Gli autori desiderano ringraziare il finanziamento parziale del progetto di ricerca FP7 NAMDIATREAM europea (NMP4-LA-2010-246479, http://www.namdiatream.eu) e il progetto INTERREG IV Francia-Svizzera NAOMI.

1. Cultura e Espansione del CES umano <ol><li> Preparare il mezzo di propagazione delle cellule (chiamata PM) contenente Knockout DMEM integrato con il 20% Knockout siero, 1% MEM non-aminoacidi essenziali, 1% di L-glutammina 200 mm, 1% penicillina-streptomicina e 3,5 microlitri β-mercaptoetanolo. </li><li> Scongelare ESC umane (hESC) in 8 ml di PM medio e centrifugare le 5 min a 115 xg per scartare DMSO-integrato medio di congelamento. </li><li> Aggiungere 1 ml di PM mezzo contenente 10 mM roccia inibitore per impedire l&#39;apoptosi e migliorare la sopravvivenza cellulare dopo lo scongelamento (solo 24 ore di incubazione). </li><li> Aggiungere fattore di crescita dei fibroblasti (bFGF) al PM alla concentrazione desiderata (4-10 ng / ml) per il mantenimento della pluripotenza. </li><li> Piatto il hESC sul umana irradiati prepuzio cellule di alimentazione (γHFF) (Figura 1A), precedentemente placcato ad una concentrazione di 2 x 10 4 cellule / cm 2. </li><li> Cambiare medio giornaliero e, se necessario, rimuovere parti di colonies iniziando a differenziarsi per aspirazione usando una forma allungata taglio deciso pipetta Pasteur. </li><li> Una volta a settimana, colonie passaggio enzimaticamente: <ol><li> Rimuovere medie e lavare le colonie con 2 ml di PBS. </li><li> Rimuovere PBS e incubare con 0.5 ml per pozzetto di caldo collagenasi IV a 1 mg / ml per 10 min a 37 ° C, 5% CO 2. </li><li> Raccogliere i frammenti colonia in 2 ml nuovo PM e centrifugare a 115 xg per 3 min. </li></ol></li><li> In alternativa, le colonie possono essere diversi passaggi meccanicamente mediante raschiatura: <ol><li> Rimuovere medie e lavare le colonie con 2 ml di PBS. </li><li> Raschiare manualmente colonie utilizzando lo strumento raschietto rullo StemPro EZPassage (Figura 1B). </li><li> Raccogliere frammenti colonia e poi centrifugare per 3 min a 115 xg per rimuovere il surnatante. </li><li> Frammenti piastra su γHFF o li elaborano per la differenziazione in corpi embrionali (EBS). </li></ol></li></ol> 2. ESC Differenziazione umanoProtocollo <ol><li> Preparare il terreno di differenziamento (DM) utilizzando Knockout DMEM supplementato con siero Hyclone 2%, 1,0% MEM non-aminoacidi essenziali, 1,0% di L-glutammina 200 mm, 1% penicillina-streptomicina, 3,5 microlitri β-mercaptoetanolo. </li><li> Rimuovere medie e lavare le colonie con 2 ml di PBS. </li><li> La formazione EB di frammenti colonia in sospensione: <ol><li> Rimuovere PBS e incubare con 0.5 ml per pozzetto di caldo collagenasi IV a 1 mg / ml per 10 min a 37 ° C, 5% CO 2. </li><li> Raccogliere i frammenti colonia in 2 ml nuovo DM e centrifugare a 115 xg per 3 min. </li><li> Li coltura in sospensione in ultra-bassa allegato 6 pozzetti (2 ml per pozzetto) per 4 giorni (Figura 1C). </li><li> Raccogliere e piastra EBS neoformati su 0,1% piastre da 24 pozzetti rivestiti di gelatina (circa 3 EBs / pozzetto) (Figura 1D). </li></ol></li><li> In alternativa, forzata aggregazione di singole cellule nel EBs usando la piastra Aggrewell: <ol><li> Rimuovere PBS edissociare le colonie nelle singole cellule utilizzando accutase (0,5 ml / pozzetto). </li><li> Centrifugare le cellule e risospendere in DM a 1.2 x 10 6 / ml. </li><li> Aggiungere 2 ml per camera Aggrewell per 1 giorno (Figura 1B &#39;). </li><li> Raccogliere EBs neoformati (Figura 1C &#39;) e piatto su 0,1% piastre da 24 pozzetti rivestiti di gelatina (circa 3 EBs / pozzetto). </li><li> Cambiare DM ogni 2-3 giorni fino alla comparsa di battere gruppi di cardiomiociti (15-30 giorni). </li></ol></li></ol> 3. Culture 3D aria-liquido di battere cluster e di etichettatura con HNPS <ol><li> Identificare e analizzare manualmente grappoli di battere cardiomiociti (Fig. 1D), di solito compaiono dopo 2-4 settimane di cultura, con una forma allungata drasticamente tagliato pipetta Pasteur. </li><li> Deposito 4 filtri in PTFE per inserto che verranno messi su una piastra da 6 pozzetti (Figura 1F). </li><li> Aggiungere 1 ml di terreno DM in cima each bene. </li><li> Aggiungi un cluster sezionato batte su ogni filtro PTFE (Figura 1E), massimo 4/well, cioè 24 aggregati per piastra (Figura 1F &#39;). </li><li> Cambiare medio DM ogni 2-3 giorni. </li><li> Per etichettare i cluster, rimuovere il filtro PTFE contenente il cluster pestaggio e metterlo su 3,5 centimetri di vetro piatto fondo (170 micron di spessore). </li><li> Aggiungere 1 ml di HNP ad una concentrazione di 50 ug / ml per 30 min. </li></ol> 4. Imaging ottico non lineare <ol><li> Preparazione del campione. Dopo l&#39;etichettatura HNP (vedi punto 3.6), trasferire sia intere che differenziano primi HeBS o cluster battito cardiaco (sezionati su comparsa di contrazione), su filtri in PTFE su un piatto fondo di vetro di spessore 170 micron compatibile con la distanza di lavoro di obiettivi di apertura numerici elevati. In alternativa, applicare un rivestimento in diretta di battere cluster per il piatto fondo di vetro pre-rivestite con 0,1% di gelatina. </li><li> Campioni di trasferimento ad unall-in-one microscopio dotato di camera di incubazione assicurare temperatura stabile, umidità e CO 2 controllo su periodi di tempo prolungati. </li><li> Ispezione iniziale, immagine dopo che il campione attraverso l&#39;imaging a grande campo sotto illuminazione a luce bianca per identificare strutture di interesse all&#39;interno EBs dissociati o cluster battitori attivi che saranno selezionati per ulteriori accertamenti. </li><li> Se autofluorescenza cellulare da NADH e SH da HNPS devono essere acquisite simultaneamente, impostare una lunghezza d&#39;onda di eccitazione laser più corta (cioè 720 nm) per abbinare l&#39;assorbimento molecolare. Utilizzare una stretta filtro passa-banda spettrale centrato a metà della lunghezza d&#39;onda di eccitazione (cioè, λ = 360 ± 10 nm) per rilevare SH, e un altro per rilevare autofluorescenza. </li><li> Innanzitutto eseguire, bassa risoluzione veloce, scansione tridimensionale per ricostruire la morfologia complessiva del cluster cardiaca e selezionare un piano dell&#39;immagine all&#39;interno del volume cluster con vari visibile HNPs. </li><li> Se necessario, modificare la lunghezza d&#39;onda di eccitazione liberamente abbinare meglio le proprietà ottiche del campione o per evitare photobleaching, danni fotografia e all&#39;immagine più in profondità i tessuti con una lunghezza d&#39;onda IR (ad esempio, 790 nm) e sostituire il secondo filtro armonico di conseguenza (ad esempio, 395 nm ). Ottimizzare le impostazioni del pre-compressore impulso laser alla lunghezza d&#39;onda di lavoro di scelta massimizzando il secondo segnale armonico di HNPS depositati sul campione. </li><li> Per monitorare in tempo reale le contrazioni cardiache del cluster, impostare lo scanner ottico microscopio in modo più veloce come modo risonante, consentendo l&#39;acquisizione ad alta velocità di immagini bidimensionali. </li><li> Scegliere le impostazioni come miglior compromesso tra contrasto dell&#39;immagine e velocità di acquisizione, come ad esempio: <ul type="disc"><li> Media Scan: 4 </li><li> Pixel tempo di sosta: 0.1 msec </li><li> Tasso Image: 3.75 fotogrammi al secondo (fps) </li></ul></li><li> Potenza laser viene regolata in base alla focale punto sizzare e velocità di scansione. 50 mW (misurata all&#39;ingresso dell&#39;obiettivo) è un valore tipico per 0,1 msec pixel tempo di sosta e il piano APO 20X NA 0.75 obiettivo preservare la vitalità cellulare. </li></ol> 5. Image Analysis <ol><li> Procedura di calibrazione assiale. Selezionare un singolo HNP depositato su un substrato nudo. Regolare il fuoco (cioè la posizione assiale della nanoparticella) per massimizzare l&#39;intensità SH. Variare in modo controllato la posizione assiale della fase microscopio avere la curva di taratura relativa intensità SH in funzione del HNP disassato rispetto al piano focale. L&#39;output di questa procedura di calibrazione per il 0,75 dell&#39;obiettivo Piano APO 20X NA è riportato in Figura 4C. </li><li> Selezionare HNPS presenti in tutti i fotogrammi video e determinare i loro movimenti periodici. Questa procedura può essere facilmente automatizzato, ad esempio mediante un codice personalizzato in cerca di massimo segnale macchie di intensità in una regione definita di interesse ecollegandoli tra i diversi fotogrammi (un esempio di codice scritto in Matlab R2009 b utilizzando la funzione &#39;puntelli regione &quot;del Toolbox Image Processing è presentato). </li><li> Valutare la quantificazione dei out-of-plane spostamenti, associati HNPS non continuo rintracciabili muove lungo l&#39;asse ottico, convertendo le loro modulazioni di intensità per tutto differenti cornici in spostamento assiale utilizzando la curva di taratura nella Fase 5.1. Si noti che questa procedura permette di accedere ai movimenti assiali, ma non può essere utilizzata per determinare la direzione assoluta (verso l&#39;alto o verso il basso). </li></ol>

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L&#39;applicazione delle nanotecnologie alla ricerca sulle cellule staminali è un settore relativamente nuovo, ma in rapida espansione. Come sottolineato da vari articoli di rassegna sull&#39;argomento, l&#39;uso di nanoparticelle può essere applicato per realizzare diversi compiti di ricerca, che vanno dalla cella di inseguimento (sia in vitro che in vivo), alla consegna intracellulare di proteine ​​e geni, non ultimo la creazione di ambienti cellulari biomimetici per preferenziale stimolazione / inibizi...

Sistemi di microscopia non lineari, grazie alle loro intrinseche capacità di sezionamento tre dimensioni, hanno sempre innescato la richiesta di fluorofori foto-stabile con bande di assorbimento a due fotoni nel vicino infrarosso. Solo negli ultimi due anni, per completare lo sviluppo di etichette a base di fluorescenza (coloranti, quantum dots, la conversione di nanoparticelle), una metodologia di imaging diverso ha sfruttato l&#39;uso di una nuova famiglia di nanoparticelle intrinsecamente non lineari come etichette, cioè nanoparticelle armonica (HNPS) che sono stati specificamente sviluppati per microscopia multi-fotone. Queste etichette, basati su cristalli noncentrosymmetric inorganici, esercitano contrasto ottico generando la SH della frequenza di eccitazione: per esempio convertendo una frazione di luce di eccitazione pulsata vicino infrarosso (λ = 800 nm) in luce blu visibile (λ / 2 = 400 nm) . Diversi autori nel recente passato hanno testato diversi materiali, tra cui iodato di ferro Fe (IO <sub&gt; 3) 3 1, niobato di potassio (KNbO 3) 2, il niobato di litio (LiNbO 3) 3, titanato di bario (batio 3) 4,5, potassio titanile fosfato (KTiOPO4, KTP) 6-8, e ossido di zinco (ZnO ) 5,9,10. Rispetto alle sonde fluorescenti, HNPS possiedono una serie di caratteristiche interessanti, come la completa assenza di candeggio e lampeggiante, bande di emissione strette, di eccitazione lunghezza d&#39;onda sintonizzabilità (da raggi ultravioletti a infrarossi), la capacità di reperimento di orientamento, e la risposta ottica coerente. Queste proprietà uniche sono state recentemente spiegato in due Opinione documenti completi 11,12. La possibilità di lavorare nella regione spettrale dell&#39;infrarosso, che aumenta la profondità di imaging minimizzando dispersione e l&#39;assorbimento, anche limita drasticamente foto campione degradazione 13,14. Inoltre, il segnale photo-stabile infinitamente garantito da HNPS li rende ideali per sonde di monitoraggio cella a lungo termine, che is particolarmente attraente per le applicazioni di medicina rigenerativa 15. In questo esperimento visualizzato, dettagli del protocollo sono previste per l&#39;etichettatura in vitro di cellule staminali embrionali umane (hESC) con HNPS unfunctionalized. La sintesi e la preparazione di sospensioni colloidali è dettagliato in una precedente pubblicazione e riferimenti ivi 16 ed è oltre la portata di questo lavoro. Metodologie di indagine hESC mediante microscopia multi-fotone e la loro differenziazione in gruppi cardiaci (mantenuti culture aria-liquido lungo termine) sono presentati. ESC umana può essere lasciato per differenziare entro cosiddetti corpi embrionali (EBS) in due modi diversi, sia per la formazione di frammenti EB colonie in sospensione o, in alternativa, l&#39;aggregazione di singole cellule nel EBs utilizzando la piastra Aggrewell forzati, come illustrato nella Figura 1A . Coltura battendo ammassi di cellule cardiache in politetrafluoroetilene (PTFE) filtri porosi facilitates loro mantenimento a lungo termine per ulteriori studi (per esempio misurazioni elettrofisiologiche dei potenziali d&#39;azione). La sorgente di eccitazione del microscopio a scansione dovrebbe essere in grado di fornire impulsi ultracorti (con durata dell&#39;impulso minore di 300 fsec al campione) per raggiungere la potenza di picco necessaria per effettuare l&#39;imaging seconda armonica di HNPS. Per esempio, il fsec-source più utilizzato per l&#39;imaging sono accordabili Ti: zaffiro laser. In alternativa, altri laser ultraveloci possono essere impiegati, ad esempio, erbio ione 17, cromo forsterite 18 Ti: zaffiro pompato infrarossi oscillatori parametrici ottici. Il microscopio può essere dotata di un obiettivo con preferibilmente una piuttosto alta apertura numerica. Molto importante, prima della misura, e ogni volta che l&#39;obiettivo viene sostituito, è obbligatorio per minimizzare la dispersione presenti nel set-up (lenti) ottimizzando le impostazioni dell&#39;impulso laser pre-compressore al lavorolunghezza d&#39;onda di scelta. Questo procedimento, descritto nel protocollo, assicura che l&#39;impulso laser è il più vicino possibile alla trasformazione durata limitata (cioè più breve possibile) in corrispondenza del piano focale e massimizza la risposta di esempio non lineare. L&#39;obiettivo dell&#39;analisi dell&#39;immagine descritto alla fine del protocollo è di identificare e tracciare in movimenti 3D HNPS associati alle contrazioni ritmiche di battere cluster cardiaci. Inseguimento nanoparticelle nel piano dell&#39;immagine è semplicemente realizzato identificando le loro posizioni in fotogrammi di film successivi. Per estrarre le informazioni sul movimento assiale, una prima taratura della risposta intensità lineare in funzione dello spostamento assiale è obbligatoria. Si noti che per misurazioni a lungo termine, un controllo interferometrico attivo della posizione assiale campione è necessario per mantenere la validità della curva di calibrazione in presenza di derive termiche e / o meccaniche. I HNPS utilizzati qui per traccellule e battendo all&#39;interno degli aggregati sono basati su ossido di niobato di potassio (KNbO 3), ma altri nanomateriali lineari disponibili sono esaminati in dettaglio nel lavoro di Staedler et al 16. Le efficienze ottici non lineari della maggior parte dei nanomateriali esaminati finora sono molto simili. La scelta per KNbO 3 era essenzialmente motivata dalla buona stabilità della soluzione colloidale e la sua buona biocompatibilità, testato su diverse linee cellulari umane anche a concentrazione piuttosto elevata e lunghi tempi di esposizione 16. Data la novità del nanomateriale impiegato per questo lavoro, le principali caratteristiche del HNPS rispetto a / luminescenti bio-marcatori fluorescenti sono mostrati in una breve animazione computer originale realizzato dagli autori.

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			<td height="22" style="height:22px;width:231px;">Microscope Incubator</td>
			<td style="width:231px;">OKO LAB</td>
			<td style="width:205px;">UNO package (top stage)</td>
			<td style="width:343px;">&#160;37&#176;C, 5% CO2, moisturized</td>
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		<tr height="21">
			<td height="64" rowspan="2" style="height:64px;width:231px;">Multiphoton microscope</td>
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		<tr height="43">
			<td height="43" style="height:43px;width:343px;">Fast scanning, four non photomultiplier descanned detectors</td>
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		<tr height="21">
			<td height="64" rowspan="3" style="height:64px;width:231px;">Filters SHG and autofluorescence</td>
			<td rowspan="3" style="width:231px;">Semrock</td>
			<td style="width:205px;">FF01-360/12-25</td>
			<td rowspan="3" style="width:343px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:205px;">FF01-395/11-25&#160;</td>
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			<td height="22" style="height:22px;width:205px;">FF02-485/20-25</td>
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			<td style="width:205px;">CFI Plan Fluor 10x</td>
			<td style="width:343px;">NA 0.30, WD 16 mm</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:205px;">CFI Plan Apo 20x</td>
			<td style="width:343px;">NA 0.75, WD 1.0 mm, VC</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:205px;">CFI Apo 40x</td>
			<td style="width:343px;">NA 1.25, WD 0.18mm &#955;S, Nano-Crystal Coat</td>
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		<tr height="43">
			<td height="43" style="height:43px;width:231px;">Rhock inhibitor&#160;</td>
			<td style="width:231px;">Sigma</td>
			<td style="width:205px;">Y-27632</td>
			<td style="width:343px;"></td>
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		<tr height="43">
			<td height="43" style="height:43px;width:231px;">Knockout DMEM</td>
			<td style="width:231px;">Invitrogen</td>
			<td style="width:205px;">10829</td>
			<td style="width:343px;"></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:231px;">Knockout Serum&#160;</td>
			<td style="width:231px;">Invitrogen</td>
			<td style="width:205px;">10828</td>
			<td style="width:343px;"></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:231px;">MEM Non-Essential Amino Acids&#160;</td>
			<td style="width:231px;">Invitrogen</td>
			<td style="width:205px;">1140</td>
			<td style="width:343px;"></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:231px;">L-glutamine 200mM&#160;</td>
			<td style="width:231px;">Invitrogen</td>
			<td style="width:205px;">25030</td>
			<td style="width:343px;"></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:231px;">Penicillin-streptomycin</td>
			<td style="width:231px;">Invitrogen</td>
			<td style="width:205px;">15140</td>
			<td style="width:343px;"></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:231px;">&#946;-mercaptoethanol&#160;</td>
			<td style="width:231px;">Sigma</td>
			<td style="width:205px;">M7522</td>
			<td style="width:343px;"></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:231px;">Collagenase IV&#160;</td>
			<td style="width:231px;">Gibco</td>
			<td style="width:205px;">17104-019</td>
			<td style="width:343px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:231px;">Roller scraper tool&#160;</td>
			<td style="width:231px;">StemPro EZPassage, Invitrogen</td>
			<td style="width:205px;">23181-010</td>
			<td style="width:343px;"></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:231px;">StemPro Accutase&#160;</td>
			<td style="width:231px;">Gibco</td>
			<td style="width:205px;">S11105-01</td>
			<td style="width:343px;"></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:231px;">Aggrewell system&#160;</td>
			<td style="width:231px;">StemCell Technologies</td>
			<td style="width:205px;">27845</td>
			<td style="width:343px;"></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:231px;">Hyclone serum&#160;</td>
			<td style="width:231px;">Thermo Scientific</td>
			<td style="width:205px;">SH30070.03</td>
			<td style="width:343px;"></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:231px;">Gelatin</td>
			<td style="width:231px;">Sigma</td>
			<td style="width:205px;">G9391</td>
			<td style="width:343px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:231px;">6-well plates&#160;</td>
			<td style="width:231px;">Falcon</td>
			<td style="width:205px;">353046</td>
			<td style="width:343px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:231px;">24-well plates&#160;</td>
			<td style="width:231px;">Nunclon</td>
			<td style="width:205px;">142475</td>
			<td style="width:343px;"></td>
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		<tr height="43">
			<td height="43" style="height:43px;width:231px;">Polytetrafluoroethylene (PTFE) filters</td>
			<td style="width:231px;">Millipore&#160;</td>
			<td style="width:205px;">NA76/25</td>
			<td style="width:343px;"></td>
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			<td height="22" style="height:22px;width:231px;">Inserts</td>
			<td style="width:231px;">Millipore</td>
			<td style="width:205px;">PICM03050</td>
			<td style="width:343px;"></td>
		</tr>
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harmonic nanoparticles for regenerative research

In questo esperimento visualizzato, dettagli del protocollo sono previste per l&#39;etichettatura in vitro di cellule staminali embrionali umane (hESC) con nanoparticelle di generazione di armoniche secondo (HNPS). Questi ultimi sono una nuova famiglia di sonde recentemente introdotte per l&#39;etichettatura di campioni biologici per l&#39;imaging multi-fotone. HNPS sono in grado di raddoppiare la frequenza della luce di eccitazione dal processo ottica non lineare di generazione di seconda armonica senza alcuna restrizione sulla lunghezza d&#39;onda di eccitazione. Multi-fotone metodologie per la differenziazione hESC nei cluster cardiaci (mantenuti culture aria-liquido lungo termine) basato sono presentati in dettaglio. In particolare, viene mostrata la prova su come massimizzare l&#39;intenso seconda armonica (SH) emissione di HNPS isolati durante il monitoraggio 3D di battere tessuto cardiaco in 3D. L&#39;analisi delle immagini risultanti per recuperare modelli 3D displacement è anche dettagliata.

Prima di valutare l&#39;attività di battitura da confocale, è stata eseguita una accurata caratterizzazione della risposta ottica non lineare dei filtri in PTFE, da soli o in presenza di HNPS ad alta concentrazione (1 mg / ml). Si è assicurato che: i) il substrato nudo due fotoni di fluorescenza eccitata è molto debole e non può impedire misurazione relativi campioni biologici, e ii) l&#39;emissione SH da HNPS isolato può essere facilmente acquisita da immagini attraverso il substrato in modo epi-rilevamento <stro...

Watch this Scientific Journal Video about Harmonic Nanoparticles for Regenerative Research at JoVE.com

Harmonic Nanoparticles for Regenerative Research

Dettagli del protocollo sono previste per l&#39;etichettatura di cellule staminali embrionali umane in vitro con la seconda nanoparticelle generano armoniche. Metodologie di indagine hESC mediante microscopia multi-fotone e la loro differenziazione in gruppi cardiache sono anche presentati.

Harmonic Nanoparticelle for Regenerative ricerca

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation ...

harmonic-nanoparticles-for-regenerative-research

Research

JoVE Journal

Bioengineering

11.2K Views.  University of Geneva.  Dettagli del protocollo sono previste per l'etichettatura di cellule staminali embrionali umane in vitro con la seconda nanoparticelle generano armoniche. Metodologie di indagine hESC mediante microscopia multi-fotone e la loro differenziazione in gruppi cardiache sono anche presentati. 

Video: Harmonic Nanoparticles for Regenerative Research

Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively for tissue regeneration, a number of variables must be taken into account. These variables include: the total volume and surface area of the implantation site, the mechanical properties of the tissue and the tissue microenvironment, which includes the amount of vascularization and the components of the extracellular matrix. Therefore, the materials being used to deliver these cells must be biocompatible with a defined chemical composition while maintaining a mechanical strength that mimics the host tissue. These materials must also be permeable to oxygen and nutrients to provide a favorable microenvironment for cells to attach and proliferate. Chitosan, a cationic polysaccharide with excellent biocompatibility, can be easily chemically modified and has a high affinity to bind with in vivo macromolecules4-5. Chitosan mimics the glycosaminoglycan portion of the extracellular matrix, enabling it to function as a substrate for cell adhesion, migration and proliferation. In this study we utilize chitosan in the form of microspheres to deliver adipose-derived stem cells (ASC) into a collagen based three-dimensional scaffold6. An ideal cell-to-microsphere ratio was determined with respect to incubation time and cell density to achieve maximum number of cells that could be loaded. Once ASC are seeded onto the chitosan microspheres (CSM), they are embedded in a collagen scaffold and can be maintained in culture for extended periods. In summary, this study provides a method to precisely deliver stem cells within a three dimensional biomaterial scaffold.

A major hurdle in current stem cell therapies is determining the most effective method to deliver these cells to host tissues. Here, we describe a chitosan-based delivery method that is efficient and simple in approach, while allowing adipose-derived stem cells to maintain their multipotency.

Costruire un idrogel collagene per la fornitura di Stem Cell-loaded Chitosan Microsfere

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively ...

Ricerca

Bioingegneria

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

Nanoparticelle virali per<em&gt; In vivo</em&gt; Imaging Tumore

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, complementary imaging techniques. These imaging agents facilitate the real-time assessment of pathways and mechanisms in vivo, which enhance both diagnostic and therapeutic efficacy. This article presents the protocol for the synthesis of biofunctionalized Prussian blue nanoparticles (PB NPs) - a novel class of agents for use in multimodal, molecular imaging applications. The imaging modalities incorporated in the nanoparticles, fluorescence imaging and magnetic resonance imaging (MRI), have complementary features. The PB NPs possess a core-shell design where gadolinium and manganese ions incorporated within the interstitial spaces of the PB lattice generate MRI contrast, both in T1 and T2-weighted sequences. The PB NPs are coated with fluorescent avidin using electrostatic self-assembly, which enables fluorescence imaging. The avidin-coated nanoparticles are modified with biotinylated ligands that confer molecular targeting capabilities to the nanoparticles. The stability and toxicity of the nanoparticles are measured, as well as their MRI relaxivities. The multimodal, molecular imaging capabilities of these biofunctionalized PB NPs are then demonstrated by using them for fluorescence imaging and molecular MRI in vitro.

This protocol describes the synthesis of biofunctionalized Prussian blue nanoparticles and their use as multimodal, molecular imaging agents. The nanoparticles have a core-shell design where gadolinium or manganese ions within the nanoparticle core generate MRI contrast. The biofunctional shell contains fluorophores for fluorescence imaging and targeting ligands for molecular targeting.

Biofunctionalized Prussian Blue Nanoparticelle per applicazioni di imaging multimodale molecolari

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, ...

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter cellular signaling and gene expression. In a previous investigation, the synthesis of ultra-small solid lipid nanoparticles (SLNs) for topical drug delivery and biomarker detection applications was demonstrated. SLNs are a well-studied example of a nanoparticle delivery system that has emerged as a promising drug delivery vehicle. In this study, SLNs were loaded with a fluorescent dye and used as a model to investigate particle-cell interactions. The phase inversion temperature (PIT) method was used for the synthesis of ultra-small populations of biocompatible nanoparticles. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylphenyltetrazolium bromide (MTT) assay was utilized in order to establish appropriate dosing levels prior to the nanoparticle-cell interaction studies. Furthermore, primary human dermal fibroblasts and mouse dendritic cells were exposed to dye-loaded SLN over time and the interactions with respect to toxicity and particle uptake were characterized using fluorescence microscopy and flow cytometry. This study demonstrated that ultra-small SLNs, as a nanoparticle delivery system, are suitable for intracellular targeting of different cell types.

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions.

Solid Lipid nanoparticelle (linfonodi sentinella) Applicazioni per intracellulare targeting

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using widefield fluorescence light microscopy and transmission electron microscopy (TEM). To demonstrate the protocol we have immunolabeled ultrathin epoxy sections of human somatostatinoma tumor using a primary antibody to somatostatin, followed by a biotinylated secondary antibody and visualization with streptavidin conjugated 585 nm cadmium-selenium (CdSe) quantum dots (QDs). The sections are mounted on a TEM specimen grid then placed on a glass slide for observation by widefield fluorescence light microscopy. Light microscopy reveals 585 nm QD labeling as bright orange fluorescence forming a granular pattern within the tumor cell cytoplasm. At low to mid-range magnification by light microscopy the labeling pattern can be easily recognized and the level of non-specific or background labeling assessed. This is a critical step for subsequent interpretation of the immunolabeling pattern by TEM and evaluation of the morphological context. The same section is then blotted dry and viewed by TEM. QD probes are seen to be attached to amorphous material contained in individual secretory granules. Images are acquired from the same region of interest (ROI) seen by light microscopy for correlative analysis. Corresponding images from each modality may then be blended to overlay fluorescence data on TEM ultrastructure of the corresponding region.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of epoxy embedded human pathology tissue. We employ commercial antibody fragment conjugated QDs that are visualized by widefield fluorescence light microscopy and transmission electron microscopy.

Correlativo luce e microscopia elettronica Utilizzando Quantum Dot nanoparticelle

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using ...

Neonatal Cardiac Scaffolds: Novel Matrices for Regenerative Studies

The only definitive therapy for end stage heart failure is orthotopic heart transplantation. Each year, it is estimated that more than 100,000 donor hearts are needed for cardiac transplantation procedures in the United States1-2. Due to the limited numbers of donors, only approximately 2,400 transplants are performed each year in the U.S.2. Numerous approaches, from cell therapy studies to implantation of mechanical assist devices, have been undertaken, either alone or in combination, in an attempt to coax the heart to repair itself or to rest the failing heart3. In spite of these efforts, ventricular assist devices are still largely used for the purpose of bridging to transplantation and the utility of cell therapies, while they hold some curative promise, is still limited to clinical trials. Additionally, direct xenotransplantation has been attempted but success has been limited due to immune rejection. Clearly, another strategy is required to produce additional organs for transplantation and, ideally, these organs would be autologous so as to avoid the complications associated with rejection and lifetime immunosuppression. Decellularization is a process of removing resident cells from tissues to expose the native extracellular matrix (ECM) or scaffold. Perfusion decellularization offers complete preservation of the three dimensional structure of the tissue, while leaving the bulk of the mechanical properties of the tissue intact4. These scaffolds can be utilized for repopulation with healthy cells to generate research models and, possibly, much needed organs for transplantation. We have exposed the scaffolds from neonatal mice (P3), known to retain remarkable cardiac regenerative capabilities,5-8 to detergent mediated decellularization and we repopulated these scaffolds with murine cardiac cells. These studies support the feasibility of engineering a neonatal heart construct. They further allow for the investigation as to whether the ECM of early postnatal hearts may harbor cues that will result in improved recellularization strategies.

In these studies, we provide methodology for novel, neonatal, murine cardiac scaffolds for use in regenerative studies.

Neonatale cardiaco Impalcature: Matrici innovativi per gli Studi rigenerativi

The only definitive therapy for end stage heart failure is orthotopic heart transplantation.  Each year, it is estimated that more than 100,000 donor ...

Production of Metal Nanoparticles by Pulsed Laser-ablation in Liquids: A Tool for Studying the Antibacterial Properties of Nanoparticles

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of medicine. In addition to efforts to identify novel small-molecule antimicrobial drugs, there has been great interest in the use of metal nanoparticles as coatings for medical devices, wound dressings, and consumer packaging, due to their antimicrobial properties. The wide variety of methods available for nanoparticle synthesis results in a broad spectrum of chemical and physical properties which can affect antibacterial efficacy. This manuscript describes the pulsed laser-ablation in liquids (PLAL) method to create nanoparticles. This approach allows for the fine tuning of nanoparticle size, composition, and stability using post-irradiation methods as well as the addition of surfactants or volume excluders. By controlling particle size and composition, a large range of physical and chemical properties of metal nanoparticles can be explored which may contribute to their antimicrobial efficacy thereby opening new avenues for antibacterial development.

The antimicrobial properties of metals such as copper and silver have been recognized for centuries. This protocol describes pulsed laser-ablation in liquids, a method of synthesizing metal nanoparticles that provides the ability to fine tune the properties of these nanoparticles to optimize their antimicrobial effects.

Produzione di nanoparticelle metalliche mediante ablazione laser pulsata nei liquidi: uno strumento per studiare le proprietà antibatteriche delle nanoparticelle

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of ...

20 mJ, 1 ps Yb:YAG Thin-disk Regenerative Amplifier

This is a report on a 100 W, 20 mJ, 1 ps Yb:YAG thin-disk regenerative amplifier. A homemade Yb:YAG thin-disk, Kerr-lens mode-locked oscillator with turn-key performance and microjoule-level pulse energy is used to seed the regenerative chirped-pulse amplifier. The amplifier is placed in airtight housing. It operates at room temperature and exhibits stable operation at a 5 kHz repetition rate, with a pulse-to-pulse stability less than 1%. By employing a 1.5 mm-thick beta barium borate crystal, the frequency of the laser output is doubled to 515 nm, with an average power of 70 W, which corresponds to an optical-to-optical efficiency of 70%. This superior performance makes the system an attractive pump source for optical parametric chirped-pulse amplifiers in the near-infrared and mid-infrared spectral range. Combining the turn-key performance and the superior stability of the regenerative amplifier, the system facilitates the generation of a broadband, CEP-stable seed. Providing the seed and pump of the optical parametric chirped-pulse amplification (OPCPA) from one laser source eliminates the demand of active temporal synchronization between these pulses. This work presents a detailed guide to set up and operate a Yb:YAG thin-disk regenerative amplifier, based on chirped-pulse amplification (CPA), as a pump source for an optical parametric chirped-pulse amplifier.

A protocol for the operation of a high-energy, high-power optical parametric chirped pulse amplifier pump source based on an Yb:YAG thin-disk regenerative amplifier is presented here.

20 mJ, 1 ps Yb: YAG Amplificatore rigenerativo a disco rigido

This is a report on a 100 W, 20 mJ, 1 ps Yb:YAG thin-disk regenerative amplifier. A homemade Yb:YAG thin-disk, Kerr-lens mode-locked oscillator with ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

Microtissues 3D per la terapia rigenerativa iniettabile e droga di High-throughput Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation purposes. The process to generate such tissues is complex, and includes an appropriate combination of specific cell types, scaffolds, and physical or biochemical stimuli to guide cell growth and differentiation. Microcarriers represent an appealing tool to expand cells in a three-dimensional (3D) microenvironment, since they provide higher surface-to volume ratios and mimic more closely the in vivo situation compared to traditional two-dimensional methods. The vascular system, supplying oxygen and nutrients to the cells and ensuring waste removal, constitutes an important building block when generating engineered tissues. In fact, most constructs fail after being implanted due to lacking vascular support. In this study, we present a protocol for endothelial cell expansion on recombinant collagen-based microcarriers under dynamic conditions in spinner flask and bioreactors, and we explain how to determine in this setting cell viability and functionality. In addition, we propose a method for cell delivery for vascularization purposes without additional detachment steps necessary. Furthermore, we provide a strategy to evaluate the cell vascularization potential in a perfusion bioreactor on a decellularized&#160;biological matrix. We believe that the use of the presented methods could lead to the development of new cell-based therapies for a large range of tissue engineering applications in the clinical practice.

We propose a cell expansion protocol on macroporous microcarriers and their use as delivery system in a perfusion bioreactor to seed a decellularized tissue matrix. We also include different techniques to determine cell proliferation and viability of cells cultured on microcarriers. Furthermore, we demonstrate functionality of cells after bioreactor cultures.

Ricombinante collagene Peptide Microcarriers per l'espansione delle cellule e loro potenziale utilizzo come sistema di consegna di cella in un bioreattore modello