This work is financially supported by the Ministry of Science and Technology, Taiwan (Contract no. MOST 103-2113-M-001 -003 -MY2) and the Research Program on Nanoscience and Nanotechnology, Academia Sinica, Taiwan.

1. Progettazione e Realizzazione della MDF Chip <ol><li> Disegnare un individuo modello strato acrilico utilizzando il software commerciale, come AutoCAD, e salvare il modello. <ol><li> Rivedere la progettazione dei quattro strati acrilico modello foglio nella figura 1A e confermare le connessioni inter-strato. </li></ol></li><li> Realizzare tutte le lastre acriliche e il nastro biadesivo per ablazione laser utilizzando una incisione laser CO 2 (figure 1B, 2A, 2B e). 16 <ol><li> Accendere il incisione laser e collegare la macchina al computer portatile di controllo. </li><li> Eseguire il software commerciale e aprire il modello progettato nel passo 1.1 cliccando su &quot;pattern file progettato.&quot; </li><li> Posizionare un pezzo di foglio bianco acrilico o nastro biadesivo sul palco XYZ di incisione laser. </li><li> Impostare il fuoco del raggio laser sulla superficie del foglio acrilico o nastro biadesivo con un auto-alignmenbastone t fornite dal produttore del incisione laser. </li><li> Inviare il modello progettato per l&#39;incisione laser per la lavorazione diretta del foglio acrilico o nastro biadesivo. </li></ol></li><li> Rimuovere la carta protettiva dalle lastre acriliche con pinze e saltare la superficie pulita con azoto. </li><li> Impilare i fogli acrilici e collegare insieme sotto una pressione di 2 kg / cm 2 in un bonder termico per 45 min a 110 ° C per formare il / assemblea canale di stimolazione elettrica di flusso. </li><li> Preparare il vetro di copertura microscopio come substrato di coltura cellulare nel chip. <ol><li> Mettere il vetro di copertura in una vaschetta di colorazione e riempire il vaso con una diluizione di dieci volte del detergente indicato nel listino materiale. </li><li> Mettere il vetro di copertura e vaschetta di colorazione in un Steri-pulitore ad ultrasuoni e pulire il vetro di copertura per 15 minuti. </li><li> Versare il detergente diluito fuori dalla vaschetta di colorazione, riempire il vaso con acqua distillata, e ripetere il punto 1.5.2 3 volte. </li><li> Asciugare il vetro di copertura pulito soffiando con azoto prima di aderire al nastro biadesivo. </li></ol></li><li> Rispettare il vetro di copertura pulita per il / gruppo elettrico canale di stimolazione del flusso con il nastro biadesivo fantasia al punto 1.2. </li><li> Rispettare 13 pezzi di adattatori acriliche alle singole aperture del livello 1 del assemblaggio di chip MDF con colla super. L&#39;assemblaggio di chip MDF è quindi completa (Figura 2D). </li><li> Sterilizzare il completo assemblaggio di chip MDF con 30 min di irradiazione UV prima dell&#39;uso. </li></ol> 2. Impostazione del Sale bridge di rete del MDF Chip <ol><li> Prima dell&#39;uso, sterilizzare tutti i tubi di plastica, dita i dadi, e tubi da microcentrifuga mostrati nella Figura 3B impostando un tempo di 15 min a 121 azienda ° C in autoclave. </li><li> Collegare i tubi fluoroplastici (Figura 3B-i) al gruppo di chip MDF attraverso l&#39;entrata mediae gli adattatori di uscita illustrati nella Figura 1A. </li><li> Collegare la conicità Luer dell&#39;ingresso medio e presa tubi di plastica in Figura 3B-i a 3 vie rubinetti. </li><li> Prendere 2,5 ml di CO 2 PBS -equilibrated (PBS) usando una siringa da 3 ml e collegare la siringa al rubinetto a 3 vie del tubo di plastica di ingresso. Collegare una siringa vuota 3 ml al rubinetto a 3 vie del tubo plastico scarico. Le due siringhe sono così interconnessi attraverso il chip MDF. NOTA: incubare il PBS in 37 ° C 5% -CO 2 cella cultura incubatore O / N di ottenere emissioni di CO 2 -equilibrated PBS. </li><li> Sigillare le aperture del blu e gli adattatori verdi (Figura 1A) sul foglio di PMMA Livello 1 con noci dito-stretto solidi bianchi (Figura 3B-iii). </li><li> Riempire i canali del ponte del sale e le camere di coltura con il CO 2 -equilibrated PBS nella siringa descritto in configurazione punto 2.4. Evitarela formazione di bolle. </li><li> Avanti, mettere la CO 2 -equilibrated PBS contenenti chip di MDF in 37 ° C 5% -CO 2 cella cultura incubatore per O / N incubazione. Questo permette l&#39;aria disciolta nel nastro biadesivo per formare bolle nelle camere. </li><li> Lavare via le bolle nei canali da un rapido flusso PBS nel canale utilizzando le due siringhe. Pompa PBS avanti e indietro se necessario. </li><li> Scaricare l&#39;PBS nei canali tramite il rubinetto a 3 vie collegato al tubo di uscita media. </li><li> Usando una nuova siringa da 3 ml, prendere 2,5 ml di CO 2 -equilibrated Dulbecco modificato (DMEM) (vedi punto 3.5), e sostituire la siringa collegata alla presa con quello nuovo. Riempire i canali con il medium. </li><li> Preparazione della rete ponte salino. <ol><li> Temporalmente rimuovere i dadi solido (Figura 3B-iii) sulle schede verdi (Figura 1A) e iniettare 3% caldo (&gt; 70 ° C) agarosio nella bri saleDGE canale attraverso l&#39;apertura nella adattatori verde (Figura 1A). NOTA: Preparazione di agarosio caldo: Sciogliere 1.5 g di polvere di agarosio in 50 ml di PBS. Sterilizzare impostando un tempo di 20 min tenuta a 121 ° C in autoclave. </li><li> Arrestare iniettando l&#39;agarosio quando il liquido riempie tre quarti della lunghezza del canale ponte salino. </li><li> Sigillare i pori sulle schede verdi (Figura 1A) avvitando i dadi solidi (Figura 3B-iii) dopo l&#39;iniezione agarosio. </li><li> Sostituire i dadi solide sulle schede blu (Figura 1A) con i dadi tubolari traslucidi dita a tenuta (Figura 3B). </li><li> Caricare il 3% preriscaldato agarosio nei dadi tubolari (Figura 3B-IV). </li><li> Incorpora gli elettrodi Ag / AgCl (Figura 3B-V) nei dadi tubolari (Figura 3B-iv) primasolidificazione del agarosio. </li></ol></li><li> Dopo aver completato la configurazione della rete ponte salino, iniettare le cellule tumorali del polmone CL1-5 nelle camere di cultura, una camera per volta. </li></ol> 3. Preparazione delle cellule tumorali e Set Up per Electrotactic Experiment <ol><li> Cultura regolare di linea di cellule cancro ai polmoni CL1-5. <ol><li> Coltivare le cellule tumorali CL1-5 polmonari, ottenuti dalla Prof. Pan-Chyr Yang, 17 in mezzo completo in un pallone di coltura cellulare a 37 75T ° C in atmosfera di CO 2 5%. Il terreno completo è composto da DMEM e siero fetale bovino 10% (FBS). Sub-cultura delle cellule ogni 3 o 4 giorni. Le cellule utilizzate per l&#39;esecuzione di esperimenti electrotaxis sono meno di 25 brani del sorgente originale. </li></ol></li><li> Aggiungere 2 ml di 0,25% tampone tripsina alle crescita esponenziale cellule CL1-5 e incubare per 2 minuti in 37 ° C 5% -CO 2 cella cultura incubatore per detachmen cellularit. </li><li> Terminare il processo di distacco con 6 ml di DMEM 10% FBS e centrifugare le cellule a 300 g per 5 minuti a temperatura ambiente. Poi scartare il medio e sospendere il pellet cellulare con 5 ml di PBS. </li><li> Contare il numero di cellule in PBS. Dai 1 x 10 6 cellule e centrifugare a 300 g per 5 minuti a temperatura ambiente. </li><li> Eliminare il PBS. Sospendere le cellule con preriscaldata CO 2 -equilibrated DMEM e regolare la densità delle cellule di essere 1 x 10 6 cellule / ml. NOTA: incubare il terreno completo in 37 ° C 5% -CO 2 cella cultura incubatore O / N per ottenere la CO 2 -equilibrated DMEM. </li></ol> 4. Impostare per Electrotactic Experiment <ol><li> Dalla presa del chip MDF, iniettare 0,3 ml, 1 × 10 6 / ml, delle cellule nel chip. </li><li> Incubare il chip MDF cellule testa di serie nell&#39;incubatrice coltura cellulare (fissato a 37 ° C e 5% CO 2) per 2-4 ore. </li><li> Configurazione di MDF microSistema fluidica. <ol><li> Installare il sistema microfluidica MDF su un calorifero trasparente di indio e stagno vetro ossido (ITO). Misurare la temperatura del chip MDF con una termocoppia di tipo K ritagliato tra il chip MDF e vetro ITO. Controllare il riscaldatore ITO con un controller proporzionale-integrale-derivativo (PID). Impostare la microfluidica temperatura del sistema di incubazione MDF a 37 ° C con il regolatore PID. NOTA: La fabbricazione del riscaldatore ITO e la messa a punto del sistema di celle di riscaldamento cultura sono descritte nelle relazioni precedenti 18,19. </li><li> Montare l&#39;assemblaggio di chip MDF temperatura controllata sul XYZ palco motorizzato controllato dal computer su un microscopio invertito. L&#39;uso dello stadio motorizzato è descritta nel passaggio 5.1. Pompare il terreno completo nelle camere di cultura attraverso le prese con una pompa a siringa a quattro canali. </li></ol></li><li> Pompa terreno completo nel chip MDF ad una portata di 20 l / h. I rifiuti cultura dal fuorilascia viene raccolto nei tubi microcentrifuga (indicati come &quot;rifiuti&quot; in Figura 3A). </li><li> Incubare le cellule per un altro 16-18 ore a 37 ° C nel chip MDF. </li><li> Per sostituire il mezzo, pompare il terreno contenente 50, 25, 5, 0 e inibitore chinasi pM, rispettivamente, in ciascuna camera di coltura ad una portata di 20 l / min per 10 min. NOTA: Preparare una soluzione madre 10 mm di Rho-associata proteina chinasi inibitore Y27632 con l&#39;aggiunta di 3 ml di acqua sterile per l&#39;inibitore della chinasi 10,6 mg. Diluire la soluzione madre 10 mm a 50, 25 e 5 micron, rispettivamente con il 10% FBS DMEM. </li><li> Incubare le cellule della chinasi per altri 60 min a 37 ° C. Impostare la portata media di 20 l / h. Utilizzare la stessa temperatura della cultura e portata sopra descritto nei punti successivi. </li><li> Utilizzare cavi elettrici per collegare un alimentatore CC al chip MDF tramite gli elettrodi Ag / AgCl sul chip. </li><li> Collegare in serie un amperometro in il circuito elettrico. Utilizzare l&#39;amperometro per controllare la corrente elettrica nel chip MDF. </li><li> Accendere l&#39;alimentazione in corrente continua e regolare la corrente di 86.94 μA amperometro regolando la tensione di alimentazione compresa tra 15 e 19 V. NOTA: Considerando la legge di Ohm, E = I / (σA eff), dove I è la corrente elettrica che scorre attraverso il materiale sfuso, cioè, il terreno di coltura nella camera electrotactic, σ (= 1,38 Ω -1 m - 1) è il conducibilità del terreno di coltura, A eff (= 0.21 mm 2 per una larghezza 3 mm x 0,07 mm di altezza) è l&#39;area della sezione trasversale efficace della camera electrotactic, e la corrente elettrica (I) necessario per generare una 300 mV / mm EF nel chip MDF è 86.94 μA. </li></ol> 5. Acquisizione e analisi di cellule Immagini <ol><li> Controllare il palco motorizzato con un programma MATLAB GUI. Con il sistema microfluidico MDF montato sul microscope, spostare il chip alle regioni di osservazione usando il tavolino motorizzato. </li><li> Immagini di cellule registrare utilizzando una fotocamera digitale reflex (SLR) montata sul microscopio. </li><li> Prendere immagini microscopiche utilizzando una lente obiettivo 4X ad intervalli di 15 minuti per 2 ore. </li><li> Analisi migrazione cellulare. <ol><li> Eseguire NIH ImageJ 1.47 V. </li><li> Analizzare la distanza dagli iniziali alle posizioni finali del baricentro della cellula. </li><li> Vai su &quot;File&quot; → &quot;Importa&quot; → &quot;Sequenza d&#39;immagini&quot;, e importare le nove immagini per l&#39;analisi migrazione cellulare. La prima immagine è il risultato del tempo zero, e l&#39;ultima immagine è il risultato di 120 min. </li><li> Andare su &quot;Analizza&quot; → &quot;Imposta misure&quot; e selezionare la casella accanto a &quot;Centroide&quot; </li><li> Fare clic sull&#39;icona &quot;a mano libera selezioni&quot;. </li><li> Descrivere il bordo delle celle selezionate nella prima immagine. </li><li> Vai su &quot;Modifica&quot; → </em&gt; &quot;Selezione&quot; → &quot;Aggiungi a Manager&quot; </li><li> Vai all&#39;immagine nono e raffigurano il bordo delle stesse celle selezionate sulla prima immagine. Le posizioni delle celle possono essere rintracciate utilizzando la seconda all&#39;ottavo immagini. </li><li> Vai su &quot;Modifica&quot; → &quot;Selezione&quot; → &quot;Aggiungi a Manager&quot; </li><li> Ripetere i passaggi da 5.4.5 a 5.4.9 per raccogliere i dati da 90 a 100 celle. </li><li> Vai &quot;Analisi&quot; → &quot;Measure&quot; per ottenere il risultato delle posizioni iniziale e finale delle celle selezionate. NOTA: La velocità di migrazione è definita come la lunghezza media movimento cellulare per ora. La direzionalità è definita come il coseno, dove è l&#39;angolo tra il vettore del dcEF (dall&#39;anodo al catodo) e il vettore dal punto di partenza di una cella per la sua posizione finale. La direzionalità è -1 per cellule migranti verso l&#39;anodo e uno per le cellule migrano verso il catodo. Per un gruppo di casualmente migrata cellule, la direzionalità è 0. 2 </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

Abbiamo trovato il processo di aderire adattatori acrilico su livello 1 del chip MDF di essere ingannevole. L&#39;applicazione di appena 1 o 2 ml di colla super è sufficiente per aderire saldamente all&#39;adattatore sul chip MDF. Grandi quantità di colla comportato un polimerizzazione incompleta del super colla e mancato adempimento. Una volta che gli adattatori acrilici erano saldamente rispettati sul chip MDF, perdite di liquido nel sistema microfluidico raramente verificato. Inoltre, O / N incubazione all&#39;inte...

Il comportamento delle cellule aderenti muovono verso un anodo o catodo in una corrente di campo elettrico diretto (dcEF) è indicato come electrotaxis. Il comportamento electrotactic delle cellule svolge un ruolo significativo nella embriogenesi, rigenerazione dei nervi, e la guarigione della ferita. 1 Le cellule tumorali come le cellule tumorali della prostata di ratto, cellule del cancro al seno 2, 3 e del polmone di cellule di adenocarcinoma 4-8 hanno dimostrato movimento electrotactic sotto un dcEF applicato . L&#39;EF fisiologica è stata misurata nei tessuti della ghiandola. 9,10 Electrotaxis è stata riportata anche in cellule tumorali ghiandola associate. 2,3 Nel loro insieme, le electrotaxis di cellule tumorali è considerata un fattore di metastasi. 11 Controllo della guida elettrica di le cellule tumorali sotto dcEF può essere un approccio potenziale per il futuro trattamento del cancro. Tuttavia, oggi, il meccanismo molecolare dettagliato della electrotaxis rimane controverso. Pertanto, un&#39;indagine sulla influence di stimolazione elettrica sulla migrazione delle cellule del cancro può facilitare lo sviluppo di strategie per il trattamento del cancro. Di recente, i dispositivi bio-microfluidica sono stati fabbricati per studiare le risposte cellulari a fluire forza di taglio, 12 gradienti chimici, 13 e stimoli elettrici 4 in vitro. La fabbricazione di dispositivi bio-microfluidica con polidimetilsilossano (PDMS) o polimetilmetacrilato (PMMA, noto anche come acrilico) ha ridotto con successo il tasso di fallimento di tali esperimenti. Inoltre, utilizzando dispositivi microfluidici base acrilica come prototipo per lo studio soggetti biologici è più semplice rispetto all&#39;utilizzo di PDMS chip. Diverse funzioni nei dispositivi a base acrilica sono stati sviluppati per electrotaxis studio. Tuttavia, nessuno dei disegni precedenti sono in grado di testare contemporaneamente gli effetti di varie condizioni chimiche e il campo elettrico sulle cellule per electrotaxis studio. Così, abbiamo sviluppato un dispositivo microfluidica-multichannel dual-campo elettrico (MDF)-chip contenente quattro canali indipendenti, cultura e otto differenti condizioni sperimentali in un unico chip. Il chip MDF base acrilica, prima volta da Hou et al., 8 integra stimolazione elettrica e diversi canali isolati chimicamente. Questi canali isolati chimicamente possono essere utilizzati per la cultura diversi tipi di cellule in un esperimento. La dcEF nei canali viene prodotta da un alimentatore elettrico. Due campi elettrici indipendenti, uno con applicazione di forza del campo elettrico (EFS) e un altro con 0 EFS, sono condotte in ciascun canale chimicamente isolato. In questo modo, il chip fornisce EF coesistenti meglio controllato e stimolazione chimica. Inoltre, i risultati della simulazione numerica della diffusione chimica all&#39;interno del chip MDF indicano che non si è verificata una contaminazione incrociata tra i canali, dopo un periodo sperimentale 24 hr. 8 Rispetto al device riportato da Li et al., 14 il chip MDF fornisce un&#39;area culturale più grande, che permette ulteriori analisi biochimica delle cellule stimolate elettricamente. Inoltre, con grande area di osservazione del chip MDF, più cellule possono essere osservati nel test, per cui l&#39;analisi della velocità di migrazione o di direzionalità delle cellule stimolate elettricamente è più accurata. I modelli di chip singolo canale di studi precedenti riportati da Huang et al. 4 e Tsai et al. 15 consentono solo un tipo di cellula o chimica da testare. Tuttavia, il chip MDF può essere usato per studiare gli effetti di varie sostanze chimiche su electrotaxis, nonché gli effetti della stimolazione elettrica su diversi tipi di cellule. In altre parole, il chip MDF consente lo studio di dose efficace delle dipendenze chimiche.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Reagent</td>
			<td ></td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >DMEM medium</td>
			<td >Gibco,Invitrogen, USA</td>
			<td >12800-017</td>
			<td></td>
		</tr>
		<tr >
			<td >Fetal Bovine Serum</td>
			<td >Gibco,Invitrogen, USA</td>
			<td >16000-044</td>
			<td></td>
		</tr>
		<tr >
			<td >Trypsin</td>
			<td >Gibco,Invitrogen, USA</td>
			<td >25200-072</td>
			<td></td>
		</tr>
		<tr >
			<td >PBS</td>
			<td >Basic Life</td>
			<td >BL2651</td>
			<td></td>
		</tr>
		<tr >
			<td >Y-27632 (hydrochloride)</td>
			<td >Cayman Chemical Co</td>
			<td >10005583</td>
			<td></td>
		</tr>
		<tr >
			<td >agarose</td>
			<td >LONZO, USA</td>
			<td >SeaKem LE AGAROSE</td>
			<td></td>
		</tr>
		<tr >
			<td >syringe</td>
			<td>Terumo</td>
			<td>3 ml with Luer taper</td>
			<td></td>
		</tr>
		<tr >
			<td >3-way stopcock</td>
			<td>Nipro</td>
			<td >with Luer taper</td>
			<td></td>
		</tr>
		<tr >
			<td >PMMA (acrylic)</td>
			<td >HiShiRon Industries CO., Ltd, Taiwan</td>
			<td ></td>
			<td >thickness 1mm, 2mm</td>
		</tr>
		<tr >
			<td >acrylic adaptor</td>
			<td >KuanMin Technology Co., Ltd, Taichung, Taiwan</td>
			<td >1/4-28 port, 10x10x6 mm</td>
			<td>customized</td>
		</tr>
		<tr >
			<td >nut</td>
			<td >Thermo Fisher Scientific Inc.</td>
			<td >UPCHURCH:P-206x, P-200x, F120x, P-659, P-315x</td>
			<td></td>
		</tr>
		<tr >
			<td >Microscope cover glass</td>
			<td >Deckgl&#228;ser, Germany</td>
			<td >24x60 mm</td>
			<td></td>
		</tr>
		<tr >
			<td >double-sided tape</td>
			<td >3M</td>
			<td >PET 8018</td>
			<td></td>
		</tr>
		<tr >
			<td >super glue</td>
			<td >3M</td>
			<td >Scotch Liquid Plus Super Glue</td>
			<td></td>
		</tr>
		<tr >
			<td >Teflon tube</td>
			<td >HENG YI ENTERPRISE CO., LTD., Taiwan</td>
			<td >UPTB_06, DUPONT TEFLON BRAND RESIN FEP TUBING</td>
			<td>outer diameter 1/16 in., inner diameter 0.03 in.; Upchurch Scienti&#64257;c</td>
		</tr>
		<tr >
			<td >TFD4 detergent</td>
			<td >Franklab, France</td>
			<td >TFD4</td>
			<td></td>
		</tr>
		<tr >
			<td >ultrasonic steri cleaner</td>
			<td >LEO ULTRASONIC CO., LTD., Taiwan</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Thermo bonder</td>
			<td >KuanMin Technology Co., Ltd, Taichung, Taiwan</td>
			<td>customized</td>
			<td></td>
		</tr>
		<tr >
			<td >CO2 laser scriber</td>
			<td >LTT group, Taiwan</td>
			<td >ISL-II</td>
			<td></td>
		</tr>
		<tr >
			<td >indium tin oxide glass (ITO glass)</td>
			<td >AimCore Technology Co., Ltd</td>
			<td >TN/STN, &#8806;10&#937;</td>
			<td></td>
		</tr>
		<tr >
			<td >proportional-integral-derivative (PID) controller</td>
			<td>JETEC Electronics Co., Japen</td>
			<td >TTM-J40-R-AB,</td>
			<td></td>
		</tr>
		<tr >
			<td >K-type thermocouple</td>
			<td >TECPEL</td>
			<td >TPK-02A</td>
			<td></td>
		</tr>
		<tr >
			<td >4-channel syringe pump</td>
			<td >KdScientific, USA</td>
			<td >250P</td>
			<td></td>
		</tr>
		<tr >
			<td >DC power supply</td>
			<td >GWInstek, Taiwan</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >X-Y-Z motor stage</td>
			<td >TanLian, E-O Co. Ltd., Taiwan</td>
			<td>customized</td>
			<td></td>
		</tr>
		<tr >
			<td >inverted microscope</td>
			<td >Olympus, Japan</td>
			<td >CKX41</td>
			<td></td>
		</tr>
		<tr >
			<td >digital SLR camera</td>
			<td >Canon, Japan</td>
			<td >60D</td>
			<td></td>
		</tr>
	</tbody>
</table>

electrotaxis studies of lung cancer cells using a multichannel dual-electric-field microfluidic chip

Il comportamento di migrazione delle cellule direzionale in una corrente di campo elettrico diretto (dcEF) è indicato come electrotaxis. Il ruolo significativo fisiologico dcEF nel guidare il movimento delle cellule durante lo sviluppo embrionale, la differenziazione cellulare e la guarigione della ferita è stata dimostrata in molti studi. Applicando chip microfluidici a un saggio electrotaxis, il processo di indagine è abbreviato e errori sperimentali sono ridotti al minimo. Negli ultimi anni, i dispositivi microfluidici fatti di sostanze polimeriche (ad esempio, polimetilmetacrilato, PMMA, o acrilico) o polidimetilsilossano (PDMS) sono stati ampiamente utilizzati nello studio delle risposte delle cellule alla stimolazione elettrica. Tuttavia, a differenza dei numerosi passaggi necessari per fabbricare un dispositivo PDMS, la costruzione semplice e rapida delle micro acrilici fl truciolo uidic rende adatto sia prototipazione dispositivo e produzione. Tuttavia nessuno dei dispositivi riportati facilitare lo studio efficace della sostanza chimica simultanea e DCEF effetti sulle cellule. In questo rapporto, descriviamo la nostra progettazione e fabbricazione di un multicanale base acrilica a doppio campo elettrico (MDF) chip per studiare l&#39;effetto concomitante di chimica e di stimolazione elettrica sulle cellule tumorali del polmone. Il chip MDF fornisce otto combinazioni di stimolazioni elettriche / chimiche in un singolo test. Il chip non solo riduce notevolmente il tempo richiesto sperimentale ma aumenta anche la precisione in electrotaxis studi.

Fabbricazione e montaggio del dispositivo MDF Un diagramma schematico del chip MDF base acrilica è mostrato nella Figura 1A. Quattro strati acrilici, un vetro di copertura, 13 adattatori acrilici, e un pezzo di nastro biadesivo sono stati utilizzati per l&#39;assemblaggio del chip MDF completato (Figura 2D). Ci sono solo quattro canali indipendenti di cultura...

Watch this Scientific Journal Video about Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip at JoVE.com

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

Many microfluidic devices have been developed for use in the study of electrotaxis. Yet, none of these chips allows the efficient study of the simultaneous chemical and electric-field (EF) effects on cells. We developed a polymethylmethacrylate-based device that offers better-controlled coexisting EF and chemical stimulation for use in electrotaxis research.

Electrotaxis Studi di polmone cellule tumorali usando un Multicanale Dual-campo elettrico Microfluidic Chip

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of ...

electrotaxis-studies-lung-cancer-cells-using-multichannel-dual

Research

JoVE Journal

Bioengineering

8.6K Views.  Academia Sinica. Many microfluidic devices have been developed for use in the study of electrotaxis. Yet, none of these chips allows the efficient study of the simultaneous chemical and electric-field (EF) effects on cells. We developed a polymethylmethacrylate-based device that offers better-controlled coexisting EF and chemical stimulation for use in electrotaxis research.

Video: Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

Metodi di rilevamento a fluorescenza per piattaforme goccia microfluidica

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

Ricerca

Bioingegneria

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and pathways as well as its ease of cultivation. Several C. elegans disease models have been reported, including neurodegenerative disorders such as Parkinson's disease (PD), which involves the degeneration of dopaminergic (DA) neurons 1. Both transgenes and neurotoxic chemicals have been used to induce DA neurodegeneration and consequent movement defects in worms, allowing for investigations into the basis of neurodegeneration and screens for neuroprotective genes and compounds 2,3.
Screens in lower eukaryotes like C. elegans provide an efficient and economical means to identify compounds and genes affecting neuronal signaling. Conventional screens are typically performed manually and scored by visual inspection; consequently, they are time-consuming and prone to human errors. Additionally, most focus on cellular level analysis while ignoring locomotion, which is an especially important parameter for movement disorders.
We have developed a novel microfluidic screening system (Figure 1) that controls and quantifies C. elegans' locomotion using electric field stimuli inside microchannels. We have shown that a Direct Current (DC) field can robustly induce on-demand locomotion towards the cathode ("electrotaxis") 4. Reversing the field's polarity causes the worm to quickly reverse its direction as well. We have also shown that defects in dopaminergic and other sensory neurons alter the swimming response 5. Therefore, abnormalities in neuronal signaling can be determined using locomotion as a read-out. The movement response can be accurately quantified using a range of parameters such as swimming speed, body bending frequency and reversal time.
Our work has revealed that the electrotactic response varies with age. Specifically, young adults respond to a lower range of electric fields and move faster compared to larvae 4. These findings led us to design a new microfluidic device to passively sort worms by age and phenotype 6.
We have also tested the response of worms to pulsed DC and Alternating Current (AC) electric fields. Pulsed DC fields of various duty cycles effectively generated electrotaxis in both C. elegans and its cousin C. briggsae 7. In another experiment, symmetrical AC fields with frequencies ranging from 1 Hz to 3 KHz immobilized worms inside the channel 8.
Implementation of the electric field in a microfluidic environment enables rapid and automated execution of the electrotaxis assay. This approach promises to facilitate high-throughput genetic and chemical screens for factors affecting neuronal function and viability.

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans&#039; Locomotion

A semi-automated micro-electro-fluidic method to induce on-demand locomotion in Caenorhabditis elegans is described. This method is based on the neurophysiologic phenomenon of worms responding to mild electric fields (&#x201C;electrotaxis&#x201D;) inside microfluidic channels. Microfluidic electrotaxis serves as a rapid, sensitive, low-cost, and scalable technique to screen for factors affecting neuronal health.

Electrotaxis microfluidica-based per On-demand analisi quantitativa della<em&gt; Caenorhabditis elegans</em&gt; &#39;Locomotion

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and ...

Microfluidic Chip Fabrication and Method to Detect Influenza

Fast and effective diagnostics play an important role in controlling infectious disease by enabling effective patient management and treatment. Here, we present an integrated microfluidic thermoplastic chip with the ability to amplify influenza A virus in patient nasopharyngeal (NP) swabs and aspirates. Upon loading the patient sample, the microfluidic device sequentially carries out on-chip cell lysis, RNA purification and concentration steps within the solid phase extraction (SPE), reverse transcription (RT) and polymerase chain reaction (PCR) in RT-PCR chambers, respectively. End-point detection is performed using an off-chip Bioanalyzer (Agilent Technologies, Santa Clara, CA). For peripherals, we used a single syringe pump to drive reagent and samples, while two thin film heaters were used as the heat sources for the RT and PCR chambers. The chip is designed to be single layer and suitable for high throughput manufacturing to reduce the fabrication time and cost. The microfluidic chip provides a platform to analyze a wide variety of virus and bacteria, limited only by changes in reagent design needed to detect new pathogens of interest.

An integrated microfluidic thermoplastic chip has been developed for use as a molecular diagnostic. The chip performs nucleic acid extraction, reverse transcriptase, and PCR. Methods for fabricating and running the chip are described.

Chip Fabrication Microfluidic e metodo per rilevare l&#39;influenza

Fast  and effective diagnostics play an important role in controlling infectious  disease by enabling effective patient management and treatment. Here, we ...

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this purpose, the cells are passively trapped in the middle of a microchamber. Upon activation of the control layer, the cell is isolated from the surrounding volume in a small chamber. The surrounding volume can then be exchanged without affecting the isolated cell. However, upon short opening and closing of the chamber, the solution in the chamber can be replaced within a few hundred milliseconds. Due to the reversibility of the chambers, the cells can be exposed to different solutions sequentially in a highly controllable fashion, e.g. for incubation, washing, and finally, cell lysis. The tightly sealed microchambers enable the retention of the lysate, minimize and control the dilution after cell lysis. Since lysis and analysis occur at the same location, high sensitivity is retained because no further dilution or loss of the analytes occurs during transport. The microchamber design therefore enables the reliable and reproducible analysis of very small copy numbers of intracellular molecules (attomoles, zeptomoles) released from individual cells. Furthermore, many microchambers can be arranged in an array format, allowing the analysis of many cells at once, given that suitable optical instruments are used for monitoring. We have already used the platform for proof-of-concept studies to analyze intracellular proteins, enzymes, cofactors and second messengers in either relative or absolute quantifiable manner.

In this article we present a microfluidic chip for single cell analysis. It allows the quantification of intracellular proteins, enzymes, cofactors, and second messengers by means of fluorescent assays or immunoassays.&#160;

Un chip microfluidica per la Versatile analisi chimica di singole cellule

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this ...

A Microfluidic Chip for ICPMS Sample Introduction

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). The chip produces monodisperse aqueous sample droplets in perfluorohexane (PFH). Size and frequency of the aqueous droplets can be varied in the range of 40 to 60 &#181;m and from 90 to 7,000&#160;Hz, respectively. The droplets are ejected from the chip with a second flow of PFH and remain intact during the ejection. A custom-built desolvation system removes the PFH and transports the droplets into the ICPMS. Here, very stable signals with a narrow intensity distribution can be measured, showing the monodispersity of the droplets. We show that the introduction system can be used to quantitatively determine iron in single bovine red blood cells. In the future, the capabilities of the introduction device can easily be extended by the integration of additional microfluidic modules.

We present a discrete droplet sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). It is based on a cheap and disposable microfluidic chip that generates highly monodisperse droplets in a size range of 40&#8722;60 &#181;m at frequencies from 90 to 7,000 Hz.

Un chip microfluidico per ICPMS introduzione del campione

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma ...

The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need for new solutions for substance testing. Many drugs that have been removed from the market due to drug-induced liver injury released their toxic potential only after several doses of chronic testing in humans. However, a controlled microenvironment is pivotal for long-term multiple dosing experiments,&#160;as even minor alterations in extracellular conditions may greatly influence the cell physiology. We focused within our research program on the generation of a microengineered bioreactor, which can be dynamically perfused by an on-chip pump and combines at least two culture spaces for multi-organ applications. This circulatory system mimics the in vivo conditions of primary cell cultures better and assures a steadier,&#160;more quantifiable extracellular relay of signals to the cells. 
For demonstration purposes, human liver equivalents, generated by aggregating differentiated HepaRG cells with human hepatic stellate cells in hanging drop plates, were cocultured with human skin punch biopsies for up to 28 days inside the microbioreactor. The use of cell culture inserts enables the skin to be cultured at an air-liquid interface, allowing topical substance exposure. The microbioreactor system is capable of supporting these cocultures at near physiologic fluid flow and volume-to-liquid ratios, ensuring stable and organotypic culture conditions. The possibility of long-term cultures enables the repeated exposure to substances. Furthermore, a vascularization of the microfluidic channel circuit using human dermal microvascular endothelial cells yields a physiologically more relevant vascular model.

Here, we present a protocol to coculture primary cells, tissue models and punch biopsies in a microfluidic multi-organ chip for up to 28 days. Human dermal microvascular endothelial cells, liver aggregates and skin biopsies were successfully combined in a common media circulation.

Il Chip Multi-organo - Una piattaforma Microfluidic per lungo termine co-coltura Multi-tessuto

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need ...

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human gut-on-a-chip microphysiological device. We recapitulate the intestinal lumen-capillary tissue interface in a microfluidic device, where physiological mechanical deformations and fluid shear flow are constantly applied to mimic peristalsis. In the lumen microchannel, human intestinal epithelial Caco-2 cells are cultured to form a &#39;germ-free&#39; villus epithelium and regenerate small intestinal villi. Pre-cultured microbial cells are inoculated into the lumen side to establish a host-microbe ecosystem. After microbial cells adhere to the apical surface of the villi, fluid flow and mechanical deformations are resumed to produce a steady-state microenvironment in which fresh culture medium is constantly supplied and unbound bacteria (as well as bacterial wastes) are continuously removed. After extended co-culture from days to weeks, multiple microcolonies are found to be randomly located between the villi, and both microbial and epithelial cells remain viable and functional for at least one week in culture. Our co-culture protocol can be adapted to provide a versatile platform for other host-microbiome ecosystems that can be found in various human organs, which may facilitate in vitro study of the role of human microbiome in orchestrating health and disease.

We describe an in vitro protocol to co-culture gut microbiome and intestinal villi for an extended period using a human gut-on-a-chip microphysiological system.

Co-cultura del vivere Microbiome con Microengineered umana intestinale Villi in un Gut-on-a-chip microfluidico dispositivo

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human ...

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An easy-to-use and low-cost sensor platform is highly desired to measure various types of analytes (e.g., biomarkers, hormones, and drugs) quantitatively and specifically. For this reason, dry film photoresist technology - enabling cheap, facile, and high-throughput fabrication - was used to manufacture the microfluidic biosensor presented here. Depending on the bioassay used afterwards, the versatile platform is capable of detecting various types of biomolecules. For the fabrication of the device, platinum electrodes are structured on a flexible polyimide (PI) foil in the only clean-room process step. The PI foil serves as a substrate for the electrodes, which are insulated with an epoxy-based photoresist. The microfluidic channel is subsequently generated by the development and lamination of dry film photoresist (DFR) foils onto the PI wafer. By using a hydrophobic stopping barrier in the channel, the channel is separated into two specific areas: an immobilization section for the enzyme-linked assay and an electrochemical measurement cell for the amperometric signal readout.
The on-chip bioassay immobilization is performed by the adsorption of the biomolecules to the channel surface. The glucose oxidase enzyme is used as a transducer for electrochemical signal generation. In the presence of the substrate, glucose, hydrogen peroxide is produced, which is detected at the platinum working electrode. The stop-flow technique is applied to obtain signal amplification along with rapid detection. Different biomolecules can quantitatively be measured by means of the introduced microfluidic system, giving an indication of different types of diseases, or, in regard to therapeutic drug monitoring, facilitating a personalized therapy.

A microfluidic biosensor platform was designed and fabricated using low-cost dry film photoresist technology for the rapid and sensitive quantification of various analytes. This single-use system allows for the electrochemical readout of on-chip-immobilized enzyme-linked assays by means of the stop-flow technique.

Film secco basati su Photoresist biosensore elettrochimico Microfluidic Platform: Fabbricazione di dispositivi, saggio su chip preparazione e funzionamento del sistema

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An ...

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means of identifying radiosensitive sites in living organisms. This paper describes a series of on-chip immobilization methods developed for the targeted microbeam irradiation of live individuals of Caenorhabditis elegans. Notably, the treatment of the polydimethylsiloxane (PDMS) microfluidic chips that we previously developed to immobilize C. elegans individuals without the need for anesthesia is explained in detail. This chip, referred to as a worm sheet, is resilient to allow the microfluidic channels to be expanded, and the elasticity allows animals to be enveloped gently. Also, owing to the self-adsorption capacity of the PDMS, animals can be sealed in the channels by covering the surface of the worm sheet with a thin cover film, in which animals are not pushed into the channels for enclosure. By turning the cover film over, we can easily collect the animals. Furthermore, the worm sheet shows water retention and allows C. elegans individuals to be subjected to microscopic observation for long periods under live conditions. In addition, the sheet is only 300 &#181;m thick, allowing heavy ions such as carbon ions to pass through the sheet enclosing the animals, thus allowing the ion particles to be detected and the applied radiation dose to be measured accurately. Because selection of the cover films used for enclosing the animals is very important for successful long-term immobilization, we conducted the selection of the suitable cover films and showed a recommended one among some films. As an application example of the chip, we introduced imaging observation of muscular activities of animals enclosing the microfluidic channel of the worm sheet, as well as the microbeam irradiation. These examples indicate that the worm sheets have greatly expanded the possibilities for biological experiments.

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

A series of immobilization methods has been established to allow the targeted irradiation of live Caenorhabditis elegans individuals using a recently developed ultra-thin polydimethylsiloxane microfluidic chip with water retention. This novel on-chip immobilization is also adequate for imaging observations. The detailed treatment and application examples of the chip are explained.

Immobilizzazione degli individui Live Caenorhabditis elegans utilizzando un Chip microfluidico Polydimethylsiloxane di ultra-sottile con ritenzione idrica

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means ...

Establishing Single-Cell Based Co-Cultures in a Deterministic Manner with a Microfluidic Chip

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases including cancer. However, it is challenging to clarify the complex mechanism of intercellular interactions in highly heterogeneous cell populations using conventional co-culture systems because the heterogeneity of the cell subpopulation is obscured by the average values; the conventional co-culture systems can only be used to describe the population signal, but are incapable of tracking individual cells behavior. Furthermore, conventional single-cell experimental methods have low efficiency in cell manipulation because of the Poisson distribution. Microfabricated devices are an emerging technology for single-cell studies because they can accurately manipulate single cells at high-throughput and can reduce sample and reagent consumption. Here, we describe the concept and application of a microfluidic chip for multiple single-cell co-cultures. The chip can efficiently capture multiple types of single cells in a culture chamber (~46%) and has a sufficient culture space useful to study the cells&#39; behavior (e.g., migration, proliferation, etc.) under cell-cell interaction at the single-cell level. Lymphatic endothelial cells and oral squamous cell carcinoma were used to perform a single-cell co-culture experiment on the microfluidic platform for live multiple single-cell interaction studies.

This report describes a microfluidic chip-based method to set up a single cell culture experiment in which high-efficiency pairing and microscopic analysis of multiple single cells can be achieved.

Stabilire coculture basate su una singola cellula in modo deterministico con un chip microfluidico

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases ...