Questo lavoro è supportato dal programma Rowland Junior Fellow. Gli autori desiderano esprimere gratitudine ai ricercatori e al personale presso l&#39;Istituto Rowland ad Harvard: Chris Stokes per il suo aiuto nello sviluppo della, configurazione controllo della pressione custom-built computer-assistita, Diane Schaak, Ph.D. per il suo ingresso per la manipolazione dei campioni biologici, Winfield Hill per sviluppare l&#39;installazione elettrica, Alavaro Sanchez, Ph.D. per concedere l&#39;accesso al citofluorimetro, Scott Bevis, Kenny Spencer e Don Rogers per la lavorazione di componenti idraulici meccanici necessari per l&#39;installazione di pressione. Maestri microfluidica sono stati realizzati presso il Centro per Nanoscale Systems (CNS) presso la Harvard University.

1 Cella Preparazione <ol><li> Tavola 1 × 10 5 cellule / ml di metastatico linea di cellule di cancro al seno MDA-MB-231 in un volume di 10 ml per tessuti pallone di coltura T75 a Leibovitz L-15 Medium supplementato con 10% (v / v) di siero fetale bovino e 1 % di penicillina-streptomicina. </li><li> Incubare MDA-MB-231 cellule in un incubatore umidificato a 37 ° C con 0% di CO 2 ambiente. </li><li> Celle di raccolta per esperimenti di 2 giorni dopo la semina trattando le cellule con il 0,25% tripsina-EDTA per 2 min e inattivano l&#39;attività enzimatica di tripsina con l&#39;aggiunta di 8 ml di terreno di coltura. </li><li> Cellule pellet di centrifugazione per 5 minuti a 200 × g e risospendere in tampone fosfato di Dulbecco (DPBS, 1x senza Ca 2 + e Mg 2 +) ad una concentrazione finale di 5 × 10 5 cellule / ml. </li></ol> 2. Dispositivo progettazione e fabbricazione NOTA: La maschera, invenzioni stampo master e ilprocesso di inclusione a microcanali devono essere condotte all&#39;interno di una stanza pulita mentre il polidimetilsilossano (PDMS) processo di fusione a microcanali può essere eseguito su un normale banco di laboratorio. <ol><li> Progettare un dispositivo di microfluidica, come illustrato nella figura 1 con AutoCAD. Il dispositivo deve essere composto da un ingresso con porte multiple di iniezione, filtri grossolani e due inerziali concentrandosi canali rettangolari parallele (L = 7 mm, L = 40 micron, e H = 70 micron). Canale rettilineo individuale si compone di 5 camere di elettroporazione, che sono posti a 800 micron a parte (W C = 400 micron), con due fori per il tramite elettrodi di alluminio. Due camere elettroporazione trasversalmente adiacenti condividono il foro per l&#39;elettrodo negativo. Due canali semplici si fondono in una presa a valle della regione elettroporazione. </li><li> Convertire il file CAD con micro-modelli in un file GDSII utilizzando LinkCAD. Scrivere le micro-pattern su un 5 &quot;x 5&quot; fotomaschera vuotoutilizzando uno scrittore maschera laser. Sviluppare la maschera seguendo il protocollo del produttore. NOTA: Il processo di sviluppo di fotoresist maschera comprende sviluppo, cromo incisione e resistere rimozione. In alternativa, micro-disegni stampati su una elevata trasparenza risoluzione (&gt; 20.000 dpi) può essere acquistato da un produttore di terze parti e utilizzato come fotomaschera. Caratteristiche microscala finale su una fotomaschera devono essere trasparenti per abbinare la polarità di un photoresist negativo. </li><li> Realizzare lo stampo colata del disegno dispositivo utilizzando tecniche di litografia soffice standard di 23 con un cappotto photoresist negativo filata su un wafer di silicio da 4 pollici a 2.000 giri per avere lo spessore finale di 70 micron. </li><li> Precottura il wafer con photoresist negativo per 20 min a 95 ° C. </li><li> Contatta l&#39;maschera con la cialda ed esporre ad una sorgente UV (17,4 mJ / cm 2) per 80 secondi usando un allineatore maschera. </li><li> Sviluppare il wafer esposto per 4 min in uno sviluppatore SU-8. </li><li> Misurare ilspessore fotoresist sviluppato utilizzando un profilometro di superficie. </li><li> Rigorosamente mescolare un rapporto 10: 1 di elastomero di agente (kit elastomero di silicone) curare e versare il composto nello stampo di fusione per generare repliche PDMS. Degasare il elastomero casting per 30 min e curare lo stampo di colata con elastomero degasata in stufa a 70 ° C per 2 ore. </li><li> Sfalda curato replica PDMS con modelli a microcanali dallo stampo e punzonatura fori per insenature, outlet e elettrodi inserimenti utilizzando una morsa pin. NOTA: Il normale diametro tagliente della morsa perno dovrebbe essere 0,76 millimetri, sufficiente per ben tenendo tubo PEEK (OD di 1/32 &quot;) ed elettrodi di alluminio (OD di 0,029&quot;) al posto. </li><li> Creare canali microfluidici racchiuso da incollaggio PDMS repliche di un vetrino trattato in un pulitore di ossigeno-plasma per 7 secondi ad una frequenza radio di potenza e pressione parziale di ossigeno di 75 W e 500 mTorr rispettivamente. </li></ol> 3. Esperimenti di flusso <ol><li> Inseriscielettrodi di alluminio (0.029 &quot;OD) e di un tubo di uscita PEEK (1/32&quot; OD) in luoghi designati attraverso fori nei microcanali (vedi Figura 3B). </li><li> Collegare l&#39;apparecchiatura elettrica 18 responsabili per la generazione di alta tensione impulsi di breve onda quadra agli elettrodi di alluminio (vedi Figura 3C) che sono a contatto con soluzioni che scorre nello stampo PDMS. NOTA: L&#39;apparecchiatura deve essere costituito da un generatore di impulsi e di un amplificatore ad alta tensione in-casa costruita 18. </li><li> Preparare quattro 50 ml provette contenenti singolarmente DPBS, soluzioni con le cellule e biomolecole, e collegare ogni tubo al suo rispettivo titolare flaconcino collegato al sistema di controllo di flusso pneumatico (Figura 3A). 18 </li><li> Collegare il tubo PEEK ingresso dai titolari delle fiale nei rispettivi fori di ingresso del dispositivo di microfluidica. </li><li> Impostare l&#39;ampiezza di impulsi ad onda quadra, V a 100 V per avere la fie elettricoforza ld, E = V / L e, attraverso la camera di elettroporazione essere equivalente a 0,7 kV / cm. NOTA: Qui, L e è la distanza tra due punti in cui gli elettrodi positivi e negativi sono in contatto con la soluzione scorrevole (vedi Figura 1). </li><li> Impostare il regolatore di pressione a 40 psi. NOTA:. Una singola fonte di azoto regolabile manualmente viene utilizzata per pressurizzare in modo uniforme tutti i vial di campioni e di un collettore ad alta velocità è utilizzata per attivare tempestivamente porte di soluzioni individuali utilizzando il software LabVIEW custom-built 18 </li><li> Per il controllo della valvola, aprire il software LabVIEW custom-built marcato Valve Runner, e fare clic su Esegui dal menu a discesa dal titolo operano. </li><li> Attivare le valvole facendo clic su ciascuna icona valvola corrispondente. NOTA: L&#39;icona valvola dovrebbe diventare verde quando viene attivata. Facendo clic sull&#39;icona attiva si disattiva e chiudere la valvola. L&#39;icona valvola chiusa dovrebbe diventare grigio. </li><li> Aprire la valvola per il serbatoio DPBS (cioè., soluzione di lavaggio) per innescare la velocità di flusso necessario per la generazione di vortice cella-trapping stabile per 1,5 min. </li><li> Passare il porto soluzione attiva dalla soluzione di lavaggio alla soluzione cella per intrappolare le cellule nella camera di elettroporazione per 30 sec (cioè, la fase cellula-trapping). </li><li> Flusso entrambe le soluzioni di lavaggio e di cella attraverso il dispositivo contemporaneamente per 10 secondi prima della fase cellula-trapping per garantire flusso undisrupted durante la fase di soluzione di commutazione. NOTA: Questa breve fase di co-flusso dovrebbe essere ripetuto ad ogni passo-soluzione di commutazione. </li><li> Accendere il porto di lavaggio e lavare il dispositivo per 20 secondi al fine di rimuovere le cellule contaminanti non intrappolati. </li><li> Iniettare la soluzione contenente la prima molecola di interesse (0,2 mg / ml di 3.000 Da molecole di destrano neutro e anionico o 1,5 mg / ml di 70.000 Da molecola destrano neutro) nel dispositivo. </li><li> Applicare cinque impulsi brevi (Dt = 30 msec, con intervalli di 2 sec) subito dopo l&#39;iniezione delsoluzione molecolare e monitorare l&#39;entità e la durata degli impulsi elettrici applicati in tempo reale con un oscilloscopio. </li><li> Ripetere passo 3.10 tante volte quanto il numero di molecole aggiuntive da consegnare. Per ogni consegna molecolare, incubare le cellule per 100 s nella soluzione molecolare poi sciacquare il dispositivo con la soluzione di lavaggio per 100 s per rimuovere le molecole in eccesso. </li><li> Rilasciare le cellule in una piastra a 96 pozzetti per l&#39;analisi a valle abbassando la pressione di esercizio inferiore a 5 psi. NOTA: Circa 100 ml di soluzione con 100 cellule sono raccolti da ogni release. </li><li> Eseguire sperimentale passi 3.9 tramite 3.16 tre volte a raccogliere abbastanza cellule per citometria a flusso. </li><li> Centrifugare la piastra a 96 pozzetti contenenti cellule lavorati per 5 min a 228 × g. </li><li> Rimuovere il surnatante che contiene molecole fluorescenti in eccesso. </li><li> Risospendere le cellule in DPBS per citometria a flusso. </li><li> Le celle di carico nel citometro a flusso per u molecolareptake analisi di efficienza. </li></ol>

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Con la nuova piattaforma elettroporazione parallelizzati, aumento di 10 volte in velocità e l&#39;efficienza della consegna multi-molecola è stata ottenuta in aggiunta a tutti i meriti che il sistema monocamerale precedentemente sviluppato fornisce. Meriti 18 Precedentemente disponibili includono (i) pre-purificazione della colpire le cellule con distribuzione uniforme di formato per la valorizzazione vitalità, (ii) un controllo preciso e individuale dosaggio molecolare, e (iii) a bassa corrente elettrica ...

Negli ultimi anni, l&#39;uso di impulsi elettrici per facilitare l&#39;erogazione citosolico di molecole extracellulari è diventato un dispositivo interessante per manipolare cellule di mammifero. 1 Questo processo, noto anche come elettroporazione, permeabilizes reversibilmente la membrana cellulare, permettendo membrana intrinsecamente molecole impermeabili per accedere di ambiente intracellulare delle cellule. Perché praticamente qualsiasi molecola può essere introdotto nel citoplasma tramite i pori temporanei creati nella membrana di qualsiasi tipo di celle utilizzando elettroporazione, la tecnica è stata segnalata come più riproducibile, universalmente applicabile, e più efficiente rispetto ad altri metodi, tra cui virus-mediata, chimica e gli approcci ottici. 2-3 Questa tecnica è stata utilizzata per introdurre molecole fluorescenti, 4 farmaci 5 e acidi nucleici 6-7, mantenendo le cellule vitali e intatto. Alla luce di questi vantaggi, l&#39;elettroporazione è stato adottato come un lavoro comunetorio tecnica per la trasfezione del DNA, in vivo terapia genica 8 e studi di vaccinazione cella. E &#39;, tuttavia, ancora difficile per i sistemi di elettroporazione convenzionali per ottenere contemporaneamente l&#39;efficienza pratica e la redditività per i campioni con grande eterogeneità in termini di dimensioni, perché la forza del campo elettrico necessario per l&#39;elettroporazione di successo è strettamente correlata con il diametro della cellula. Inoltre, tali sistemi non consentono un controllo preciso delle somme molecolari multipli consegnato a causa di affidamento sulle bulk processo stocastico consegna molecolare. 9 Al fine di risolvere questi problemi, molti gruppi hanno sviluppato piattaforme di elettroporazione microfluidica, offrendo il vantaggio di tensioni poration inferiori, migliore efficienza di trasfezione, una forte riduzione della mortalità delle cellule, e la capacità di fornire più molecole. 10-13 Tali vantaggi sono stati resi possibili grazie alle piccole orme dei sistemi di elettroporazione microscala il cui elettrodo passolunghezze sono sub-millimetri, drammaticamente diminuendo le tensioni necessarie per la consegna di successo. Inoltre, questi sistemi di elettroporazione microscala possono raggiungere una distribuzione uniforme del campo elettrico e rapidamente dissipare il calore generato, producendo una ridotta mortalità cellulare, migliorando l&#39;efficienza di consegna. L&#39;utilizzo di materiali trasparenti per questi microchip ulteriori permette osservazione in situ del processo di elettroporazione per la modifica dei parametri del prompt. 2,12 Tuttavia, il controllo preciso dosaggio e controllo molecular- e cellulare-dipendenti dei parametri, necessari per la ricerca e le applicazioni terapeutiche, 6 emergenti, 14-16 rimangono ancora irrisolti. Questo lavoro presenta un sistema di elettroporazione microfluidica vortice assistita, in grado di erogare più molecole sequenzialmente in una popolazione preselezionata identica di cellule bersaglio. Celle con distribuzione granulometrica uniforme sono isolati prima di elettroporazione utilizzando riportato in precedenza SImeccanismo ze-selettivi di cattura. 17-18 Avendo una distribuzione uniforme dimensioni, meno variazione di efficienza elettroporazione e una maggiore redditività per data intensità di campo elettrico sono stati raggiunti. 19 Inoltre, agitando continuamente cellule intrappolate utilizzando vortici microscala permesso per la consegna uniforme di molecole attraverso la intero citosol, in accordo con i risultati riportati in precedenza con un&#39;altra piattaforma elettroporazione vortice-assistita. 20 Per dimostrare che questo sistema sarebbe applicabile a una vasta gamma di molecole comunemente utilizzati in applicazioni biologiche, macromolecole con una vasta gamma di pesi molecolari sono state consegnate a cellule del carcinoma mammario metastatico. Inoltre, con l&#39;aiuto di monitoraggio del processo in tempo reale, questo lavoro fornisce ulteriori prove per mettere fine al lungo dibattito in piedi per quanto riguarda il meccanismo di consegna molecolare durante electrporation, essendo prevalentemente elettroforesi-mediata contro la diffusione mediata. 14 </sup&gt; A differenza di altri sistemi di elettroporazione, questa piattaforma offre unicamente i vantaggi combinati di preciso consegna multi-molecola, ad alta efficienza di consegna molecolare, mortalità cellulare minimale, per un&#39;ampia gamma di dimensioni e accuse di molecole consegnato, così come la visualizzazione in tempo reale della elettroporazione processo. Date queste funzionalità, il sistema di elettroporazione sviluppato ha un potenziale pratico come uno strumento versatile per gli studi di riprogrammazione cellulare, applicazioni di consegna della droga 6,14,21-22 10,19 e le applicazioni che richiedono per la comprensione approfondita dei meccanismi di erogazione di elettroporazione molecolari.

<table border="0" cellpadding="0" cellspacing="0" width="1056">
	<tbody>
		<tr height="20">
			<td height="20" style="height:21px;width:475px;">MDA-MB-231 cancer cell line</td>
			<td style="width:297px;">American Type Culture Collection (ATCC)</td>
			<td style="width:119px;">HTB-26</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Leibovitz&#8217;s L-15 Medium</td>
			<td style="width:297px;">Cellgro, Mediatech, Inc.</td>
			<td style="width:119px;">10-045-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">fetal bovine serum (FBS)</td>
			<td style="width:297px;">Gibco, Life Technologies</td>
			<td style="width:119px;">16000-044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">penicillin-streptomycin</td>
			<td style="width:297px;">Sigma-Aldrich</td>
			<td style="width:119px;">P4333</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:475px;">Dulbecco&#39;s phosphate buffered saline (DPBS)</td>
			<td style="width:297px;">Cellgro, Mediatech, Inc.</td>
			<td style="width:119px;">21-030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Trypsin</td>
			<td style="width:297px;">Gibco, Life Technologies</td>
			<td style="width:119px;">25200-056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Flow Cytometer easyCyte HT</td>
			<td style="width:297px;">Millipore</td>
			<td style="width:119px;">0500-4008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Oxygen Plasma Cleaner</td>
			<td style="width:297px;">Technics Micro-RIE</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Dektak 6M surface profiler</td>
			<td style="width:297px;">Veeco</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">KMPR 1050</td>
			<td style="width:297px;">Microchem</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">SYLGARD 184 SILICONE ELASTOMER KIT</td>
			<td style="width:297px;">Dow Corning</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Compressed Nitrogen gas</td>
			<td style="width:297px;">Airgas</td>
			<td style="width:119px;">NI 300</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:475px;">High Pressure Regulator</td>
			<td style="width:297px;">McMaster-Carr</td>
			<td>6162K22</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Downstream regulator</td>
			<td style="width:297px;">McMaster-Carr</td>
			<td style="width:119px;">4000K563</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">high-speed 3/2way-8 valve manifold</td>
			<td style="width:297px;">Festo</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Inline Check Valve</td>
			<td style="width:297px;">Idex Health and Science</td>
			<td style="width:119px;">CV3320</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:475px;">5/32&#34; OD x 3/32&#34;ID Polyurethan tubes</td>
			<td style="width:297px;">Pneumadyne</td>
			<td>PU-156F-0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">1/4&#34; OD X 0.17&#34; ID Polyurethan tubes</td>
			<td style="width:297px;">Pneumadyne</td>
			<td style="width:119px;">PU-250PB-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">1/16&#34; PEEK tubings</td>
			<td style="width:297px;">Festo</td>
			<td style="width:119px;">P1533</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">1/32&#34; PEEK tubings</td>
			<td style="width:297px;">Idex Health and Science</td>
			<td style="width:119px;">P1569</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">PEEK tubing unions</td>
			<td style="width:297px;">Idex Health and Science</td>
			<td style="width:119px;">P881</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Pulse Generator</td>
			<td style="width:297px;">HP</td>
			<td style="width:119px;">8110A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Aluiminum Wire</td>
			<td style="width:297px;">Bob Martin Company</td>
			<td style="width:119px;">6061 ALUM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">oscilloscope</td>
			<td style="width:297px;">Agilent</td>
			<td style="width:119px;">DSO3062A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">50 mL centrifuge tubes</td>
			<td style="width:297px;">VWR</td>
			<td style="width:119px;">21008-178</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">15 mL centrifuge tube</td>
			<td style="width:297px;">VWR</td>
			<td style="width:119px;">21008-216</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">T75 culture flask</td>
			<td style="width:297px;">VWR</td>
			<td style="width:119px;">82050-862</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Dextran, Tetramethylrhodamine, 3000 MW, Anionic</td>
			<td style="width:297px;">Gibco, Life Technologies</td>
			<td style="width:119px;">D3307</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Dextran, Tetramethylrhodamine, 70,000 MW, Neutral&#160;</td>
			<td style="width:297px;">Gibco, Life Technologies</td>
			<td style="width:119px;">D1819</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Dextran, Texas Red, 3000 MW, Neutral</td>
			<td style="width:297px;">Gibco, Life Technologies</td>
			<td style="width:119px;">D3329</td>
			<td></td>
		</tr>
	</tbody>
</table>

microscale vortex-assisted electroporator for sequential molecular delivery

Elettroporazione ha ricevuto crescente attenzione negli ultimi anni, perché è una tecnica molto potente per introdurre fisicamente non permeanti sonde molecolari esogeni in cellule. Questo lavoro riporta una piattaforma elettroporazione microfluidica in grado di effettuare la consegna molecola multiplo per cellule di mammifero con un controllo preciso e molecolare-dipendente parametro. La capacità del sistema di isolare le cellule con distribuzione di dimensione uniforme permette per meno variazione di efficienza elettroporazione per data intensità di campo elettrico; quindi una maggiore redditività del campione. Inoltre, la sua funzione di visualizzazione di processo consente di osservazione del processo di assorbimento molecolare fluorescente in tempo reale, che permette rapide regolazioni dei parametri consegna molecolari in situ per il miglioramento dell&#39;efficienza. Per mostrare le vaste funzionalità della piattaforma segnalati, macromolecole con diverse dimensioni e cariche elettriche (ad esempio, Destrano con MW di 3.000 e 70.000 Da) sono staticonsegnato alle cellule del cancro al seno metastatico con elevate efficienze di consegna (&gt; 70%) per tutte le molecole testate. La piattaforma sviluppata ha dimostrato il suo potenziale per l&#39;uso nella espansione dei campi di ricerca in cui on-chip tecniche di elettroporazione può essere utile.

Il elettroporatore microfluidica parallelo sviluppato consegnato macromolecole con dimensioni diverse e cariche elettriche nelle cellule viventi con carcinoma mammario metastatico. Consegna molecolare di successo è stato qualitativamente determinata monitorando i cambiamenti di intensità di fluorescenza delle cellule orbitanti elettroporate in situ e confermato da misurazioni quantitative mediante citometria a flusso. Figura 4A mostra che il 90% delle cellule trattate uptake 70.000 Da destran...

Watch this Scientific Journal Video about Microscale Vortex-assisted Electroporator for Sequential Molecular Delivery at JoVE.com

Microscale Vortex-assisted Electroporator for Sequential Molecular Delivery

Un vortice microfluidica piattaforma elettroporazione assistito è stato sviluppato per la consegna sequenziale di più molecole in popolazioni di cellule identiche, con un controllo preciso e indipendente il dosaggio. Formato basato fase di purificazione delle cellule bersaglio precedente elettroporazione del sistema aiutato a migliorare l&#39;efficienza di consegna molecolare e la vitalità cellulare trasformati.

Microscala Vortex-assistita Elettroporatore per sequenziale molecolare di consegna

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant ...

microscale-vortex-assisted-electroporator-for-sequential-molecular

Research

JoVE Journal

Bioengineering

8.4K Views.  Harvard University.  Un vortice microfluidica piattaforma elettroporazione assistito è stato sviluppato per la consegna sequenziale di più molecole in popolazioni di cellule identiche, con un controllo preciso e indipendente il dosaggio. Formato basato fase di purificazione delle cellule bersaglio precedente elettroporazione del sistema aiutato a migliorare l'efficienza di consegna molecolare e la vitalità cellulare trasformati. 

Video: Microscale Vortex-assisted Electroporator for Sequential Molecular Delivery

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

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

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

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

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

Ricerca

Bioingegneria

Skin Tattooing As A Novel Approach For DNA Vaccine Delivery

Nucleic acid-based vaccination is a topic of growing interest, especially plasmid DNA (pDNA) encoding immunologically important antigens. After the engineered pDNA is administered to the vaccines, it is transcribed and translated into immunogen proteins that can elicit responses from the immune system. Many ways of delivering DNA vaccines have been investigated; however each delivery route has its own advantages and pitfalls. Skin tattooing is a novel technique that is safe, cost-effective, and convenient. In addition, the punctures inflicted by the needle could also serve as a potent adjuvant. Here, we a) demonstrate the intradermal delivery of plasmid DNA encoding enhanced green fluorescent protein (pCX-EGFP) in a mouse model using a tattooing device and b) confirm the effective expression of EGFP in the skin cells using confocal microscopy.

Skin tattooing is a potent and safe way to delivery DNA vaccine intradermally. Here, a DNA plasmid encoding EGFP is delivered by tattooing to the skin of a laboratory mouse, and the expression of EGFP in the skin cells is then inspected by confocal microscopy.

Pelle tatuaggio come un nuovo approccio per la consegna del DNA vaccino

Nucleic acid-based vaccination is a topic of growing interest, especially plasmid DNA (pDNA) encoding immunologically important antigens. After the ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

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 ...

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

Spremere cellulare come un robusto, Microfluidic intracellulare Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro

The development of more predictive and biologically relevant in vitro assays is predicated on the advancement of versatile cell culture systems which facilitate the functional assessment of the seeded cells. To that end, microscale cantilever technology offers a platform with which to measure the contractile functionality of a range of cell types, including skeletal, cardiac, and smooth muscle cells, through assessment of contraction induced substrate bending. Application of multiplexed cantilever arrays provides the means to develop moderate to high-throughput protocols for assessing drug efficacy and toxicity, disease phenotype and progression, as well as neuromuscular and other cell-cell interactions. This manuscript provides the details for fabricating reliable cantilever arrays for this purpose, and the methods required to successfully culture cells on these surfaces. Further description is provided on the steps necessary to perform functional analysis of contractile cell types maintained on such arrays using a novel laser and photo-detector system. The representative data provided highlights the precision and reproducible nature of the analysis of contractile function possible using this system, as well as the wide range of studies to which such technology can be applied. Successful widespread adoption of this system could provide investigators with the means to perform rapid, low cost functional studies in vitro, leading to more accurate predictions of tissue performance, disease development and response to novel therapeutic treatment.

Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro

This protocol describes the use of microscale silicon cantilevers as pliable culture surfaces for measuring the contractility of muscle cells in vitro. Cellular contraction causes cantilever bending, which can be measured, recorded, and converted into readouts of force, providing a non-invasive and scalable system for measuring contractile function in vitro.

L&#39;utilizzo di microscala Silicon cantilever per valutare Cellular contrattile Funzione<em&gt; In Vitro</em

The development of more predictive and biologically relevant in vitro assays is predicated on the advancement of versatile cell culture systems which ...

Porous Silicon Microparticles for Delivery of siRNA Therapeutics

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the successful implementation of siRNA therapy requires the use of delivery platforms that can overcome the major challenges of siRNA delivery, such as enzymatic degradation, low intracellular uptake and lysosomal entrapment. Here, a protocol for the preparation and use of a biocompatible and effective siRNA delivery system is presented. This platform consists of polyethylenimine (PEI) and arginine (Arg)-grafted porous silicon microparticles, which can be loaded with siRNA by performing a simple mixing step. The silicon particles are gradually degraded over time, thereby triggering the formation of Arg-PEI/siRNA nanoparticles. This delivery vehicle provides a means for protecting and internalizing siRNA, without causing cytotoxicity. The major steps of polycation functionalization, particle characterization, and siRNA loading are outlined in detail. In addition, the procedures for determining particle uptake, cytotoxicity, and transfection efficacy are also described.

Delivery remains the main challenge for the therapeutic implementation of small interfering RNA (siRNA). This protocol involves the use of a multifunctional and biocompatible siRNA delivery platform, consisting of arginine and polyethylenimine grafted porous silicon microparticles.

Poroso Silicon microparticelle per consegna di siRNA Therapeutics

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the ...

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, ...

Analyzing Mixing Inhomogeneity in a Microfluidic Device by Microscale Schlieren Technique

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren system is constructed from a Hoffman modulation contrast microscope, which provides easy access to the rear focal plane of the objective lens, by removing the slit plate and replacing the modulator with a knife-edge. The working principle of microscale schlieren technique relies on detecting light deflection caused by variation of refractive index1-3. The deflected light either escapes or is obstructed by the knife-edge to produce a bright or a dark band, respectively. If the refractive index of the mixture varies linearly with its composition, the local change in light intensity in the image plane is proportional to the concentration gradient normal to the optical axis. The micro-schlieren image gives a two-dimensional projection of the disturbed light produced by three-dimensional inhomogeneity.
To accomplish quantitative analysis, we describe a calibration procedure that mixes two fluids in a T-microchannel. We carry out a numerical simulation to obtain the concentration gradient in the T-microchannel that correlates closely with the corresponding micro-schlieren image. By comparison, a relationship between the grayscale readouts of the micro-schlieren image and the concentration gradients presented in a microfluidic device is established. Using this relationship, we are able to analyze the mixing inhomogeneity from associate micro-schlieren image and demonstrate the capability of microscale schlieren technique with measurements in a microfluidic oscillator4. For optically transparent fluids, microscale schlieren technique is an attractive diagnostic tool to provide instantaneous full-field information that retains the three-dimensional features of the mixing process.

Herein, we describe a procedure that employs microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. Through calibration, distribution of concentration gradient can be derived from the micro-schlieren image.

Analizzando disomogeneità Mescolando in un dispositivo a microfluidi da Tecnica microscala Schlieren

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren ...

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

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

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

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

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

Four-Dimensional CT Analysis Using Sequential 3D-3D Registration

Four-dimensional computed tomography (4DCT) provides a series of volume data and visualizes joint motions. However, numerical analysis of 4DCT data remains difficult because segmentation in all volumetric frames is time-consuming. We aimed to analyze joint kinematics using a sequential 3D-3D registration technique to provide the kinematics of the moving bone with respect to the fixed bone semiautomatically using 4DCT DICOM data and existing software. Surface data of the source bones are reconstructed from 3DCT. The trimmed surface data are respectively matched with surface data from the first frame in 4DCT. These trimmed surfaces are sequentially matched until the last frame. These processes provide positional information for target bones in all frames of the 4DCT. Once the coordinate systems of the target bones are decided, translation and rotation angles between any two bones can be calculated. This 4DCT analysis offers advantages in kinematic analyses of complex structures such as carpal or tarsal bones. However, fast or large-scale motions cannot be traced because of motion artifacts.

We analyzed joint kinematics from four-dimensional computed tomography data. The sequential 3D-3D registration method semiautomatically provides the kinematics of the moving bone with respect to the subject bone from four-dimensional computed tomography data.

Analisi CT quadridimensionale mediante registrazione sequenziale 3D-3D

Four-dimensional computed tomography (4DCT) provides a series of volume data and visualizes joint motions. However, numerical analysis of 4DCT data ...