This work was financially supported by the Ministry of Science and Technology of Taiwan under Contract No. MOST 104-2311-B-002-026 (K. Y. Lo), No. MOST 104-2112-M-030-002 (Y. S. Sun), and National Taiwan University Career Development Project (103R7888) (K. Y. Lo). The authors also thank the Center for Emerging Material and Advanced Devices, National Taiwan University, for the use of the cell culture room.

1. Chip progettazione e fabbricazione <ol><li> Disegnare i modelli da ablazione su substrati PMMA e nastri doppio lato utilizzando software commerciale 24. <ol><li> Per studiare gli effetti di concentrazioni chimiche e sforzi di taglio, disegnare un pattern &quot;albero di Natale&quot; con una larghezza variabile alla sua estremità in ciascuna delle cinque aree culturali (Figure 1A e 1B). </li><li> Per studiare gli effetti delle concentrazioni chimiche e campi elettrici, disegnare un modello di &quot;albero di Natale&quot; con due canali fluidici più di ponti salini (Figura 2a e 2b). </li></ol></li><li> Scribe un modello individuale su un foglio di PMMA o un nastro a doppio lato caricando il file corrispondente nella incisione laser CO 2. <ol><li> Accendere il incisione laser, e controllare il suo collegamento al computer. Aprire il modello da ablated utilizzando il software commerciale. </li><li> Mettere un foglio PMMA o un nastro biadesivo on cima alla fase della punta per tracciare. Regolare il fuoco del laser CO 2, se necessario, utilizzando la barra di calibrazione e il visibile laser He-Ne. </li><li> Caricare il modello in scriber per l&#39;ablazione sul foglio PMMA o il nastro. </li><li> Sollevare il foglio di fantasia o nastro, rimuovere pezzi indesiderati, e pulire la superficie con soffiaggio di azoto. NOTA: Lo spessore della lastra PMMA è 1 mm, e quello del nastro biadesivo è 0,07 millimetri o 0,22 mm. </li></ol></li></ol> 2. assemblaggio di chip <ol><li> Con super colla, collegare gli adattatori acriliche (lunghezza x larghezza x altezza 10 mm x 10 mm x 5 mm, con filettature in mezzo) al livello più alto del chip allineando i filettature ei fori sulla parte superiore -La maggior parte strato. Queste schede servono come mezzo di ingressi / uscite e connettori ponte salino. </li><li> Assemblare il chip microfluidico all&#39;interno di una cappa a flusso laminare. </li><li> Montare il chip microfluidica stress chimico-shear (circuito integrato CSS). <ol><li> UNttach 3 fogli di lastre in PMMA tracciatura con 2 pezzi di descritto nastro biadesivo. (Figura 1A e 1B). </li><li> Aggiungere un altro pezzo di nastro biadesivo spessore 0,07 millimetri al fondo del chip e collegare il chip ad un diametro di 10 cm Petri. </li><li> Mettere il chip di assemblaggio nella camera a vuoto per una notte. </li></ol></li><li> Per montare il chip campo chimico-elettrico (circuito integrato CEF), allegare il foglio di PMMA ad un nastro biadesivo di spessore = 0,22 millimetri (Figura 2A e 2B). <ol><li> Attaccare il foglio di PMMA descritto e nastro biadesivo per un diametro di 10 cm Petri utilizzando questo stesso pezzo di nastro adesivo. </li></ol></li><li> Lasciare il chip montato all&#39;interno della cappa ed esporlo ai raggi UV per 30 minuti per la sterilizzazione. </li><li> Collegare gli ingressi a due siringhe da 3 ml con tubi di plastica e dita i dadi. Collegare l&#39;uscita di un tubo di scarico tramite un tubo di plastica ed un dado a bloccarlo. NOTA: Autoclavetutti i tubi e dadi a 121 ° C per 15 min prima dell&#39;uso. </li><li> Premere lentamente lo stantuffo della siringa per primi canali fluidici con PBS. Spingere siringhe avanti e indietro per rimuovere le bolle. </li><li> Mettere le fiches in un incubatore notte a 37 ° C sotto 5% di CO 2. NOTA: Questi due passaggi sono finalizzate a lavare il chip e rimuovere eventuali bolle rimanenti all&#39;interno del chip. </li><li> Prendere il chip fuori dell&#39;incubatore. </li><li> Preparare ponti salini agar per la generazione di campi elettrici nel chip CEF. <ol><li> Sciogliere 3% agarosio punto basso punto di fusione in tampone fosfato 1x salina tamponata (PBS) utilizzando un forno a microonde. </li><li> Iniettare la soluzione-phased agarosio nel canale ponte salino e inserire gli elettrodi prima soluzione si solidifica. </li><li> Posizionare elettrodi argento / argento cloruro nei dadi tubolari. </li></ol></li><li> Collegare le siringhe alle pompe siringa, e il flusso continuo del terreno di coltura (Dulbecco Modified Eagler17; s media, DMEM) nel canale fluidica per 10 minuti a una portata di 20 microlitri / min per sostituire PBS. </li></ol> 3. cellulare Preparazione e apparato sperimentale NOTA: Pre-caldo 1x PBS, terreno di coltura (DMEM più FBS), e tripsina in un bagno d&#39;acqua C 37 ° prima dell&#39;uso. <ol><li> Piastra 2 x 10 di cancro al polmone 5 celle CL1-5 25,26 in una capsula di Petri 10 centimetri fornito con DMEM più siero fetale bovino al 10% (FBS). Incubare le cellule all&#39;interno di un incubatore sotto 5% di CO 2 a 37 ° C fino al 90% di confluenza. </li><li> Aspirare il medio e lavare le cellule una volta con pre-riscaldato 1x PBS. Aggiungere 2 ml di tampone tripsina 0,05% per le cellule e attendere 2 a 3 min a 37 ° C per staccare le cellule. </li><li> Trasferire le cellule ad un 15 ml tubo di centrifugazione sterile e aggiungere 6 ml di mezzo di coltura nel tubo. invertire delicatamente il tubo per la miscelazione ed estrarre 5 microlitri del mezzo cella contenente per contare il numero di cellule in un hemocytometer. </li><li> Centrifugare la provetta a 300 x g per 5 min. Sospendere 10 6 cellule in 1 ml di mezzo di coltura e posizionare il mezzo cella contenente in una siringa da 1 ml. </li><li> Infondere medio cella contenente nel chip microfluidico dalla presa e assicurarsi che la soluzione distribuisce attraverso tutte le cinque aree culturali. Incubare il chip all&#39;interno di un incubatore sotto 5% di CO 2 a 37 ° C per 2 ore. </li></ol> 4. Setup sperimentale <ol><li> Prendere il chip fuori dell&#39;incubatore e posizionarlo in cima ad un riscaldatore di vetro di ossido di indio e stagno trasparente (ITO). NOTA: Il vetro ITO è collegato ad un regolatore proporzionale-integrale-derivativa (PID) per mantenere la temperatura a 37 ± 0,5 ° C tramite feedback da un accoppiatore termica fissata saldamente tra il riscaldatore ITO e il chip. </li><li> Mettere il gruppo di chip-riscaldatore sulla cima di una fase XY motorizzato di un microscopio invertito per il monitoraggio migrazione cellulare o uno stadio XY fissoun microscopio a fluorescenza invertito per misurare la produzione di ROS. </li><li> Per generare differenti concentrazioni chimiche, riempire due siringhe con 5 ml di soluzioni chimiche di concentrazioni relative 0 e 1 (disciolti nel mezzo di coltura) e pompare nel chip a portate desiderate: nel chip CSS, ad un flusso di 0,3 ml / min per 1 ora; nel chip CEF, a una portata di 20 microlitri / min per i primi 20 min e una velocità di flusso di 20 ml / h per altri 120 minuti (tempo totale = 2 ore). </li><li> Nel chip CEF, per il monitoraggio della migrazione delle cellule, il programma lo stadio XY del microscopio per ripetere scattare foto, tramite una reflex digitale (DSLR), di certo campo di vista (FOV) all&#39;interno di aree culturali di ogni 15 min per 2 hr. </li><li> Per misurare la produzione di ROS, utilizzare l&#39;indicatore basato sulla fluorescenza 2&#39;-7&#39;-dichlorodihydrofluoresce diacetato (2&#39;-7&#39;-DCFDA). <ol><li> Preparare il brodo di 2&#39;-7&#39;-DCFDA a 10 mm di biologia molecolare grado disolfossido metilico (DMSO). Diluire DCFDA in DMEM senza siero solo (5 micron di DMEM). Il potenziale deacetilasi potrebbe aumentare i segnali di fondo e diminuire i segnali nelle cellule. </li><li> Dopo 1 h di shear stimolo stress nel chip CSS o dopo 2 ore di EF stimolo chip CEF, pompare 2&#39;-7&#39;-DCFDA (5 pM in DMEM) nel chip ad una portata di 20 microlitri / min per il primo 20 min e una velocità di flusso di 20 ml / h per altri 20 min. Per il lavaggio, pompa DMEM nel chip ad una portata di 20 l / h per 20 min. </li><li> Scatta foto, tramite una fotocamera del dispositivo ad accoppiamento di carica (CCD), di alcune FOV all&#39;interno di aree culturali per analizzare le intensità fluorescenti. </li></ol></li></ol> 5. I calcoli di concentrazioni chimiche, sforzi di taglio, e campi elettrici <ol><li> Sia il chip CSS e il chip CEF, calcolare le concentrazioni chimiche nelle cinque aree culturali. Ad esempio, iniettando H 2 O 2 di Conczioni 0 e 200 pM dalle due insenature, concentrazioni di 0, 25, 100, 175, e 200 pM vengono generati. NOTA: partendo dal presupposto che tutti i liquidi a flusso separato senza intoppi e in modo uniforme intorno alla forcella, le concentrazioni relative nelle cinque aree culturali sono 0, 1/8, 1/2, 7/8, e 1 rispettivamente. </li><li> Nel circuito integrato CSS, calcolare la sollecitazione di taglio (τ) in ciascuna delle aree di coltura usando <img alt="Equazione 1" src="/files/ftp_upload/54397/54397eq1.jpg" /> 27, in cui Q è la portata volumetrica, η è la viscosità fluidico, h è l&#39;altezza del canale, e w è la larghezza del canale. NOTA: Impostando Q = 0,3 ml / min a ciascun ingresso (Q = 0,12 ml / min in ciascuna area culturale), η = 0.0008 Pa · s per mezzo di coltura, h = 1 mm, w = 1 ~ 4 mm, la sollecitazione di taglio viene calcolata variare da 0,0048 Pa (regione larghezza di 4 mm) a 0,0192 Pa (regione di larghezza 1 mm). </li><li> Inil chip CEF, calcolare la forza EF entro ciascuna delle zone di coltura utilizzando E = I / (σA eff) (legge di Ohm), dove I è la corrente elettrica che scorre attraverso il canale fluidico, σ è la conducibilità elettrica del terreno di coltura, e A eff è l&#39;area della sezione trasversale effettiva del canale. NOTA: Utilizzando σ = 1,38 Ω -1 m -1 per mezzo di coltura e A eff = 0,22 millimetri 2 (larghezza = 1 mm ed altezza = 0,22 millimetri), la forza EF è calcolato per essere E (mV / mm) = I ( A) × 3.3 × 10 6. <ol><li> Come mostrato in figura 2D, trattare il circuito equivalente di cinque circuiti a sezione a C con quattro (8 + 35 + 8) segmenti e uno (5 + 35 + 5) segmento. NOTA: Analizzando questo circuito parallelo secondo la legge la tensione di Kirchhoff e la legge di Ohm, corrente che fluisce attraverso la cultura sono cinquecome, I 1 a I 5 dal basso verso l&#39;alto, sono calcolati a circa 0,49 I (zona 1), 0,25 mi (zona 2), 0,13 mi (zona 3), 0,08 mi (zona 4), ​​e 0,05 mi (area 5) rispettivamente, dove I è la totale corrente continua (cc). Con una corrente continua applicata di 0,157 mA, EF di 254, 130, 67, 41, e 26 mV / mm sono generate nei cinque aree culturali. NOTA: Per un&#39;analisi elettrico semplificato della rete microfluidica, tutti i segmenti fluidici sono considerate come resistori con resistenza proporzionale alla loro lunghezza. </li></ol></li></ol> Analisi 6. Dati Nota: L&#39;analisi dei dati viene effettuata utilizzando il software ImageJ. <ol><li> Analizzare la produzione di ROS. <ol><li> Eseguire il software ImageJ. Vai su &quot;File&quot; → Apri per caricare un&#39;immagine fluorescente da analizzare. </li><li> Vai a &quot;Immagine&quot; → &quot;Tipo&quot; → &quot;16 bit&quot; per cambiare l&#39;imetà per una scala di grigi. </li><li> Disegnare un poligono per racchiudere una cella. Vai a &quot;Analisi&quot; → &quot;Measure&quot; per misurare l&#39;intensità di fluorescenza media della cella. </li><li> Ripetere 6.1.3 per raccogliere intensità da almeno 50 cellule di tre esperimenti indipendenti, e calcolare l&#39;intensità media con errore standard della media (SEM). </li><li> Ripetere 6.1.1-6.1.4 per ogni condizione sperimentale. </li></ol></li><li> Analizzare Migrazione cellulare. <ol><li> Eseguire il software ImageJ. Vai su &quot;File&quot; → Apri per caricare un&#39;immagine scattata al tempo = 0 da analizzare. </li><li> Disegnare un poligono per racchiudere una cella. Vai a &quot;Analisi&quot; → &quot;Measure&quot; per misurare il centro di massa della cella come (x 1, y 1). </li><li> Ripetere 6.2.1- 6.2.2 per misurare il centro di massa della stessa cella (x 2, y 2) da un&#39;altra immagine prendere al tempo = t. </li><li> Calcolare il tasso di migrazione (in micron / ora) diquesta cella come <img alt="Equazione 2" src="/files/ftp_upload/54397/54397eq2.jpg" /> . </li><li> Ripetere 6.2.1-6.2.4 per raccogliere i tassi di migrazione da almeno 50 cellule di tre esperimenti indipendenti, e calcolare il tasso di migrazione media con errore standard della media (SEM). </li><li> Da 6.2.2 e 6.2.3, calcolare la direzionalità migrazione di questa cellula come coseno θ o <img alt="Equazione 3" src="/files/ftp_upload/54397/54397eq3.jpg" /> , Dove θ è l&#39;angolo tra il vettore della EF applicata (da positivo a negativo) e il vettore dall&#39;inizio alla posizione finale della cella (Figura 2G). </li><li> Ripetere 6.2.6 per raccogliere directedness migrazione da almeno 50 cellule di tre esperimenti indipendenti, e calcolare la direzionalità di migrazione media con errore standard della media (SEM). </li><li> Calcolare il tasso di migrazione media con SEM e la direzionalità di migrazione media con SEM per ogni condizione sperimentale. NOTA: A directednessdi +1 indica che tutte le cellule migrano verso il catodo, e un valore -1 indica che tutte le cellule migrano verso l&#39;anodo. La direzionalità di un gruppo di cellule in modo casuale in movimento è vicino a 0. </li></ol></li><li> Analizzare Allineamento celle. <ol><li> Eseguire il software ImageJ. Vai su &quot;File&quot; → &quot;Apri&quot; per caricare un&#39;immagine da analizzare. </li><li> Trattare la cella come un&#39;ellisse e tracciare una linea per indicare l&#39;asse longitudinale di una cella. Vai a &quot;Analisi&quot; → &quot;Measure&quot; per misurare l&#39;angolo β tra la linea e la direzione EF orizzontale. </li><li> Ripetere 6.3.2 per raccogliere β da almeno 50 cellule di tre esperimenti indipendenti, e calcolare il coseno media β con errore standard della media (SEM). </li><li> Ripetere 6.3.1-6.3.3 per ogni condizione sperimentale. NOTA: A cos β di +1 indica che tutte le celle siano allineati in parallelo alla applicata EF, e un valore 0 indica che tutte le cellule siano allineati perpendicularly al applicata EF. </li></ol></li></ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+shear+stresses+and+antioxidant+concentrations+on+the+production+of+reactive+oxygen+species+in+lung+cancer+cells.">Effects of shear stresses and antioxidant concentrations on the production of reactive oxygen species in lung cancer cells.</a> Biomicrofluidics. 7, 64108 (2013).</li><li>Cheng, J. Y., Wei, C. W., Hsu, K. H., Young, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct-write+laser+micromachining+and+universal+surface+modification+of+PMMA+for+device+development.">Direct-write laser micromachining and universal surface modification of PMMA for device development.</a> Sensor Actuat B-Chem. 99, 186-196 (2004).</li><li>Chen, J. J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+analysis+of+gene+expression+in+invasion+by+a+lung+cancer+model.">Global analysis of gene expression in invasion by a lung cancer model.</a> Cancer Res. 61, 5223-5230 (2001).</li><li>Shih, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collapsin+response+mediator+protein-1+and+the+invasion+and+metastasis+of+cancer+cells.">Collapsin response mediator protein-1 and the invasion and metastasis of cancer cells.</a> J Natl Cancer I. 93, 1392-1400 (2001).</li><li>Lu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+shear+devices+for+quantitative+analysis+of+cell+adhesion.">Microfluidic shear devices for quantitative analysis of cell adhesion.</a> Anal Chem. 76, 5257-5264 (2004).</li><li>Fried, L. E., Arbiser, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Honokiol,+a+Multifunctional+Antiangiogenic+and+Antitumor+Agent.">Honokiol, a Multifunctional Antiangiogenic and Antitumor Agent.</a> Antioxid Redox Sign. 11, 1139-1148 (2009).</li><li>Chisti, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrodynamic+damage+to+animal+cells.">Hydrodynamic damage to animal cells.</a> Crit Rev Biotechnol. 21, 67-110 (2001).</li><li>Davies, P. F., Remuzzi, A., Gordon, E. J., Dewey, C. F., Gimbrone, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Turbulent+fluid+shear+stress+induces+vascular+endothelial+cell+turnover+in+vitro.">Turbulent fluid shear stress induces vascular endothelial cell turnover in vitro.</a> Proc Natl Acad Sci U S A. 83, 2114-2117 (1986).</li><li>Zoro, B. J., Owen, S., Drake, R. A., Hoare, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+process+stress+on+suspended+anchorage-dependent+mammalian+cells+as+an+indicator+of+likely+challenges+for+regenerative+medicines.">The impact of process stress on suspended anchorage-dependent mammalian cells as an indicator of likely challenges for regenerative medicines.</a> Biotechnol Bioeng. 99, 468-474 (2008).</li><li>Chin, L. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+reactive+oxygen+species+in+endothelial+cells+under+different+pulsatile+shear+stresses+and+glucose+concentrations.">Production of reactive oxygen species in endothelial cells under different pulsatile shear stresses and glucose concentrations.</a> Lab Chip. 11, 1856-1863 (2011).</li><li>Tsai, H. F., Peng, S. W., Wu, C. Y., Chang, H. F., Cheng, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrotaxis+of+oral+squamous+cell+carcinoma+cells+in+a+multiple-electric-field+chip+with+uniform+flow+field.">Electrotaxis of oral squamous cell carcinoma cells in a multiple-electric-field chip with uniform flow field.</a> Biomicrofluidics. 6, 34116 (2012).</li></ol>

The authors have nothing to disclose.

chip PMMA-based sono fabbricati con ablazione laser e la scrittura, che sono metodi meno costosi e più facili rispetto ai chip PDMS-based che richiedono più complicato litografia soft. Dopo aver progettato un chip microfluidica, la fabbricazione e l&#39;assemblaggio può essere fatto nel giro di 5 minuti. Ci sono alcuni passaggi critici che l&#39;attenzione dovrebbe essere prestata alla nello svolgimento dell&#39;esperimento. Il primo è la questione &quot;assemblaggio&quot;. Gli adattatori devono essere incollati cor...

In cellule in vivo sono circondate da una varietà di biomolecole compreso matrice extracellulare (ECM), carboidrati, lipidi, e altre cellule. Essi funzionalizzare rispondendo agli stimoli micro-ambientali quali le interazioni con ECM e le risposte alle gradienti chimici di vari fattori di crescita. Tradizionalmente, studi cellulare in vitro sono condotti in piatti di coltura di cellule in cui il consumo di cellule e reagenti è grande e le cellule crescono in modo statico (non circolanti) ambiente. Recentemente, dispositivi micro-fabbricato integrati con componenti fluidici hanno fornito una piattaforma alternativa a studi su cellule in un modo più controllabile. Tali dispositivi sono in grado di creare una precisa microambiente di stimoli chimici e fisici riducendo al minimo il consumo di cellule e reagenti. Questi chip microfluidici possono essere fatte di substrati di vetro, silicio, polidimetilsilossano polimeri (PDMS), polimetilmetacrilato (PMMA) substrati, o polyethylenetereftalato (PET) substrati 1-3. dispositivi PDMS basati sono trasparenti, biocompatibili, e permeabile ai gas, che li rende adatti per colture cellulari a lungo termine e studi. substrati PMMA e PET sono economici e facili da lavorare con ablazione laser e la scrittura. dispositivi microfluidici dovrebbero fornire cellule con un micro-ambiente stabile e controllabile in cui le cellule sono soggette a differenti stimoli chimici e fisici. Ad esempio, i chip microfluidici sono utilizzati per studiare chemiotassi di cellule. Invece di metodi tradizionali che impiegano camera di Boyden e capillare di 4,5 questi dispositivi fluidici miniaturizzati in grado di generare precise gradienti chimici per lo studio dei comportamenti cellule &#39;1,6,7. Un altro esempio è quello di studiare la migrazione direzionale cellule &#39;in campi elettrici (EFS), un fenomeno chiamato electrotaxis. Comportamenti Electrotactic di cellule sono stati segnalati per essere collegato con la rigenerazione dei nervi 8, lo sviluppo embrionale 9,e ferita guarigione 10,11. E molti studi sono stati condotti per indagare il electrotaxis di vari tipi di cellule, tra cui le cellule tumorali 12,13, i linfociti 14,15, cellule leucemiche 11, e cellule staminali 16. Convenzionalmente, capsule di Petri e bicchieri di copertura sono utilizzati per la costruzione di camere di electrotactic per la generazione di EF 17. Tali configurazioni semplici pongono problemi di media evaporazione e EF imprecise, ma possono essere superati dai dispositivi microfluidici di canali fluidici chiuso, ben definiti 12,18,19. Per studiare sistematicamente risposte cellulari sotto precise stimoli chimici ed elettrici controllabili,, sarebbe di grande utilità per sviluppare dispositivi microfluidici grado di fornire cellule con stimoli multipli contemporaneamente. Ad esempio, Li et al. riferito un dispositivo microfluidica PDMS-based per la creazione di una o coesistenti gradienti chimici ed EFS 20. Kao et al. devfuggì un chip microfluidica simile a modulare la chemiotassi delle cellule tumorali del polmone da EF 6. Inoltre, per aumentare la produttività, Hou et al. progettato e fabbricato un chip multicanale-dual-campo elettrico PMMA per poter fornire le cellule con 8 diversi stimoli combinati, (concentrazioni 2 punti di forza EF x 4 chimici) essendo 21. Per aumentare ulteriormente il tutto e aggiungere lo stimolo sforzo di taglio, abbiamo sviluppato due dispositivi microfluidici PMMA-based per studiare le risposte cellulari sotto chimici singola o coesistenti / stimoli di stress / taglio elettrici. Segnalato da Lo et al. 22,23, questi dispositivi contengono cinque canali di coltura cellulare indipendenti soggetti a continuo fluido che scorre, imitando il sistema circolatorio in vivo. Nel primo chip (chip chimico-shear stress o il chip CSS), cinque concentrazioni relative di 0, 1/8, 1/2, 7/8 e 1 sono generati nelle regioni di coltura, e un gradiente di sforzo di taglio è produced all&#39;interno di ciascuno dei cinque aree culturali. Nella seconda piastrina (chip chimico-elettrico campo o il chip CEF), utilizzando un&#39;unica serie di elettrodi e 2 pompe a siringa, cinque resistenze EF si generano in aggiunta a cinque diverse concentrazioni chimiche all&#39;interno di queste aree di coltura. calcoli numerici e simulazioni sono eseguite per una migliore progettazione e funzionano questi chip, e cellule tumorali coltivate all&#39;interno di questi dispositivi sono soggetti a stimoli singoli o coesistenti per osservare le loro risposte rispetto alla produzione di specie reattive dell&#39;ossigeno (ROS), il tasso di migrazione, e la direzione di migrazione. Questi chip sono dimostrati di essere risparmio di tempo,-throughput elevato e dispositivi affidabili per indagare come le cellule rispondono a stimoli diversi micro-ambientali.

<table border="0" cellpadding="0" cellspacing="0" width="1070">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td style="width:213px;">Gibco</td>
			<td style="width:119px;">11965-092</td>
			<td style="width:460px;">Cell culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Trypsin</td>
			<td style="width:213px;">Gibco</td>
			<td style="width:119px;">25300-054</td>
			<td>detach cell from the dish</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Fetal bovine serum (FBS)</td>
			<td style="width:213px;">Gibco</td>
			<td style="width:119px;">10082147</td>
			<td>Cell culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">10-cm cell culture Petri dish</td>
			<td style="width:213px;">Nunc</td>
			<td style="width:119px;">150350</td>
			<td>Cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Bright-Line&#160;Hemacytometer</td>
			<td style="width:213px;">Sigma</td>
			<td>Z359629</td>
			<td>Cell Counting Equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">PMMA</td>
			<td style="width:213px;">Customized</td>
			<td style="width:119px;">Customized</td>
			<td>Microfluidic chip</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Adaptor</td>
			<td style="width:213px;">Customized</td>
			<td style="width:119px;">Customized</td>
			<td>Microfluidic chip</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.07/0.22 mm double-sided tape&#160;</td>
			<td>3M</td>
			<td>8018/9088</td>
			<td>Microfluidic chip</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Low melting point agarose</td>
			<td style="width:213px;">Sigma</td>
			<td style="width:119px;">A9414</td>
			<td>Salt bridge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2&#39;-7&#39;-dichlorodihydrofluoresce diacetate</td>
			<td>Sigma</td>
			<td style="width:119px;">D6883</td>
			<td>Intracellular ROS measurement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Indium tin oxide (ITO) glass</td>
			<td style="width:213px;">Merck</td>
			<td>300739</td>
			<td>Heater</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Proportional-integral-derivative controller&#160;</td>
			<td>JETEC Electronics Co.</td>
			<td>TTM-J4-R-AB</td>
			<td>Temperature controller</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermal coupler</td>
			<td>TECPEL</td>
			<td style="width:119px;">TPK-02A</td>
			<td>Temperature controller</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">CO2 laser scriber</td>
			<td>Laser Tools &#38; Technics Corp.</td>
			<td style="width:119px;">ILS2</td>
			<td>Microfluidic chip fabrication</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringe pumps</td>
			<td>New Era</td>
			<td style="width:119px;">NE-300</td>
			<td>Pumping medium and chemicals into the chip</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Power supply</td>
			<td>Major Science&#160;</td>
			<td style="width:119px;">MP-300V</td>
			<td>Supplying direct currents</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Inverted microscope</td>
			<td>Olympus</td>
			<td>CKX41</td>
			<td>Monitoring cell migration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Inverted fluorescent microscope</td>
			<td style="width:213px;">Nikon</td>
			<td style="width:119px;">TS-100</td>
			<td>Monitoring cell migartion and fluorescencent signals</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DSLR camera</td>
			<td>Canon</td>
			<td>60D</td>
			<td>Recording bright-field images&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CCD camera</td>
			<td>Nikon</td>
			<td style="width:119px;">DS-Qi1</td>
			<td>Recording fluorescent images&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">super glue</td>
			<td style="width:213px;">3M Scotch</td>
			<td style="width:119px;">7004</td>
			<td>Attaching adaptors to PMMA substrates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AutoCAD</td>
			<td style="width:213px;">Autodesk Inc.</td>
			<td style="width:119px;"></td>
			<td>Designing microfluidic chips</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">DMSO</td>
			<td style="width:213px;">Sigma</td>
			<td style="width:119px;">D8418</td>
			<td>Dissolving DCFDA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">ImgeJ</td>
			<td style="width:213px;">National Institutes of Health</td>
			<td style="width:119px;"></td>
			<td>Quantifying fluorescent intensities and cell migration</td>
		</tr>
	</tbody>
</table>

designing microfluidic devices for studying cellular responses under single or coexisting chemical/electrical/shear stress stimuli

dispositivi microfluidici sono capaci di creare una precisa e controllabile cellulare microambiente di pH, temperatura, concentrazione salina, e altri stimoli fisici o chimici. Essi sono stati comunemente utilizzati per studi cellulari in vitro, fornendo in vivo come i dintorni. In particolare, come le cellule di risposta a gradienti chimici, campi elettrici, e sforzi di taglio ha attirato molti interessi in quanto questi fenomeni sono importanti per comprendere le proprietà e funzioni cellulari. Questi chip microfluidici possono essere fatte di substrati di vetro, silicio, polimeri polidimetilsilossano (PDMS), polimetilmetacrilato (PMMA), substrati o substrati di polietilentereftalato (PET). Di questi materiali, substrati PMMA sono economici e possono essere facilmente trattati con ablazione laser e la scrittura. Anche se alcuni dispositivi microfluidici sono stati progettati e fabbricati per la generazione multipla, coesistenti stimoli elettrici e chimici, nessuno di loro è stato consideratosufficientemente efficace nel ridurre le ripetizioni sperimentali, particolari a fini di screening. In questo rapporto, descriviamo la nostra progettazione e fabbricazione di due chip microfluidica PMMA-based per indagare le risposte cellulari, per la produzione di specie reattive dell&#39;ossigeno e la migrazione, sotto chimici / stimoli di stress / shear elettrici singoli o coesistenti. Il primo chip genera cinque concentrazioni relative di 0, 1/8, 1/2, 7/8 e 1 nelle regioni di coltura, insieme con un gradiente di sforzo di taglio prodotta all&#39;interno di ciascuna di queste aree. Il secondo chip genera le stesse concentrazioni relativi, ma con cinque diversi punti di forza del campo elettrico creato all&#39;interno di ogni area culturale. Questi dispositivi non solo forniscono le cellule con un preciso, controllabile micro-ambiente, ma anche di aumentare notevolmente il throughput sperimentale.

Il stress chimico-taglio (CSS) Chip Il chip CSS è costituito da tre lastre in PMMA, ciascuno di 1 mm di spessore, collegati tra loro tramite due nastri biadesivi, ciascuno dello spessore 0,07 millimetri (Figura 1A e 1B). La struttura &quot;ad albero di Natale&quot; genera cinque concentrazioni relative di 0, 1/8, 1/2, 7/8, e 1 nelle cinque aree culturali. Progettando l&#39;area cultura come u...

Watch this Scientific Journal Video about Designing Microfluidic Devices for Studying Cellular Responses Under Single or Coexisting Chemical/Electrical/Shear Stress Stimuli at JoVE.com

Designing Microfluidic Devices for Studying Cellular Responses Under Single or Coexisting Chemical/Electrical/Shear Stress Stimuli

Micro-fabricated devices integrated with fluidic components provide an in vitro platform for cell studies mimicking the in vivo micro-environment. We developed polymethylmethacrylate-based microfluidic chips for studying cellular responses under single or coexisting chemical/electrical/shear stress stimuli.

Progettazione di dispositivi microfluidici per lo studio Risposte cellulare in singolo o Coesistenza chimico / elettrico / Shear stress stimoli

Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other ...

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JoVE Journal

Bioengineering

8.8K Views.  National Taiwan University. Micro-fabricated devices integrated with fluidic components provide an in vitro platform for cell studies mimicking the in vivo micro-environment. We developed polymethylmethacrylate-based microfluidic chips for studying cellular responses under single or coexisting chemical/electrical/shear stress stimuli.

Video: Designing Microfluidic Devices for Studying Cellular Responses Under Single or Coexisting Chemical/Electrical/Shear Stress Stimuli

A Microfluidic-based Hydrodynamic Trap for Single Particles

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science. Methods for particle trapping based on optical, magnetic, electrokinetic, and acoustic techniques have led to major advancements in physics and biology ranging from the molecular to cellular level. In this article, we introduce a new microfluidic-based technique for particle trapping and manipulation based solely on hydrodynamic fluid flow. Using this method, we demonstrate trapping of micro- and nano-scale particles in aqueous solutions for long time scales. The hydrodynamic trap consists of an integrated microfluidic device with a cross-slot channel geometry where two opposing laminar streams converge, thereby generating a planar extensional flow with a fluid stagnation point (zero-velocity point). In this device, particles are confined at the trap center by active control of the flow field to maintain particle position at the fluid stagnation point. In this manner, particles are effectively trapped in free solution using a feedback control algorithm implemented with a custom-built LabVIEW code. The control algorithm consists of image acquisition for a particle in the microfluidic device, followed by particle tracking, determination of particle centroid position, and active adjustment of fluid flow by regulating the pressure applied to an on-chip pneumatic valve using a pressure regulator. In this way, the on-chip dynamic metering valve functions to regulate the relative flow rates in the outlet channels, thereby enabling fine-scale control of stagnation point position and particle trapping. The microfluidic-based hydrodynamic trap exhibits several advantages as a method for particle trapping. Hydrodynamic trapping is possible for any arbitrary particle without specific requirements on the physical or chemical properties of the trapped object. In addition, hydrodynamic trapping enables confinement of a "single" target object in concentrated or crowded particle suspensions, which is difficult using alternative force field-based trapping methods. The hydrodynamic trap is user-friendly, straightforward to implement and may be added to existing microfluidic devices to facilitate trapping and long-time analysis of particles. Overall, the hydrodynamic trap is a new platform for confinement, micromanipulation, and observation of particles without surface immobilization and eliminates the need for potentially perturbative optical, magnetic, and electric fields in the free-solution trapping of small particles.

In this article, we present a microfluidic-based method for particle confinement based on hydrodynamic flow. We demonstrate stable particle trapping at a fluid stagnation point using a feedback control mechanism, thereby enabling confinement and micromanipulation of arbitrary particles in an integrated microdevice.

Un Microfluidic basato Trappola idrodinamico per particelle singole

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science.  Methods for ...

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Bioingegneria

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are available for microfluidic devices using soft lithography techniques 1-3. Microfluidic devices have been used for sub-cellular imaging 4,5, in vivo laser microsurgery 2,6 and cellular imaging 4,7. In vivo imaging requires immobilization of organisms. This has been achieved using suction 5,8, tapered channels 6,7,9, deformable membranes 2-4,10, suction with additional cooling 5, anesthetic gas 11, temperature sensitive gels 12, cyanoacrylate glue 13 and anesthetics such as levamisole 14,15. Commonly used anesthetics influence synaptic transmission 16,17 and are known to have detrimental effects on sub-cellular neuronal transport 4. In this study we demonstrate a membrane based poly-dimethyl-siloxane (PDMS) device that allows anesthetic free immobilization of intact genetic model organisms such as Caenorhabditis elegans (C. elegans), Drosophila larvae and zebrafish larvae. These model organisms are suitable for in vivo studies in microfluidic devices because of their small diameters and optically transparent or translucent bodies. Body diameters range from ~10 &mu;m to ~800 &mu;m for early larval stages of C. elegans and zebrafish larvae and require microfluidic devices of different sizes to achieve complete immobilization for high resolution time-lapse imaging. These organisms are immobilized using pressure applied by compressed nitrogen gas through a liquid column and imaged using an inverted microscope. Animals released from the trap return to normal locomotion within 10 min.
We demonstrate four applications of time-lapse imaging in C. elegans namely, imaging mitochondrial transport in neurons, pre-synaptic vesicle transport in a transport-defective mutant, glutamate receptor transport and Q neuroblast cell division. Data obtained from such movies show that microfluidic immobilization is a useful and accurate means of acquiring in vivo data of cellular and sub-cellular events when compared to anesthetized animals (Figure 1J and 3C-F 4).
Device dimensions were altered to allow time-lapse imaging of different stages of C. elegans, first instar Drosophila larvae and zebrafish larvae. Transport of vesicles marked with synaptotagmin tagged with GFP (syt.eGFP) in sensory neurons shows directed motion of synaptic vesicle markers expressed in cholinergic sensory neurons in intact first instar Drosophila larvae. A similar device has been used to carry out time-lapse imaging of heartbeat in ~30 hr post fertilization (hpf) zebrafish larvae. These data show that the simple devices we have developed can be applied to a variety of model systems to study several cell biological and developmental phenomena in vivo.

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

A simple microfluidic device has been developed to perform anesthetic free in vivo imaging of C. elegans, intact Drosophila larvae and zebrafish larvae. The device utilizes a deformable PDMS membrane to immobilize these model organisms in order to perform time lapse imaging of numerous processes such as heart beat, cell division and sub-cellular neuronal transport. We demonstrate the use of this device and show examples of different types of data collected from different model systems.

Semplici dispositivi microfluidici per<em&gt; In vivo</em&gt; Imaging di<em&gt; C. elegans</em&gt;,<em&gt; Drosophila</em&gt; E Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are ...

Microfluidic Mixers for Studying Protein Folding

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason for this is that folding takes place over a wide range of timescales, from nanoseconds to seconds or longer, depending on the protein1. Conventional stopped-flow mixers have allowed measurement of folding kinetics starting at about 1 ms. We have recently developed a microfluidic mixer that dilutes denaturant ~100-fold in ~8 &mu;s2. Unlike a stopped-flow mixer, this mixer operates in the laminar flow regime in which turbulence does not occur. The absence of turbulence allows precise numeric simulation of all flows within the mixer with excellent agreement to experiment3-4.
Laminar flow is achieved for Reynolds numbers Re &le;100. For aqueous solutions, this requires micron scale geometries. We use a hard substrate, such as silicon or fused silica, to make channels 5-10 &mu;m wide and 10 &mu;m deep (See Figure 1). The smallest dimensions, at the entrance to the mixing region, are on the order of 1 &mu;m in size. The chip is sealed with a thin glass or fused silica coverslip for optical access. Typical total linear flow rates are ~1 m/s, yielding Re~10, but the protein consumption is only ~0.5 nL/s or 1.8 &mu;L/hr. Protein concentration depends on the detection method: For tryptophan fluorescence the typical concentration is 100 &mu;M (for 1 Trp/protein) and for FRET the typical concentration is ~100 nM.
The folding process is initiated by rapid dilution of denaturant from 6 M to 0.06 M guanidine hydrochloride. The protein in high denaturant flows down a central channel and is met on either side at the mixing region by buffer without denaturant moving ~100 times faster (see Figure 2). This geometry causes rapid constriction of the protein flow into a narrow jet ~100 nm wide. Diffusion of the light denaturant molecules is very rapid, while diffusion of the heavy protein molecules is much slower, diffusing less than 1 &mu;m in 1 ms. The difference in diffusion constant of the denaturant and the protein results in rapid dilution of the denaturant from the protein stream, reducing the effective concentration of the denaturant around the protein. The protein jet flows at a constant rate down the observation channel and fluorescence of the protein during folding can be observed using a scanning confocal microscope5.

In this work we explain the fabrication and use of a microfluidic mixer capable of mixing two solutions in ~8 &mu;s. We also demonstrate the use of these mixers with spectroscopic detection using UV fluorescence and fluorescence resonance energy transfer (FRET).

Miscelatori per lo studio microfluidici Protein Folding

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason ...

A Microfluidic Device for Studying Multiple Distinct Strains

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a comparative manner. Multiwell plates are routinely used to compare many different strains or cell lines, but allow limited control over the environment dynamics. Microfluidic devices, on the other hand, allow exquisite dynamic control over the surrounding conditions, but it is challenging to image and distinguish more than a few strains in them. Here we describe a method to easily and rapidly manufacture a microfluidic device capable of applying dynamically changing conditions to multiple distinct yeast strains in one channel. The device is designed and manufactured by simple means without the need for soft lithography. It is composed of a Y-shaped flow channel attached to a second layer harboring microwells. The strains are placed in separate microwells, and imaged under the exact same dynamic conditions. We demonstrate the use of the device for measuring protein localization responses to pulses of nutrient changes in different yeast strains.

We present a simple method to produce microfluidic devices capable of applying similar dynamic conditions to multiple distinct strains, without the need for a clean room or soft lithography.

Un dispositivo a microfluidi per lo studio più ceppi distinti

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a ...

Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D culture of cells in collagen type I gels helps to stabilize cell morphology and function, which is necessary for creating microfluidic tissue models in microdevices. Translating traditional 3D culture techniques for tissue culture plates to microfluidic devices is often difficult because of the limited channel dimensions. In this method, we describe a technique for modifying native type I collagen to generate polycationic and polyanionic collagen solutions that can be used with layer-by-layer deposition to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These thin collagen layers stabilize cell morphology and function, as shown using primary hepatocytes as an example cell, allowing for the long term culture of microtissues in microfluidic devices.

The creation of functional microtissues within microfluidic devices requires the stabilization of cell phenotypes by adapting traditional cell culture techniques to the limited spatial dimensions in microdevices. Modification of collagen allows the layer-by-layer deposition of ultrathin collagen assemblies that can stabilize primary cells, such as hepatocytes, as microfluidic tissue models.

Layer-by-layer collagene Deposizione nel Microfluidic Dispositivi per microtessuto Stabilizzazione

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D ...

Using Adhesive Patterning to Construct 3D Paper Microfluidic Devices

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol adhesive through a metal stencil, the overall amount of adhesive used in assembling paper microfluidic devices can be significantly reduced. We show on a simple 4-layer planar paper microfluidic device that the optimal adhesive application technique and device construction style depends heavily on desired performance characteristics. By moderately increasing the overall area of a device, it is possible to dramatically decrease the wicking time and increase device success rates while also reducing the amount of adhesive required to keep the device together. Such adhesive application also causes the adhesive to form semi-permanent bonds instead of permanent bonds between paper layers, enabling single-use devices to be non-destructively disassembled after use. Nonplanar 3D origami devices also benefit from the semi-permanent bonds during folding, as it reduces the likelihood that unrelated faces may accidently stick together. Like planar devices, nonplanar structures see reduced wicking times with patterned adhesive application vs uniformly applied adhesive.

We demonstrate the use of patterned aerosol adhesives to construct 3D paper microfluidic devices. This method of adhesive application forms semi-permanent bonds between layers, enabling single-use devices to be non-destructively disassembled after use and to ease folding complex nonplanar structures.

Utilizzando adesivo Patterning per costruire dispositivi microfluidici di carta 3D

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol ...

A Method for Evaluating Timeliness and Accuracy of Volitional Motor Responses to Vibrotactile Stimuli

Artificial sensory feedback (ASF) systems can be used to compensate for lost proprioception in individuals with lower-limb impairments. Effective design of these ASF systems requires an in-depth understanding of how the parameters of specific feedback mechanism affect user perception and reaction to stimuli. This article presents a method for applying vibrotactile stimuli to human participants and measuring their response. Rotating mass vibratory motors are placed at pre-defined locations on the participant's thigh, and controlled through custom hardware and software. The speed and accuracy of participants' volitional responses to vibrotactile stimuli are measured for researcher-specified combinations of motor placement and vibration frequency. While the protocol described here uses push-buttons to collect a simple binary response to the vibrotactile stimuli, the technique can be extended to other response mechanisms using inertial measurement units or pressure sensors to measure joint angle and weight bearing ratios, respectively. Similarly, the application of vibrotactile stimuli can be explored for body segments other than the thigh.

This article describes a technique for applying vibrotactile stimuli to the thigh of a human participant, and measuring the accuracy and reaction time of the participant&#39;s volitional response for various combinations of stimulation location and frequency.

Un metodo per la valutazione tempestività e l&#39;accuratezza delle risposte volitivo motore a vibrotattili stimoli

Artificial sensory feedback (ASF) systems can be used to compensate for lost proprioception in individuals with lower-limb impairments.  Effective design ...

Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to entry for non-specialists who would benefit greatly from the ability to develop their own microfluidic devices to address research questions. Particularly lacking has been the open dissemination of protocols related to photolithography, a key step in the development of a replica mold for the manufacture of polydimethylsiloxane (PDMS) devices. While the fabrication of single height silicon masters has been explored extensively in literature, fabrication steps for more complicated photolithography features necessary for many interesting device functionalities (such as feature rounding to make valve structures, multi-height single-mold patterning, or high aspect ratio definition) are often not explicitly outlined. 
Here, we provide a complete protocol for making multilayer microfluidic devices with valves and complex multi-height geometries, tunable for any application. These fabrication procedures are presented in the context of a microfluidic hydrogel bead synthesizer and demonstrate the production of droplets containing polyethylene glycol (PEG diacrylate) and a photoinitiator that can be polymerized into solid beads. This protocol and accompanying discussion provide a foundation of design principles and fabrication methods that enables development of a wide variety of microfluidic devices. The details included here should allow non-specialists to design and fabricate novel devices, thereby bringing a host of recently developed technologies to their most exciting applications in biological laboratories.

Multilayer microfluidic devices often involve the fabrication of master molds with complex geometries for functionality. This article presents a complete protocol for multi-step photolithography with valves and variable height features tunable to any application. As a demonstration, we fabricate a microfluidic droplet generator capable of producing hydrogel beads.

Multi-step altezza variabile fotolitografia per Valvolati multistrato microfluidici Devices

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to ...

Fabrication of Three-dimensional Paper-based Microfluidic Devices for Immunoassays

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and directed within a layer of paper. Moreover, stacking multiple layers of patterned paper creates sophisticated three-dimensional microfluidic networks that can support the development of analytical and bioanalytical assays. Paper-based microfluidic devices are inexpensive, portable, easy to use, and require no external equipment to operate. As a result, they hold great promise as a platform for point-of-care diagnostics. In order to properly evaluate the utility and analytical performance of paper-based devices, suitable methods must be developed to ensure their manufacture is reproducible and at a scale that is appropriate for laboratory settings. In this manuscript, a method to fabricate a general device architecture that can be used for paper-based immunoassays is described. We use a form of additive manufacturing (multi-layer lamination) to prepare devices that comprise multiple layers of patterned paper and patterned adhesive. In addition to demonstrating the proper use of these three-dimensional paper-based microfluidic devices with an immunoassay for human chorionic gonadotropin (hCG), errors in the manufacturing process that may result in device failures are discussed. We expect this approach to manufacturing paper-based devices will find broad utility in the development of analytical applications designed specifically for limited-resource settings.

We detail a method to fabricate three-dimensional paper-based microfluidic devices for use in the development of immunoassays. Our approach to device assembly is a type of multilayer, additive manufacturing. We demonstrate a sandwich immunoassay to provide representative results for these types of paper-based devices.

Fabbricazione di dispositivi microfluidici basati su carta tridimensionale per gli immunodosaggi

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and ...

Polydimethylsiloxane-polycarbonate Microfluidic Devices for Cell Migration Studies Under Perpendicular Chemical and Oxygen Gradients

This paper reports a microfluidic device made of polydimethylsiloxane (PDMS) with an embedded polycarbonate (PC) thin film to study cell migration under combinations of chemical and oxygen gradients. Both chemical and oxygen gradients can greatly affect cell migration in vivo; however, due to technical limitations, very little research has been performed to investigate their effects in vitro. The device developed in this research takes advantage of a series of serpentine-shaped channels to generate the desired chemical gradients and exploits a spatially confined chemical reaction method for oxygen gradient generation. The directions of the chemical and oxygen gradients are perpendicular to each other to enable straightforward migration result interpretation. In order to efficiently generate the oxygen gradients with minimal chemical consumption, the embedded PC thin film is utilized as a gas diffusion barrier. The developed microfluidic device can be actuated by syringe pumps and placed into a conventional cell incubator during cell migration experiments to allow for setup simplification and optimized cell culture conditions. In cell experiments, we used the device to study migrations of adenocarcinomic human alveolar basal epithelial cells, A549, under combinations of chemokine (stromal cell-derived factor, SDF-1&#945;) and oxygen gradients. The experimental results show that the device can stably generate perpendicular chemokine and oxygen gradients and is compatible with cells. The migration study results indicate that oxygen gradients may play an essential role in guiding cell migration, and cellular behavior under combinations of gradients cannot be predicted from those under single gradients. The device provides a powerful and practical tool for researchers to study interactions between chemical and oxygen gradients in cell culture, which can promote better cell migration studies in more in vivo-like microenvironments.

The control of chemical and oxygen gradients is essential for cell cultures. This paper reports a polydimethylsiloxane-polycarbonate (PDMS-PC) microfluidic device capable of reliably generating combinations of chemical and oxygen gradients for cell migration studies, which can be practically utilized in biological labs without sophisticated instrumentation.

Polidimetilsilossano-policarbonato dispositivi microfluidici per Cell Migration Studies Sotto perpendicolare chimica e gradienti di ossigeno

This paper reports a microfluidic device made of polydimethylsiloxane (PDMS) with an embedded polycarbonate (PC) thin film to study cell migration under ...