Name: microscale vortex-assisted electroporator for sequential molecular delivery
Uploaded: 2014-08-07T20:15:00.000+00:00
Duration: 10 min 51 s
Description: Watch this Scientific Journal Video about Microscale Vortex-assisted Electroporator for Sequential Molecular Delivery at JoVE.com

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><tbody><tr><td>MDA-MB-231 cancer cell line</td><td>American Type Culture Collection (ATCC)</td><td>HTB-26</td><td></td></tr><tr><td>Leibovitz&amp;rsquo;s L-15 Medium</td><td>Cellgro, Mediatech, Inc.</td><td>10-045-CV</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Gibco, Life Technologies</td><td>16000-044</td><td></td></tr><tr><td>Penicillin-streptomycin</td><td>Sigma-Aldrich</td><td>P4333</td><td></td></tr><tr><td>Dulbecco&#039;s phosphate buffered saline (DPBS)</td><td>Cellgro, Mediatech, Inc.</td><td>21-030</td><td></td></tr><tr><td>Trypsin</td><td>Gibco, Life Technologies</td><td>25200-056</td><td></td></tr><tr><td>Flow Cytometer easyCyte HT</td><td>Millipore</td><td>0500-4008</td><td></td></tr><tr><td>Oxygen Plasma Cleaner</td><td>Technics Micro-RIE</td><td></td><td></td></tr><tr><td>Dektak 6M surface profiler</td><td>Veeco</td><td></td><td></td></tr><tr><td>KMPR 1050</td><td>Microchem</td><td></td><td></td></tr><tr><td>SYLGARD 184 SILICONE ELASTOMER KIT</td><td>Dow Corning</td><td></td><td></td></tr><tr><td>Compressed Nitrogen gas</td><td>Airgas</td><td>NI 300</td><td></td></tr><tr><td>High Pressure Regulator</td><td>McMaster-Carr</td><td>6162K22</td><td></td></tr><tr><td>Downstream regulator</td><td>McMaster-Carr</td><td>4000K563</td><td></td></tr><tr><td>High-speed 3/2way-8 valve manifold</td><td>Festo</td><td></td><td></td></tr><tr><td>Inline Check Valve</td><td>Idex Health and Science</td><td>CV3320</td><td></td></tr><tr><td>5/32&quot; OD x 3/32&quot; ID Polyurethan tubes</td><td>Pneumadyne</td><td>PU-156F-0</td><td></td></tr><tr><td>1/4&quot; OD X 0.17&quot; ID Polyurethan tubes</td><td>Pneumadyne</td><td>PU-250PB-4</td><td></td></tr><tr><td>1/16&quot; PEEK tubings</td><td>Festo</td><td>P1533</td><td></td></tr><tr><td>1/32&quot; PEEK tubings</td><td>Idex Health and Science</td><td>P1569</td><td></td></tr><tr><td>PEEK tubing unions</td><td>Idex Health and Science</td><td>P881</td><td></td></tr><tr><td>Pulse Generator</td><td>HP</td><td>8110A</td><td></td></tr><tr><td>Aluminum Wire</td><td>Bob Martin Company</td><td>6061 ALUM</td><td></td></tr><tr><td>Oscilloscope</td><td>Agilent</td><td>DSO3062A</td><td></td></tr><tr><td>50 ml centrifuge tubes</td><td>VWR</td><td>21008-178</td><td></td></tr><tr><td>15 ml centrifuge tube</td><td>VWR</td><td>21008-216</td><td></td></tr><tr><td>T75 culture flask</td><td>VWR</td><td>82050-862</td><td></td></tr><tr><td>Dextran, Tetramethylrhodamine, 3,000 MW, Anionic</td><td>Gibco, Life Technologies</td><td>D3307</td><td></td></tr><tr><td>Dextran, Tetramethylrhodamine, 70,000 MW, Neutral&amp;nbsp;</td><td>Gibco, Life Technologies</td><td>D1819</td><td></td></tr><tr><td>Dextran, Texas Red, 3,000 MW, Neutral</td><td>Gibco, Life Technologies</td><td>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

8.5K 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

Taking Advantage of Reduced Droplet-surface Interaction to Optimize Transport of Bioanalytes in Digital Microfluidics

Digital microfluidics (DMF), a technique for manipulation of droplets, is a promising alternative for the development of &#8220;lab-on-a-chip&#8221; platforms. Often, droplet motion relies on the wetting of a surface, directly associated with the application of an electric field; surface interactions, however, make motion dependent on droplet contents, limiting the breadth of applications of the technique.
Some alternatives have been presented to minimize this dependence. However, they rely on the addition of extra chemical species to the droplet or its surroundings, which could potentially interact with droplet moieties. Addressing this challenge, our group recently developed Field-DW devices to allow the transport of cells and proteins in DMF, without extra additives.
Here, the protocol for device fabrication and operation is provided, including the electronic interface for motion control. We also continue the studies with the devices, showing that multicellular, relatively large, model organisms can also be transported, arguably unaffected by the electric fields required for device operation.

The protocol for fabrication and operation of field dewetting devices (Field-DW) is described, as well as the preliminary studies of the effects of electric fields on droplet contents.

Approfittando di ridotta interazione Droplet-superficie per ottimizzare il trasporto di Bioanalytes in microfluidica digitale

Digital microfluidics (DMF), a technique for manipulation of droplets, is a promising alternative for the development of &#8220;lab-on-a-chip&#8221; ...

Ricerca

Ingegneria

High-Throughput DNA Plasmid Multiplexing and Transfection Using Acoustic Nanodispensing Technology

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often performed at low throughput, it is moreover time-consuming and error-prone, even more so when multiplexing several plasmids. We developed an easy, fast, and accurate method to perform cell transfection in a 384-well plate layout using acoustic droplet ejection (ADE) technology. The nanodispenser device used in this study&#160;is based on this technology and allows precise nanovolume delivery at high speed from a source well plate to a destination one. It can dispense and multiplex DNA and transfection reagent according to a predesigned spreadsheet. Here we present an optimal protocol to perform ADE-based high-throughput plasmid transfection which makes it possible to reach an efficiency of up to 90% and a nearly 100% cotransfection in cotransfection experiments. We extend initial work by proposing a user-friendly spreadsheet-based macro, able to manage up to four plasmids/wells from a library containing up to 1,536 different plasmids, and a tablet-based pipetting guide application. The macro designs the necessary template(s) of the source plate(s) and generates the ready-to-use files for the nanodispenser and tablet-based application. The four-steps transfection protocol involves i) a diluent dispense with a classical liquid handler, ii) plasmid distribution and multiplexing, iii) a transfection reagent dispense by the nanodispenser, and iv) cell plating on the prefilled wells. The described software-based control of ADE plasmid multiplexing and transfection allows even nonspecialists in the field to perform a reliable cell transfection in a fast and safe way. This method enables rapid identification of optimal settings for a given cell type and can be transposed to higher-scale and manual approaches. The protocol eases applications, such as human ORFeome protein (set of open reading frames [ORFs] in a genome) expression or CRISPR-Cas9-based gene function validation, in nonpooled screening strategies.

This protocol describes high-throughput plasmid transfection of mammalian cells in a 384-well plate using acoustic droplet ejection technology. The time-consuming, error-prone DNA dispensing and multiplexing, but also the transfection reagent dispensing, are software-driven and performed by a nanodispenser device. The cells are then seeded in these prefilled wells.

Plasmid di DNA ad alto throughput e trasfezione utilizzando la tecnologia di nanoedispensing acustico

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often ...

Genetica

High Speed Droplet-based Delivery System for Passive Pumping in Microfluidic Devices

A novel microfluidic system has been developed that uses the phenomenon of passive pumping along with a user controlled droplet based fluid delivery system. Passive pumping is the phenomenon by which surface tension induced pressure differences drive fluid movement in closed channels. The automated fluid delivery system consists of a set of voltage controlled valves with micro-nozzles connected to a fluid reservoir and a control system. These voltage controlled valves offer a volumetrically precise way to deliver fluid droplets to the inlet of a microfluidic device in a high frequency manner. Based on the  dimensions demonstrated in the current study example, the system is capable of flowing 4 milliliters per minute (through a 2.2mm by 260um cross-sectional channel). Based on these same channel dimensions, fluid exchange of a point inside the channel can be achieved in as little as eight milliseconds. It is observed that there is interplay between momentum of the system (imparted by a combination of the droplets created by the valves and the fluid velocity in the channel), and the surface tension of the liquid. Where momentum provides velocity to the fluid flow (or vice-versa), equilibration of surface tension at the inlet provides a sudden stop to any flow. This sudden stop allows the user to control the flow characteristics of the channel and opens the door for a variety of biological applications, ranging anywhere from reagent delivery to drug-cell studies. It is also observed that when nozzles are aimed at the inlet at shallow angles, the droplet momentum can cause additional interesting fluid phenomena, such as mixing of multiple droplets in the inlet.

A novel microfluidic system has been developed using the phenomenon of passive pumping and a user controlled fluid delivery system. This microfluidic system has the potential to be used in a wide variety of biological applications given its low cost, ease of use, volumetric precision, high speed, repeatability and automation.

Alta velocità Droplet basato Delivery System per pompaggio passiva in dispositivi microfluidici

A novel microfluidic system has been developed that uses the phenomenon of passive pumping along with a user controlled droplet based fluid delivery ...

Biologia

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of cellular heterogeneity in biological systems. However, single-cell sequencing of large populations has been hampered by limitations in processing genomes for sequencing. In this paper, we describe a method for single-cell genome sequencing (SiC-seq) which uses droplet microfluidics to isolate, amplify, and barcode the genomes of single cells. Cell encapsulation in microgels allows the compartmentalized purification and tagmentation of DNA, while a microfluidic merger efficiently pairs each genome with a unique single-cell oligonucleotide barcode, allowing &gt;50,000 single cells to be sequenced per run. The sequencing data is demultiplexed by barcode, generating groups of reads originating from single cells. As a high-throughput and low-bias method of single-cell sequencing, SiC-seq will enable a broader range of genomic studies targeted at diverse cell populations.

Single-cell sequencing reveals genotypic heterogeneity in biological systems, but current technologies lack the throughput necessary for the deep profiling of community composition and function. Here, we describe a microfluidic workflow for sequencing &gt;50,000 single-cell genomes from diverse cell populations.

Una piattaforma di Microfluidic ad altissima velocità di trasmissione per il sequenziamento del genoma di cella singola

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of ...

Bioingegneria

A Droplet-Based Microfluidic Approach and Microsphere-PCR Amplification for Single-Stranded DNA Amplicons

Droplet-based microfluidics enable the reliable production of homogeneous microspheres in the microfluidic channel, providing controlled size and morphology of the obtained microsphere. A microsphere copolymerized with an acrydite-DNA probe was successfully fabricated. Different methods such as asymmetric PCR, exonuclease digestion, and isolation on streptavidin-coated magnetic beads can be used to synthesize single-stranded DNA (ssDNA). However, these methods cannot efficiently use large amounts of highly purified ssDNA. Here, we describe a microsphere-PCR protocol detailing how ssDNA can be efficiently amplified and separated from dsDNA simply by pipetting from a PCR reaction tube. The amplification of ssDNA can be applied as potential reagents for the DNA microarray and DNA-SELEX (Systematic evolution of ligands by exponential enrichment) processes.

This work provides a method for the fabrication of droplet-based microfluidic platforms and the application of polyacrylamide microspheres for microsphere-PCR amplification. The microsphere-PCR method makes it possible to obtain single-stranded DNA amplicons without separating double-stranded DNA.

Un approccio basato su goccia di microfluidica e microsfere-PCR amplificazione per ampliconi di DNA Single-Stranded

Droplet-based microfluidics enable the reliable production of homogeneous microspheres in the microfluidic channel, providing controlled size and ...

A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic regulatory circuits. The analysis of synthetic genetic regulatory networks in vivo is time consuming and suffers from a lack of environmental control, with exogenous synthetic components interacting with host processes resulting in undesired behavior. To overcome these issues, cell-free characterization of novel circuitry is becoming more prevalent. In vitro transcription and translation (IVTT) mixtures allow the regulation of the experimental environment and can be optimized for each unique system. The protocols presented here detail the fabrication of a multilayer microfluidic device that can be utilized to sustain IVTT reactions for prolonged durations. In contrast to batch reactions, where resources are depleted over time and (by-) products accumulate, the use of microfluidic devices allows the replenishment of resources as well as the removal of reaction products. In this manner, the cellular environment is emulated by maintaining an out-of-equilibrium environment in which the dynamic behavior of gene circuits can be investigated over extended periods of time. To fully exploit the multilayer microfluidic device, hardware and software have been integrated to automate the IVTT reactions. By combining IVTT reactions with the microfluidic platform presented here, it becomes possible to comprehensively analyze complex network behaviors, furthering our understanding of the mechanisms that regulate cellular processes.

The fabrication process of a PDMS-based, multilayer, microfluidic device that allows in vitro transcription and translation (IVTT) reactions to be performed over prolonged periods is described. Furthermore, a comprehensive overview of the hardware and software required to automate and maintain these reactions for prolonged durations is provided.

Una piattaforma microfluidica multistrato per la conduzione di un'espressione genica prolungata senza cellule

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic ...

Electroporation of Craniofacial Mesenchyme

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. For example, electroporation has been used with great success to study neural and retinal development in Xenopus, chicken and mouse 1-10. However, it is important to note that in all of these studies, investigators were not targeting soft tissues. Because we are interested in craniofacial development, we adapted a method to target facial mesenchyme.
When we searched the literature, we found, to our surprise, very few reports of successful gene transfer into cartilaginous tissue. The majority of these studies were gene therapy studies, such as siRNA or protein delivery into chondrogenic cell lines, or, animal models of arthritis 11-13. In other systems, such as chicken or mouse, electroporation of facial mesenchyme has been challenging (personal communications, Dept of Craniofacial Development, KCL). We hypothesized that electroporation into procartilaginous and cartilaginous tissues in Xenopus might work better. In our studies, we show that gene transfer into the facial cartilages occurs efficiently at early stages (28), when the facial primordium is still comprised of soft tissue prior to cartilage differentiation.
Xenopus is a very accessible vertebrate system for analysis of craniofacial development. Craniofacial structures are more readily visible in Xenopus than in any other vertebrate model, primarily because Xenopus embryos are fertilized externally, allowing analyses of the earliest stages, and facilitating live imaging at single cell resolution, as well as reuse of the mothers 14. Among vertebrate models developing externally, Xenopus is more useful for craniofacial analysis than zebrafish, as Xenopus larvae are larger and easier to dissect, and the developing facial region is more accessible to imaging than the equivalent region in fish. In addition, Xenopus is evolutionarily closer to humans than zebrafish (&tilde;100 million years closer) 15. Finally, at these stages, Xenopus tadpoles are transparent, and concurrent expression of fluorescent proteins or molecules will allow easy visualization of the developing cartilages. We anticipate that this approach will allow us to rapidly and efficiently test candidate molecules in an in vivo model system.

Craniofacial cartilages develop in close contact with other tissues and are difficult to manipulate in live animals. We are using electroporation to deliver molecular tools during growth of the craniofacial skeleton while bypassing early embryonic effects. This approach will allow us to efficiently test candidate molecules in vivo.

Elettroporazione di Mesenchima craniofacciale

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. ...

Electricity-Free, Sequential Nucleic Acid and Protein Isolation

Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern for governments, industries and medical professionals worldwide. For example, EHECs, combined with Shigella, are responsible for the deaths of approximately 325,000 children each year and are particularly prevalent in the developing world where laboratory-based identification, common in the United States, is unavailable 1. The development and distribution of low cost, field-based, point-of-care tools to aid in the rapid identification and/or diagnosis of pathogens or disease markers could dramatically alter disease progression and patient prognosis. We have developed a tool to isolate nucleic acids and proteins from a sample by solid-phase extraction (SPE) without electricity or associated laboratory equipment 2. The isolated macromolecules can be used for diagnosis either in a forward lab or using field-based point-of-care platforms. Importantly, this method provides for the direct comparison of nucleic acid and protein data from an un-split sample, offering a confidence through corroboration of genomic and proteomic analysis.
Our isolation tool utilizes the industry standard for solid-phase nucleic acid isolation, the BOOM technology, which isolates nucleic acids from a chaotropic salt solution, usually guanidine isothiocyanate, through binding to silica-based particles or filters 3. CUBRC's proprietary solid-phase extraction chemistry is used to purify protein from chaotropic salt solutions, in this case, from the waste or flow-thru following nucleic acid isolation4.
By packaging well-characterized chemistries into a small, inexpensive and simple platform, we have generated a portable system for nucleic acid and protein extraction that can be performed under a variety of conditions. The isolated nucleic acids are stable and can be transported to a position where power is available for PCR amplification while the protein content can immediately be analyzed by hand held or other immunological-based assays. The rapid identification of disease markers in the field could significantly alter the patient's outcome by directing the proper course of treatment at an earlier stage of disease progression. The tool and method described are suitable for use with virtually any infectious agent and offer the user the redundancy of multi-macromolecule type analyses while simultaneously reducing their logistical burden.

A tool and chemistries are described to sequentially isolate nucleic acids followed by proteins from a sample without the need for electricity. The tool consists of a sorbent held within a transfer pipette while the isolation chemistries are based on solid-phase extraction principles. The isolated macromolecules can be analyzed by immuno-based and PCR-based assays.

Elettricità-Free, Nucleic Acid sequenziale e isolamento delle proteine

Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern ...

The Fabrication and Operation of a Continuous Flow, Micro-Electroporation System with Permeabilization Detection

Current therapeutic innovations, such as CAR-T cell therapy, are heavily reliant on viral-mediated gene delivery. Although efficient, this technique is accompanied by high manufacturing costs, which has brought about an interest in using alternative methods for gene delivery. Electroporation is an electro-physical, non-viral approach for the intracellular delivery of genes and other exogenous materials. Upon the application of an electric field, the cell membrane temporarily allows molecular delivery into the cell. Typically, electroporation is performed on the macroscale to process large numbers of cells. However, this approach requires extensive empirical protocol development, which is costly when working with primary and difficult-to-transfect cell types. Lengthy protocol development, coupled with the requirement of large voltages to achieve sufficient electric-field strengths to permeabilize the cells, has led to the development of micro-scale electroporation devices. These micro-electroporation devices are manufactured using common microfabrication techniques and allow for greater experimental control with the potential to maintain high throughput capabilities. This work builds off a microfluidic-electroporation technology capable of detecting the level of cell membrane permeabilization at a single-cell level under continuous flow. However, this technology was limited to 4 cells processed per second, and thus a new approach for increasing the system throughput is proposed and presented here. This new technique, denoted as cell-population-based feedback control, considers the cell permeabilization response to a variety of electroporation pulsing conditions and determines the best-suited electroporation pulse conditions for the cell type under test. A higher-throughput mode is then used, where this 'optimal' pulse is applied to the cell suspension in transit. The steps for fabricating the device, setting up and running the microfluidic experiments, and analyzing the results are presented in detail. Finally, this micro-electroporation technology is demonstrated by delivering a DNA plasmid encoding for green fluorescent protein (GFP) into HEK293 cells.

This protocol describes the microfabrication techniques required to build a lab-on-a-chip, microfluidic electroporation device. The experimental setup performs controlled, single-cell-level transfections in a continuous flow and can be extended to higher throughputs with population-based control. An analysis is provided showcasing the ability to electrically monitor the degree of cell membrane permeabilization in real-time.

La fabbricazione e il funzionamento di un sistema di micro-elettroporazione a flusso continuo con rilevamento di permeabilizzazione

Current therapeutic innovations, such as CAR-T cell therapy, are heavily reliant on viral-mediated gene delivery. Although efficient, this technique is ...

Dispositivo microfluidico per l'isolamento continuo di vescicole extracellulari dall'urina umana

Single Step Isolation of Extracellular Vesicles from Large-Volume Samples with a Bifurcated A4F Microfluidic Device

Extracellular vesicles (EVs) hold immense potential for various biomedical applications, including diagnostics, drug delivery, and regenerative medicine. Nevertheless, the current methodologies for isolating EVs present significant challenges, such as complexity, time consumption, and the need for bulky equipment, which hinders their clinical translation. To address these limitations, we aimed to develop an innovative microfluidic system based on cyclic olefin copolymer-off-stoichiometry thiol-ene (COC-OSTE) for the efficient isolation of EVs from large-volume samples in a continuous manner. By utilizing size and buoyancy-based separation, the technology used in this study achieved a significantly narrower size distribution compared to existing approaches from urine and cell media samples, enabling the targeting of specific EV size fractions in future applications. Our innovative COC-OSTE microfluidic device design, utilizing bifurcated asymmetric flow field-flow fractionation technology, offers a straightforward and continuous EV isolation approach for large-volume samples. Furthermore, the potential for mass manufacturing of this microfluidic device offers scalability and consistency, making it feasible to integrate EV isolation into routine clinical diagnostics and industrial processes, where high consistency and throughput are essential requirements.

Autore in primo piano: Frazionamento asimmetrico del flusso di campo per l'integrazione di bioreattori

Extracellular vesicles hold immense promise for biomedical applications, but current isolation methods are time-consuming and impractical for clinical use. In this study, we present a microfluidic device that enables the direct isolation of extracellular vesicles from large volumes of biofluids in a continuous manner with minimal steps.

Isolamento in un'unica fase di vescicole extracellulari da campioni di grande volume con un dispositivo microfluidico A4F biforcato

Extracellular vesicles (EVs) hold immense potential for various biomedical applications, including diagnostics, drug delivery, and regenerative medicine. ...