="width:71px;">Iwaki</td>
			<td style="width:137px;">3000035</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:195px;">10 ml syringe</td>
			<td style="width:71px;">Becton Dickenson</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;"></td>
			<td style="width:71px;"></td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td style="width:71px;"></td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:195px;">3T3 Fibroblast cells</td>
			<td style="width:71px;">European Collection of Cell Cultures</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:195px;">PLGA</td>
			<td style="width:71px;">Lakeshore Biomaterials</td>
			<td>7525 DLG 7E</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Pyridinium formate</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td>P8535</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Trypan blue</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td>T8154</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Sodium phosphate monobasic</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td>S9638</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Sodium phosphate dibasic</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td>S5136</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">HCl</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td>320331</td>
			<td style="width:79px;"></td>
		</tr>
	</tbody>
</table>

self-reporting scaffolds for 3-dimensional cell culture

Celle di coltura in 3D su ponteggi adeguati è pensato per imitare al meglio il microambiente in vivo e aumentare le interazioni cellula-cellula. La risultante costrutto cellulare 3D può essere spesso più rilevante per studiare gli eventi molecolari e le interazioni cellula-cellula di esperimenti simili studiati in 2D. Per creare culture efficaci 3D con la vitalità delle cellule elevata in tutto il patibolo le condizioni di coltura come ossigeno e pH bisogno di essere attentamente controllata come gradienti di concentrazione di analiti possono esistere in tutto il costrutto 3D. Qui si descrivono i metodi di preparazione biocompatibili pH reattivo nanosensori sol-gel e la loro incorporazione in poli (acido lattico-co-glicolico) (PLGA) ponteggi elettrofilate insieme a loro successiva preparazione per la coltura di cellule di mammifero. I ponteggi reattivo pH possono essere utilizzati come strumenti per determinare microambientali pH all&#39;interno di un costrutto cellulare 3D. Inoltre, tendiamo particolare la consegna di pH nanose reattivansors per l&#39;ambiente intracellulare di cellule di mammifero la cui crescita è stata sostenuta da elettrofilate scaffold PLGA. La posizione citoplasmatica dei nanosensori reattive pH può essere utilizzato per monitorare pH intracellulare (PHI) durante la sperimentazione in corso.

La distribuzione dimensionale delle pH nanosensori reattivi preparati è stata caratterizzata mediante SEM, dove la popolazione di nanosensori impressionate stati misurati e risultano avere dimensioni nanometriche nell&#39;intervallo 240-470 nm (Figura 1A). Il raggiungimento di un diametro ridotto e ragionevolmente piccolo è coerente con l&#39;utilizzo del metodo Stöber per preparare nanoparticelle. È stato trovato che utilizzando un ambiente basico pH durante la sintesi di nanoparticelle IE...

Watch this Scientific Journal Video about Self-reporting Scaffolds for 3-Dimensional Cell Culture at JoVE.com

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Biocompatibili pH reattivo nanosensori sol-gel possono essere incorporati in poli (acido lattico-co-glicolico) (PLGA) ponteggi elettrofilate. Le impalcature self-reporting prodotti possono essere utilizzati per il monitoraggio in situ delle condizioni microambientali mentre coltura di cellule sul patibolo. Questo è vantaggioso in quanto il costrutto cellulare 3D può essere monitorato in tempo reale senza disturbare l&#39;esperimento.

Ponteggi self-reporting per coltura cellulare 3-Dimensional

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

self-reporting-scaffolds-for-3-dimensional-cell-culture

Research

JoVE Journal

Bioengineering

13.1K Views.  University of Nottingham.  Biocompatibili pH reattivo nanosensori sol-gel possono essere incorporati in poli (acido lattico-co-glicolico) (PLGA) ponteggi elettrofilate. Le impalcature self-reporting prodotti possono essere utilizzati per il monitoraggio in situ delle condizioni microambientali mentre coltura di cellule sul patibolo. Questo è vantaggioso in quanto il costrutto cellulare 3D può essere monitorato in tempo reale senza disturbare l'esperimento. 

Video: Self-reporting Scaffolds for 3-Dimensional Cell Culture

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and increasing obesity1. Currently, bypass surgery using autologous vessels, allografts, and synthetic grafts are known as a commonly used for arterial substitutes2. However, these grafts have limited applications when an inner diameter of arteries is less than 6 mm due to low availability, thrombotic complications, compliance mismatch, and late intimal hyperplasia3,4. To overcome these limitations, tissue engineering has been successfully applied as a promising alternative to develop small-diameter arterial constructs that are nonthrombogenic, robust, and compliant. Several previous studies have developed small-diameter arterial constructs with tri-lamellar structure, excellent mechanical properties and burst pressure comparable to native arteries5,6. While high tensile strength and burst pressure by increasing collagen production from a rigid material or cell sheet scaffold, these constructs still had low elastin production and compliance, which is a major problem to cause graft failure after implantation. Considering these issues, we hypothesized that an elastometric biomaterial combined with mechanical conditioning would provide elasticity and conduct mechanical signals more efficiently to vascular cells, which increase extracellular matrix production and support cellular orientation.
The objective of this report is to introduce a fabrication technique of porous tubular scaffolds and a dynamic mechanical conditioning for applying them to arterial tissue engineering. We used a biodegradable elastomer, poly (glycerol sebacate) (PGS)7 for fabricating porous tubular scaffolds from the salt fusion method. Adult primary baboon smooth muscle cells (SMCs) were seeded on the lumen of scaffolds, which cultured in our designed pulsatile flow bioreactor for 3 weeks. PGS scaffolds had consistent thickness and randomly distributed macro- and micro-pores. Mechanical conditioning from pulsatile flow bioreactor supported SMC orientation and enhanced ECM production in scaffolds. These results suggest that elastomeric scaffolds and mechanical conditioning of bioreactor culture may be a promising method for arterial tissue engineering.

Elastomeric PGS scaffolds with vascular smooth muscle cells cultured in a pulsatile flow bioreactor may lead to promising small-diameter arterial constructs with native ECM production in a relatively short culture period.

PGS elastomerico Ponteggi in Ingegneria tessuto arterioso

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and ...

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A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro follicle techniques provide a tool to model follicle development in order to investigate basic biology, and are further being developed as a technique to preserve fertility in the clinic1-4. Our in vitro culture system employs hydrogels in order to mimic the native ovarian environment by maintaining the 3D follicular architecture, cell-cell interactions and paracrine signaling that direct follicle development 5. Previously, follicles were successfully cultured in alginate, an inert algae-derived polysaccharide that undergoes gelation with calcium ions6-8. Alginate hydrogels formed at a concentration of 0.25% w/v were the most permissive for follicle culture, and retained the highest developmental competence 9. Alginate hydrogels are not degradable, thus an increase in the follicle diameter results in a compressive force on the follicle that can impact follicle growth10. We subsequently developed a culture system based on a fibrin-alginate interpenetrating network (FA-IPN), in which a mixture of fibrin and alginate are gelled simultaneously. This combination provides a dynamic mechanical environment because both components contribute to matrix rigidity initially; however, proteases secreted by the growing follicle degrade fibrin in the matrix leaving only alginate to provide support. With the IPN, the alginate content can be reduced below 0.25%, which is not possible with alginate alone 5. Thus, as the follicle expands, it will experience a reduced compressive force due to the reduced solids content. Herein, we describe an encapsulation method and an in vitro culture system for ovarian follicles within a FA-IPN. The dynamic mechanical environment mimics the natural ovarian environment in which small follicles reside in a rigid cortex and move to a more permissive medulla as they increase in size11. The degradable component may be particularly critical for clinical translation in order to support the greater than 106-fold increase in volume that human follicles normally undergo in vivo .

A new method for ovarian follicle encapsulation in a 3D fibrin-alginate interpenetrating network is described. This system combines structural support with proteolytic degradation to support the development of immature follicles to produce mature oocytes. This method may be applied to culture cell aggregates to maintain cell-cell contacts without limiting expansion.

Un metodo per l&#39;incapsulamento follicolo ovarico e la cultura in un proteoliticamente degradabile 3 Sistema dimensionale

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro  follicle techniques provide ...

Shape Memory Polymers for Active Cell Culture

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent shape upon the application of a stimulus such as heat1-5. In a typical shape memory cycle, the SMP is first deformed at an elevated temperature that is higher than its transition temperature, Ttrans [either the melting temperature (Tm) or the glass transition temperature (Tg)]. The deformation is elastic in nature and mainly leads to a reduction in conformational entropy of the constituent network chains (following the rubber elasticity theory). The deformed SMP is then cooled to a temperature below its Ttrans while maintaining the external strain or stress constant. During cooling, the material transitions to a more rigid state (semi-crystalline or glassy), which kinetically traps or "freezes" the material in this low-entropy state leading to macroscopic shape fixing. Shape recovery is triggered by continuously heating the material through Ttrans under a stress-free (unconstrained) condition. By allowing the network chains (with regained mobility) to relax to their thermodynamically favored, maximal-entropy state, the material changes from the temporary shape to the permanent shape.
Cells are capable of surveying the mechanical properties of their surrounding environment6. The mechanisms through which mechanical interactions between cells and their physical environment control cell behavior are areas of active research. Substrates of defined topography have emerged as powerful tools in the investigation of these mechanisms. Mesoscale, microscale, and nanoscale patterns of substrate topography have been shown to direct cell alignment, cell adhesion, and cell traction forces7-14. These findings have underscored the potential for substrate topography to control and assay the mechanical interactions between cells and their physical environment during cell culture, but the substrates used to date have generally been passive and could not be programmed to change significantly during culture. This physical stasis has limited the potential of topographic substrates to control cells in culture.
Here, active cell culture (ACC) SMP substrates are introduced that employ surface shape memory to provide programmed control of substrate topography and deformation. These substrates demonstrate the ability to transition from a temporary grooved topography to a second, nearly flat memorized topography. This change in topography can be used to control cell behavior under standard cell culture conditions.

A method for developing cell culture substrates with the ability to change topography during culture is described. The method makes use of smart materials known as shape memory polymers that have the ability to memorize a permanent shape. This concept is adaptable to a wide range of materials and applications.

Shape Memory Polymers per la Cultura cella attiva

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent ...

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and metastasis of tumor cells. Migration has been studied on glass coverslips in order to make cellular dynamics amenable to investigation by light microscopy. However, it has become clear that many aspects of cell migration depend on features of the local environment including its elasticity, protein composition, and pore size, which are not faithfully represented by rigid two dimensional substrates such as glass and plastic 1. Furthermore, interaction with other cell types, including stromal fibroblasts 2 and immune cells 3, has been shown to play a critical role in promoting the invasion of cancer cells. Investigation at the molecular level has increasingly shown that molecular dynamics, including response to drug treatment, of identical cells are significantly different when compared in vitro and in vivo 4. 
Ideally, it would be best to study cell migration in its naturally occurring context in living organisms, however this is not always possible. Intermediate tissue culture systems, such as cell derived matrix, matrigel, organotypic culture (described here) tissue explants, organoids, and xenografts, are therefore important experimental intermediates. These systems approximate certain aspects of an in vivo environment but are more amenable to experimental manipulation such as use of stably transfected cell lines, drug treatment regimes, long term and high-resolution imaging. Such intermediate systems are especially useful as proving grounds to validate probes and establish parameters required to image the dynamic response of cells and fluorescent reporters prior to undertaking imaging in vivo 5. As such, they can serve an important role in reducing the need for experiments on living animals.

A method is described for the preparation of a 3-dimensional matrix consisting of collagen type I and primary human fibroblasts. This organotypic gel serves as a useful substrate to assess invasive cell migration because it mimics basic features of tissue stroma and is amenable to many forms of microscopy.

Organotipiche Collagene I test: una piattaforma malleabile per valutare Comportamento cella in un 3-dimensionale Contesto

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.
The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13.
We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. 
In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.

Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.

Rotazione Cultura sistemi cellulari per colture cellulari umane: cellule del trofoblasto umano come modello

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

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 ...

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

The influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally neuronal differentiation is of great interest in order to find new methods for cell-based and standardised therapies in neurological disorders or neurodegenerative diseases. 3D structures are expected to provide an environment much closer to the in vivo situation than 2D cultures. In the context of regenerative medicine, the combination of biomaterial scaffolds with neural stem and progenitor cells holds great promise as a therapeutic tool.1-5 Culture systems emulating a three dimensional environment have been shown to influence proliferation and differentiation in different types of stem and progenitor cells. Herein, the formation and functionalisation of the 3D-microenviroment is important to determine the survival and fate of the embedded cells.6-8 Here we used PuraMatrix9,10 (RADA16, PM), a peptide based hydrogel scaffold, which is well described and used to study the influence of a 3D-environment on different cell types.7,11-14 PuraMatrix can be customised easily and the synthetic fabrication of the nano-fibers provides a 3D-culture system of high reliability, which is in addition xeno-free.
Recently we have studied the influence of the PM-concentration on the formation of the scaffold.13 In this study the used concentrations of PM had a direct impact on the formation of the 3D-structure, which was demonstrated by atomic force microscopy. A subsequent analysis of the survival and differentiation of the hNPCs revealed an influence of the used concentrations of PM on the fate of the embedded cells. However, the analysis of survival or neuronal differentiation by means of immunofluorescence techniques posses some hurdles. To gain reliable data, one has to determine the total number of cells within a matrix to obtain the relative number of e.g. neuronal cells marked by &beta;III-tubulin. This prerequisites a technique to analyse the scaffolds in all 3-dimensions by a confocal microscope or a comparable technique like fluorescence microscopes able to take z-stacks of the specimen. Furthermore this kind of analysis is extremely time consuming. 
Here we demonstrate a method to release cells from the 3D-scaffolds for the later analysis e.g. by flow cytometry. In this protocol human neural progenitor cells (hNPCs) of the ReNcell VM cell line (Millipore USA) were cultured and differentiated in 3D-scaffolds consisting of PuraMatrix (PM) or PuraMatrix supplemented with laminin (PML). In our hands a PM-concentration of 0.25% was optimal for the cultivation of the cells13, however the concentration might be adapted to other cell types.12 The released cells can be used for e.g. immunocytochemical studies and subsequently analysed by flow cytometry. This speeds up the analysis and more over, the obtained data rest upon a wider base, improving the reliability of the data.

Here we describe the use of a self-assembling 3-dimensional scaffold to culture human neural progenitor cells. We present a protocol to release the cells from the scaffolds to be analysed subsequently e.g. by flow cytometry. This protocol might be adapted to other cell types to perform detailed mechanistically studies.

La coltivazione di cellule progenitrici neurali umane in un 3-dimensionale di auto-assemblaggio Peptide Hydrogel

The  influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally  neuronal differentiation is of great interest in order to find new ...

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.

Cartilage repair represents an unmet medical challenge and cell-based approaches to engineer human articular cartilage are a promising solution. Here, we describe three-dimensional (3D) biomimetic hydrogels as an ideal tool for the expansion and maturation of human articular chondrocytes.

3D Hydrogel Ponteggi per articolare condrociti, Cultura e Cartilagine Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer ...

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model substrates for organ-scale tissue development. Sprague Dawley rat kidneys are cannulated by inserting a catheter into the renal artery and perfused with a series of low-concentration detergents (Triton X-100 and sodium dodecyl sulfate (SDS)) over 26 hr to derive intact, whole-kidney scaffolds with intact perfusable vasculature, glomeruli, and renal tubules. Following decellularization, the renal scaffold is placed inside a custom-designed perfusion bioreactor vessel, and the catheterized renal artery is connected to a perfusion circuit consisting of: a peristaltic pump; tubing; and optional probes for pH, dissolved oxygen, and pressure. After sterilizing the scaffold with peracetic acid and ethanol, and balancing the pH (7.4), the kidney scaffold is prepared for seeding via perfusion of culture medium within a large-capacity incubator maintained at 37 &#176;C and 5% CO2. Forty million renal cortical tubular epithelial (RCTE) cells are injected through the renal artery, and rapidly perfused through the scaffold under high flow (25 ml/min) and pressure (~230 mmHg) for 15 min before reducing the flow to a physiological rate (4 ml/min). RCTE cells primarily populate the tubular ECM niche within the renal cortex, proliferate, and form tubular epithelial structures over seven days of perfusion culture. A 44 &#181;M resazurin solution in culture medium is perfused through the kidney for 1 hr during medium exchanges to provide a fluorometric, redox-based metabolic assessment of cell viability and proliferation during tubulogenesis. The kidney perfusion bioreactor permits non-invasive sampling of medium for biochemical assessment, and multiple inlet ports allow alternative retrograde seeding through the renal vein or ureter. These protocols can be used to recellularize kidney scaffolds with a variety of cell types, including vascular endothelial, tubular epithelial, and stromal fibroblasts, for rapid evaluation within this system.

This protocol describes decellularization of Sprague Dawley rat kidneys by antegrade perfusion of detergents through the vasculature, producing acellular renal extracellular matrices that serve as templates for repopulation with human renal epithelial cells. Recellularization and use of the resazurin perfusion assay to monitor growth is performed within specially-designed perfusion bioreactors.

Ripopolamento cellule epiteliali e preparazione di roditori matrice extracellulare Ponteggi per lo sviluppo del tessuto renale

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model ...

Synthesis of Biocompatible Liquid Crystal Elastomer Foams as Cell Scaffolds for 3D Spatial Cell Cultures

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block co-polymers featuring cholesterol units as side-chain pendant groups, resulting in smectic-A (SmA) liquid crystal elastomers (LCEs). Foam-like scaffolds, prepared using metal templates, feature interconnected microchannels, making them suitable as 3D cell culture scaffolds. The combined properties of the regular structure of the metal foam and of the elastomer result in a 3D cell scaffold that promotes not only higher cell proliferation compared to conventional porous templated films, but also better management of mass transport (i.e., nutrients, gases, waste, etc.). The nature of the metal template allows for the easy manipulation of foam shapes (i.e., rolls or films) and for the preparation of scaffolds of different pore sizes for different cell studies while preserving the interconnected porous nature of the template. The etching process does not affect the chemistry of the elastomers, preserving their biocompatible and biodegradable nature. We show that these smectic LCEs, when grown for extensive time periods, enable the study of clinically relevant and complex tissue constructs while promoting the growth and proliferation of cells.

This study presents a methodology to prepare 3D, biodegradable, foam-like cell scaffolds based on biocompatible side-chain liquid crystal elastomers (LCEs). Confocal microscopy experiments show that foam-like LCEs allow for cell attachment, proliferation, and the spontaneous alignment of C2C12s myoblasts.

Sintesi di biocompatibili a cristalli liquidi in elastomero Schiume come impalcature cellulari per 3D spaziali colture cellulari

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block ...