Questa ricerca è stata finanziata dal National Institute of numeri di sovvenzione Salute R01NS050452 e R01EB009429. Fabbricazione dei chip a sbalzo è stata eseguita esternamente da collaboratori presso l&#39;impianto di nanofabbricazione situato presso la Cornell University. Tutte le apparecchiature utilizzate nel processo di fabbricazione a sbalzo era situato in questa struttura. Un ringraziamento speciale a Mandy Esch e Jean-Matthieu Prot per la loro assistenza con cantilever micro-fabbricazione. Animazione Video di funzionalità cantilever è stato generato da Charles Hughes, Alex Zelenin ed Eric Imperiale dal Laboratorio di Realtà sintetica a UCF.

1 Cantilever Chip Fabrication Dettagli illustrati delle fasi di fabbricazione descritti sono forniti in Figura 1. <ol><li> Posizionare wafer di silicio su isolante (SOI) in un forno e cuocere a 125 ° C per 20 min a disidratarsi loro. </li><li> Depositare uno spesso strato di ossido di silicio 1,5 micron sullo strato maniglia del wafer SOI disidratato usando un potenziato a plasma Chemical Vapor Deposition (PECVD) dell&#39;utensile. </li><li> Posizionare la cialda sul mandrino di spin coater con lo strato dispositivo rivolto verso l&#39;alto. Assicurarsi wafer è centrata, dispensare 2 ml di P20 Primer sul centro della fetta, e centrifuga a 3000 rpm per 60 sec a un&#39;accelerazione di 1.000 giri / sec. P20 Primer promuove l&#39;adesione del fotoresist allo strato dispositivo. </li><li> Mentre il wafer è ancora sul mandrino di spin coater, dispensare 2 ml di S1818 fotoresist sul centro della fetta, e centrifuga a 3000 rpm per 60 sec a un&#39;accelerazione di 1.000 giri / sec obtain uno spesso strato di resina fotosensibile 1,5 micron. </li><li> Inserire la cialda su una piastra calda ed effettuare una cottura morbida a 115 ° C per 1 min. </li><li> Eseguire un&#39;esposizione contatto UV disco utilizzando un contatto fotolitografia maschera allineatore per trasferire il disegno dalla maschera sbalzo per il fotoresist sullo strato del dispositivo. Calcolare il tempo di esposizione in base al valore energetico di esposizione di 125 mJ / cm 2 per S1818 photoresist. </li><li> Sviluppare il photoresist immergendo la fetta in una MIF autore fotoresist 726 per 1 min, agitando delicatamente il wafer avanti e indietro. </li><li> Sciacquare il wafer con (DI) di acqua deionizzata e asciugare, preferibilmente utilizzando uno spin risciacquo biancheria (SRD) strumento. </li><li> Rimuovere il fotoresist dal bordo del wafer (fino a 5 mm dal bordo) usando un batuffolo di cotone imbevuto di acetone. </li><li> Caricare il wafer in un Reactive Ion Etch (DRIE) strumento profonda, con il lato fotosensibile rivolto verso l&#39;alto, ed eseguire una ricetta per incidere lo strato di silicio modellato attraverso la devistrato di ce. Le funzioni di strato di ossido sepolto come un arresto etch, quindi eseguire un attacco con il 50% al 100% più cicli che si prevede sarà necessario per garantire una completa etch. </li><li> Posizionare la cialda in una soluzione bagno photoresist caldo per 20 minuti a nudo il photoresist. Trasferire la cialda di un rapido-dump-sciacquatrice (QDR) per sciacquare la cialda con acqua deionizzata. Dopo la striscia di photoresist, caricare la cialda in uno strumento di dominio di risorse condivise per sciacquare e asciugare il wafer. Controllare il wafer al microscopio per assicurare lo schema cantilever visualizza come previsto. </li><li> Depositare uno strato di ossido di silicio drogato un-1 micron sullo strato periferica del wafer con un attrezzo PECVD. Funzioni di questo strato di ossido, come uno strato protettivo per i cantilever durante ulteriori fasi di lavorazione. </li><li> Posizionare il wafer sul mandrino rotazione coater con lo strato rivolto verso l&#39;alto la maniglia per iniziare l&#39;elaborazione del lato posteriore del wafer. Garantire wafer è centrata, dispensare 2 ml di P20 Primer sul centro della fetta, e centrifuga a 3000 rpmper 60 sec a un&#39;accelerazione di 1.000 giri / sec. </li><li> Mentre il wafer è ancora sul mandrino di spin coater, dispensare 2 ml di SPR220-4.5 fotoresist sul centro della fetta, e centrifugare a 2000 rpm per 45 sec a un&#39;accelerazione di 1.000 giri / sec per ottenere uno spesso strato di 6,5 micron photoresist. </li><li> Inserire la cialda su una piastra calda vicinanza effettuare una cottura morbida a 115 ° C per 2 min. </li><li> Utilizzando un contatto fotolitografia allineatore grado di anteriore / posteriore allineamento, allineare la maschera finestra posteriore al modello cantilever lato anteriore ed eseguire un hard esposizione ai raggi UV contatto per trasferire il disegno dalla maschera finestra posteriore per il fotoresist sullo strato maniglia. Utilizzare un tempo di esposizione calcolato in base al valore energetico di esposizione di 480 mJ / cm 2 per SPR220-4.5 photoresist. </li><li> Dopo l&#39;esposizione ai raggi UV, lasciare che i wafer rimangono in una zona scura per 30 minuti prima della prossima fase di lavorazione. </li><li> Sviluppare il photoresist immergendo il wafer inuno sviluppatore 726 MIF photoresist per 2 minuti mentre delicatamente scuotendo il wafer avanti e indietro. </li><li> Sciacquare il wafer con (DI) di acqua deionizzata e asciugare, preferibilmente utilizzando uno spin risciacquo biancheria (SRD) strumento. </li><li> Posizionare i wafer in un forno a 90 ° C per 12 ore. </li><li> Etch lo strato di ossido di silicio sul retro del wafer utilizzando un sistema RIE con gas fluorurati e una ricetta per incisione ossido. L&#39;ossido modellata sul retro della fetta agisce come una maschera di attacco per l&#39;ulteriore elaborazione. Controllare i wafer al microscopio per assicurare che l&#39;ossido di silicio nella finestra esposta è completamente inciso. </li><li> Rimuovere il photoresist sul bordo del wafer (fino a 5 mm dal bordo) usando una camera tampone pulito imbevuto in acetone. </li><li> Caricare il wafer in un utensile con DRIE ed eseguire una ricetta per incidere lo strato maniglia modellata ad una profondità di 500 micron con il funzionamento strato di ossido sepolto come arresto etch. Dividere questo etch in più piste per impedire un eccessivo riscaldamento delwafer, che provoca incisione incoerente del silicio. Questo passaggio etch rimuove completamente il silicio sotto i cantilever. </li><li> Eseguire una fase di attacco umido utilizzando il 25% diluito HF mordenzante, per togliere lo strato di ossido sepolto sotto i cantilever e lo strato di ossido protettivo sulla parte superiore del cantilever. NOTA: Questo passaggio rilascia la superficie inferiore dei cantilever in silicio e apre una finestra sotto per fornire un accesso per sondare la mensola con il laser. </li><li> Sciacquare la cialda con una serie di bagni di acqua deionizzata e asciugare accuratamente con azoto. Dal momento che i cantilever sono supportate solo alle loro basi, non utilizzare spray forti d&#39;acqua DI o gas inerte direttamente sui cantilever. </li><li> Cleave i singoli chip dal wafer lungo le linee Cleave prodotti durante la fase maniglia strato etch. </li></ol> Cultura 2. cellulare <ol><li> Preparare 13F coprioggetto secondo i metodi precedentemente pubblicati 17. NOTA: Se 13F coverslips non sono disponibili, qualsiasi superficie idrofobica con un angolo di contatto sopra 95 ° può essere utilizzato. </li><li> Sterilizzare chip a sbalzo e 13F coprioggetto in una soluzione di etanolo al 70% e lasciare asciugare in una cappa a flusso. </li><li> Posizionare singoli chip a sbalzo sulla parte superiore del 13F vetrini all&#39;interno di una piastra standard 12 bene. </li><li> Rivestire le cantilever con la modifica biopolimero o superficie ottimizzata per il tipo di cellule utilizzate secondo i protocolli standard di coltura cellulare. </li><li> Risospendere le cellule nel loro terreno di crescita specifico alla concentrazione desiderata. NOTA: Questo protocollo si tradurrà in un notevole numero di cellule che cadono attraverso la finestra a sbalzo e non aderenti alla superficie cantilever desiderato. Preparazioni di cellule devono pertanto essere 3-4 volte più concentrato di quello per le preparazioni coprioggetto standard. Ad esempio, la semina di cellule satelliti muscolari scheletrico di ratto è tipicamente effettuata con una densità di semina di 500 e 700 cellule / quadrato mm 15,16, E sono usati con supporti a sbalzo a 2.000 cellule / mm quadrato. Esperimenti di ottimizzazione devono essere effettuate con le nuove fonti di cellule per accertare una adeguata densità di semina. </li><li> Pipettare 200 microlitri della sospensione cellulare sulla superficie del chip cantilever, assicurando la bolla di media copre le finestre a sbalzo interamente. Se il mezzo assorbe attraverso la finestra e non forma una bolla statica sulla parte superiore del cantilever sostituire il coprioggetto 13F e ritentare il fasciame. </li><li> Trasferire il piatto contenente i chip di un incubatore e permettono alle cellule di aderire per almeno 1 ora (preferibilmente 2-3 ore). </li><li> Dopo questo periodo di placcatura, utilizzare pinze sterili per trasferire il chip di un ben pulito, senza coprioggetto 13F, e rabboccare la cultura con 1 ml di terreno di coltura. </li><li> Riportare la piastra per l&#39;incubatore. </li><li> Mantenere le cellule in base alla loro protocollo standard per la manutenzione in vitro su vetrini. Per le cellule del muscolo scheletrico,un interruttore di media composizione per indurre la formazione di miotubi una volta le cellule diventano confluenti sarà necessario. </li></ol> 3 Configurazione di hardware e software per l&#39;analisi sbalzo Flessione <ol><li> Posizionare un piatto di coltura riscaldato nella fase di un microscopio elettrofisiologia in posizione verticale. </li><li> Aggiungere 3 ml del mezzo di alimentazione delle celle sono attualmente sospesi in (HEPES mM + 10) alla fase microscopio riscaldata. </li><li> Montare elettrodi inox sulla parte interna del piatto cultura riscaldata ad una distanza di 15 mm e collegarli ad un generatore di impulsi, in grado di produrre impulsi di stimolazione campo di varia intensità, frequenza e forma d&#39;onda, per permettere al sistema di produrre stimolazione del campo delle cellule quando appropriato. </li><li> Fissare un laser a elio-neon, montato su fasi di traslazione XY, alla parte inferiore del tavolo microscopio e regolare in modo che il fascio laser è diretto attraverso la base del piatto cultura riscaldata a 30 ° relativposta al piano del cantilever. </li><li> Fissare un modulo foto-rilevatore quadrante, montato su fasi di traslazione XY, alla parte inferiore del palco microscopio e regolare la posizione in modo che i riflessi terre raggio laser al centro dei quattro quadranti. Figura 2 fornisce una panoramica dell&#39;hardware istituito necessarie per l&#39;attuazione del protocollo descritto. </li><li> Scrivere un programma software per controllare gli attuatori lineari che scandiscono attraverso i cantilever. Scrivere il programma software con riferimento al diagramma di flusso illustrato nella Figura 3. L&#39;interfaccia grafica programmato per l&#39;uso con questo sistema è fornito in Figura 4. </li></ol> 4 Registrazione Cantilever deflessione dei dati <ol><li> Accendere l&#39;hardware di analisi cantilever e software associato. </li><li> Inserire il palco termistore riscaldato nel mezzo e attendere che la lettura a 37 ° C. </li><li> Inserire il chip a sbalzo in fase con il cantilevers orientati verso il lato destro del palco. </li><li> Accendere la sorgente di luce del microscopio. </li><li> Mettere a fuoco il microscopio per portare i bordi dei cantilever in vista e utilizzare il software di controllo laser / foto-rilevatore per posizionare il raggio laser sulla punta del cantilever 1 NOTA: Supponendo che i cantilever sono orientati alla destra del palco, cantilever 1 è quello posizionato in alto a sinistra della matrice e numeri scendono a 16 in basso a sinistra. Mensola 17 è nella posizione in alto a destra e corre a 32 in basso a destra (Figura 5A). </li><li> Premere il tasto &quot;play&quot; sul software di registrazione. </li><li> Posizionare la foto-rivelatore in modo che il segnale indica &quot;0&quot; in entrambi x ed y fotogrammi regolando i motori passo-passo che controllano la foto-rivelatore. </li><li> Impostare cantilever 1 posizione nel software di controllo laser / foto-rilevatore (Figura 4). </li><li> Spostare il laser alla punta del cantilever 16, ripetere il punto 4.7., E impostare cantilever 16 posizione nel software di controllo laser / foto-rivelatore. </li><li> Spostare il laser alla punta del cantilever 32, ripetere il punto 4.7., E impostare sbalzo 32 posizione nel software di controllo laser / foto-rivelatore. </li><li> Spostare il laser alla punta del cantilever 17, ripetere il punto 4.7., E impostare sbalzo 17 posizione nel software di controllo laser / foto-rivelatore. </li><li> Spegnere la fonte di luce microscopio e la plafoniera in laboratorio. </li><li> Premere il tasto &quot;record&quot; sul software di registrazione. </li><li> Impostare l&#39;hardware generatore di impulsi di 40 msec, 5 V impulsi ad una frequenza di 1 Hz, e accendere la macchina. NOTA: In alternativa, impiegare protocolli di stimolazione ottimizzate per fonti di cellule specifiche, a questo punto, le impostazioni indicate sono linee guida basate su dati raccolti utilizzando fonti umane e cellule di roditore 12,13,15,16. </li><li> Utilizzando il software di controllo laser / foto-rivelatore, impostare l&#39;hardware per eseguire la scansione attraverso la matrice 32 cantilever, fermandosi per 5 secondi ad ogni sue. </li><li> Quando la scansione dei 32 cantilever è completata, spegnere lo stimolatore, quindi arrestare il software di registrazione e aprire il file di dati. </li><li> Esaminare la traccia registrata da ogni sbalzo per prove di attività contrattile. Prendere nota di ciascuna mensola con le risposte positive. Una contrazione è definito come un picco se la deviazione è di almeno 0,1 V sopra la linea di base. </li><li> Rimuovere qualsiasi cantilever che non rispondono dal protocollo di scansione sul software di controllo laser / foto-rivelatore. </li><li> I cantilever attivi possono quindi essere riesaminati senza stimolazione al fine di ottenere una lettura di spontanea attività contrattile della cellula. </li><li> Eseguire scansioni con o senza stimolazione elettrica vasto campo, dopo aggiunta di un composto terapeutico al mezzo per osservare il suo effetto sull&#39;uscita funzionale delle cellule in coltura. </li><li> Effettuare valutazioni fatica stimolando elettricamente le cellule per lunghi periodi e livelli di scansione di contr<html> actility per misurare quanto tempo ci vuole per forza di picco a scendere al di sotto di una determinata soglia. </li><li> Negli esperimenti in cui i neuroni motori sono mantenuti in co-coltura con il muscolo, misurare la formazione di giunzione neuromuscolare attraverso il trattamento dei motoneuroni-myotube sbalzo co-culture con uno stimolante neuronale (come il glutammato) o inibitore sinaptica (ad esempio, D-tubocurarina) e scansione per aumenti e diminuzioni di attività spontanea, rispettivamente 16. </li><li> Eseguire scansioni specifiche dettate dalle esigenze degli esperimenti pianificati. </li></ol> 5 Analisi di sbalzo deflessione dati <ol><li> Usa modificato equazioni di Stoney 15 (di seguito dettagliate) per convertire i dati cantilever grezzi deflessione (in Volt) in una lettura di stress nello strato di cellule o miotubi (in Pascal), e una misura diretta della forza contrattile cellulare (in nano-newton): annuncio / 51866 / 51866eq1.jpg &quot;/&gt; Equazione 1 <img alt="Equazione 2" fo:content-width="2in" width="300" src="/files/ftp_upload/51866/51866eq2.jpg" /> Equazione 2 </li><li> Dove δ = sbalzo punta flessione e lo stress prodotti dalla myotube, σ c = lo stress prodotto dal myotube, assumendo un film di spessore uniforme l&#39;intera larghezza del cantilever, C rivelatore = coefficiente specifica del sistema relativa tensione di posizione laser sulla foto -detector, θ = l&#39;angolo del laser e rivelatore rispetto al piano del cantilever, E Si = modulo elastico di silicio, t Si = spessori del cantilever, t f = spessore miotubi, v Si = coefficiente di veleno silicio, L = lunghezza a sbalzo, lunghezza P = percorso del laser dalla punta a sbalzo a rivelatore, e w <sub&gt; Si = la larghezza del cantilever. Uno schema che spiega i termini utilizzati in queste equazioni è fornita in Figura 6. </li><li> Assumendo il miotubi è un film uniforme, la forza nella miotubi è uguale alla forza del film, portando a equazione 3, uguagliando il calcolo della forza da stress e l&#39;area della sezione trasversale cella assunto che è stato utilizzato per l&#39;applicazione di Stoney di equazione. <img alt="Equazione 3" fo:content-width="1.5in" src="files/ftp_upload/51866/51866eq3.jpg" /> Equazione 3 </li><li> Dopo la raccolta dei dati funzionali, fissare i chip a sbalzo per l&#39;analisi immunocitochimica o utilizzare le celle per proteine ​​o DNA analisi utilizzando tecniche standard. </li><li> Facoltativamente, tornare le cellule al incubatore invece di preparare per l&#39;analisi molecolare, al fine di rivalutare le prestazioni funzionali in un momento-momento successivo. </li></ol>

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A., 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=Coplanar+molecular+assemblies+of+amino-+and+perfluorinated+alkylsilanes:+characterization+and+geometric+definition+of+mammalian+cell+adhesion+and+growth.">Coplanar molecular assemblies of amino- and perfluorinated alkylsilanes: characterization and geometric definition of mammalian cell adhesion and growth.</a> Journal of the American Chemical Society. 114, 8435-8442 (1992).</li><li>Das, M., Rumsey, J. W., Bhargava, N., Stancescu, M., Hickman, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+defined+long-term+in+vitro+tissue+engineered+model+of+neuromuscular+junctions.">A defined long-term in vitro tissue engineered model of neuromuscular junctions.</a> Biomaterials. 31, 4880-4888 (2010).</li><li>Rumsey, J. W., Das, M., Bhalkikar, A., Stancescu, M., Hickman, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+engineering+the+mechanosensory+circuit+of+the+stretch+reflex+arc:+Sensory+neuron+innervation+of+intrafusal+muscle+fibers.">Tissue engineering the mechanosensory circuit of the stretch reflex arc: Sensory neuron innervation of intrafusal muscle fibers.</a> Biomaterials. 31, 8218-8227 (2010).</li><li>Das, M., Rumsey, J. W., Bhargava, N., Stancescu, M., Hickman, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle+tissue+engineering:+A+maturation+model+promoting+long-term+survival+of+myotubes,+structural+development+of+the+excitation-contraction+coupling+apparatus+and+neonatal+myosin+heavy+chain+expression.">Skeletal muscle tissue engineering: A maturation model promoting long-term survival of myotubes, structural development of the excitation-contraction coupling apparatus and neonatal myosin heavy chain expression.</a> Biomaterials. 30, 5392-5402 (2009).</li><li>Das, M., 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=Developing+a+novel+serum-free+cell+culture+model+of+skeletal+muscle+differentiation+by+systematically+studying+the+role+of+different+growth+factors+in+myotube+formation.">Developing a novel serum-free cell culture model of skeletal muscle differentiation by systematically studying the role of different growth factors in myotube formation.</a> In Vitro Cell Dev Biol Anim. 45, 378-387 (2009).</li><li>Stenger, D. A., Pike, C. J., Hickman, J. J., Cotman, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+determinants+of+neuronal+survival+and+growth+on+self-assembled+monolayers+in+culture.">Surface determinants of neuronal survival and growth on self-assembled monolayers in culture.</a> Brain research. 630, 136-147 (1993).</li><li>Hickman, J. J., 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=Rational+pattern+design+for+in+vitro+cellular+networks+using+surface+photochemistry.">Rational pattern design for in vitro cellular networks using surface photochemistry.</a> Journal of Vacuum Scienc., &amp; Technology A: Vacuum, Surfaces, and Films. 12, 607-616 (1994).</li><li>Das, M., Molnar, P., Devaraj, H., Poeta, M., Hickman, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+and+morphological+characterization+of+rat+embryonic+motor+neurons+in+a+defined+system.">Electrophysiological and morphological characterization of rat embryonic motor neurons in a defined system.</a> Biotechnology progress. 19, 1756-1761 (2003).</li><li>Rumsey, 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=Node+of+Ranvier+formation+on+motor+neurons+in+vitro.">Node of Ranvier formation on motor neurons in vitro.</a> Biomaterials. 30, 3567-3572 (2009).</li><li>Murugan, R., Molnar, P., Rao, K. P., Hickman, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomaterial+Surface+patterning+of+self+assembled+monolayers+for+controlling+neuronal+cell+behavior.">Biomaterial Surface patterning of self assembled monolayers for controlling neuronal cell behavior.</a> International journal of biomedical engineering and technology. 2, 104-134 (2009).</li><li>Guo, X., Johe, K., Molnar, P., Davis, H., Hickman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+human+fetal+spinal+cord+stem+cell+line,+NSI-566RSC,+and+its+induction+to+functional+motoneurons.">Characterization of a human fetal spinal cord stem cell line, NSI-566RSC, and its induction to functional motoneurons.</a> Journal of Tissue Engineering and Regenerative Medicine. 4, 181-193 (2010).</li><li>Varghese, 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=A+new+target+for+amyloid+beta+toxicity+validated+by+standard+and+high-throughput+electrophysiology.">A new target for amyloid beta toxicity validated by standard and high-throughput electrophysiology.</a> PLoS ONE. 5, e8643 (2010).</li><li>Natarajan, A., 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=Patterned+cardiomyocytes+on+microelectrode+arrays+as+a+functional,+high+information+content+drug+screening+platform.">Patterned cardiomyocytes on microelectrode arrays as a functional, high information content drug screening platform.</a> Biomaterials. 32, 4267-4274 (2011).</li><li>Davis, 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=Rat+Cortical+Oligodendrocyte-Embryonic+Motoneuron+Co-Culture:+An+Axon-Oligodendrocyte+Interaction+Model.">Rat Cortical Oligodendrocyte-Embryonic Motoneuron Co-Culture: An Axon-Oligodendrocyte Interaction Model.</a> Journal of biomaterials and tissue. 2, 206-214 (2012).</li><li>Natarajan, A., DeMarse, T., Molnar, P., Hickman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+In+Vitro+Feed-Forward+Networks.">Engineered In Vitro Feed-Forward Networks.</a> J Biotechnol Biomater. 3, 2 (2013).</li></ol>

The authors have nothing to disclose.

Le fasi critiche analizzando cantilever microscala per prove di contrazione cellulare sono il posizionamento del chip a sbalzo all&#39;interno della fase di microscopio, e il successivo allineamento del laser e foto-rivelatore con la punta del cantilever angolari nella matrice. Se questo non viene fatto accuratamente, quindi il software sarà in grado di estrapolare le posizioni dei rimanenti cantilever nella matrice, potenzialmente conseguente accumulo di falsi negativi durante la raccolta dei dati. Gli operatori devon...

The in vitro culture of muscle cells from both human and rodent sources has been possible for decades1,2. However, while standard coverslip preparations are useful for biochemical assessment, they do not facilitate analysis of the cell&#8217;s primary functional output (contractility), and therefore are of somewhat limited value as a means to assess cellular maturation and performance. In order to maximize the amount of data obtainable from such in vitro cultures, it is necessary to advance the development of systems capable of housing such cells in configurations that permit the real-time assessment of their functional performance. The establishment of a multitude of three dimensional muscle models has made some progress toward fulfilling this need, and such systems have been used in a number of publications as a means to assess the contractile capacity of cultured muscle cells in vitro3-5. While such systems are invaluable for tissue modeling and reconstruction studies, they are limited in their applicability for studies of single cell responses. In such cases where single fiber studies are necessary, complex and labor intensive ex vivo methodologies remain the only option6-10. Furthermore, current movement toward the development of complex, multi-organ platforms for drug development and screening protocols requires the establishment of systems which are non-invasive, easily scalable and which integrate readily with supporting cells and tissue models11. 
Microscale cantilevers offer a simple method for assessing the functional contractile capacity of single cells/small populations of cells12,13. The technique is based on modified Atomic Force Microscopy (AFM) technology14, and uses a laser and photo-detector system to measure microscale cantilever deflection in response to cultured myotube contractile activity. Modified Stoney&#8217;s equations are then used to calculate stress in the myotube, and the force exerted by the myotube in order to generate the observed substrate deflection15. A scanning program has been written which enables simultaneous assessment of multiplexed cantilever arrays, offering potential moderate to high through-put applications for drug toxicity/efficacy studies15,16. Such technology may prove invaluable in the development of functional, pre-clinical assays for predicting drug efficacy in vivo. Furthermore, fabrication of cantilever chips in silicon does not impede post analysis processing of cells for standard biomolecular assays such as immunostaining, western blotting and PCR. 
This manuscript provides detailed instructions on the fabrication and preparation of microscale silicon cantilevers, the hardware and software set-up, and the operating guidelines for assessing the functionality of contractile cells cultured on these chips. Standard cell culture techniques can be implemented for plating and maintenance of cells on these surfaces, hence any contractile cell type for which reliable culture parameters exist should be able to integrate with this device with ease. The relatively simple 2D culture parameters utilized in this system makes integration of other cell models or addition of cell types that can interact with muscle (such as innervating neurons) straight-forward, greatly increasing the applicability of this model in the development of more complex functional in vitro assays and multi-organ models of mammalian systems.

<table border="0" cellpadding="0" cellspacing="0" width="690">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:299px;">Name of material/ equipment</td>
			<td style="width:128px;">Company</td>
			<td style="width:108px;">Catalog number</td>
			<td style="width:156px;">Comments/ Description</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Primary rat muscle growth medium</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Neurobasal medium</td>
			<td dir="LTR" style="width:128px;">Life Technologies</td>
			<td dir="LTR" style="width:108px;">21103-049&#160;</td>
			<td dir="LTR" style="width:156px;">N/A</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">B27 (50x)</td>
			<td dir="LTR" style="width:128px;">Life Technologies</td>
			<td dir="LTR" style="width:108px;">17504044</td>
			<td dir="LTR" style="width:156px;">1x</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Glutamax (100x)</td>
			<td dir="LTR" style="width:128px;">Life Technologies</td>
			<td dir="LTR" style="width:108px;">35050061</td>
			<td dir="LTR" style="width:156px;">1x</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">G5 supplement</td>
			<td dir="LTR" style="width:128px;">Life Technologies</td>
			<td dir="LTR" style="width:108px;">17503-012&#160;</td>
			<td dir="LTR" style="width:156px;">1x</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Glial-Derived Neurotrophic Factor</td>
			<td dir="LTR" style="width:128px;">Cell sciences</td>
			<td dir="LTR" style="width:108px;">CRG400B</td>
			<td dir="LTR" style="width:156px;">20 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Brain-Derived Neurotrophic Factor</td>
			<td dir="LTR" style="width:128px;">Cell sciences</td>
			<td dir="LTR" style="width:108px;">CRB600B</td>
			<td dir="LTR" style="width:156px;">20 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Ciliary Neurotrophic Factor</td>
			<td dir="LTR" style="width:128px;">Cell sciences</td>
			<td dir="LTR" style="width:108px;">CRC400A</td>
			<td dir="LTR" style="width:156px;">40 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Neurotrophin-3</td>
			<td dir="LTR" style="width:128px;">Cell sciences</td>
			<td dir="LTR" style="width:108px;">CRN500B</td>
			<td dir="LTR" style="width:156px;">20 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Neurotrophin-4</td>
			<td dir="LTR" style="width:128px;">Cell sciences</td>
			<td dir="LTR" style="width:108px;">CRN501B</td>
			<td dir="LTR" style="width:156px;">20 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Acidic Fibroblast Growth Factor</td>
			<td dir="LTR" style="width:128px;">Life Technologies</td>
			<td dir="LTR" style="width:108px;">13241-013&#160;</td>
			<td dir="LTR" style="width:156px;">25 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Vascular Endothelial Growth Factor</td>
			<td dir="LTR" style="width:128px;">Life Technologies</td>
			<td dir="LTR" style="width:108px;">P2654</td>
			<td dir="LTR" style="width:156px;">20 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Cardiotrophin-1</td>
			<td dir="LTR" style="width:128px;">Cell sciences</td>
			<td dir="LTR" style="width:108px;">CRC700B</td>
			<td dir="LTR" style="width:156px;">20 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Heparin Sulphate</td>
			<td dir="LTR" style="width:128px;">Sigma</td>
			<td dir="LTR" style="width:108px;">D9809&#160;</td>
			<td dir="LTR" style="width:156px;">100 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Leukemia Inhibitory Factor</td>
			<td dir="LTR" style="width:128px;">Sigma</td>
			<td dir="LTR" style="width:108px;">L5158&#160;</td>
			<td dir="LTR" style="width:156px;">20 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Vitronectin</td>
			<td dir="LTR" style="width:128px;">Sigma</td>
			<td dir="LTR" style="width:108px;">V0132</td>
			<td dir="LTR" style="width:156px;">100 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;"></td>
			<td dir="LTR" style="width:128px;"></td>
			<td dir="LTR" style="width:108px;"></td>
			<td dir="LTR" style="width:156px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Primary rat muscle differentiation medium</td>
			<td dir="LTR" style="width:128px;"></td>
			<td dir="LTR" style="width:108px;"></td>
			<td dir="LTR" style="width:156px;"></td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">NB Activ 4</td>
			<td dir="LTR" style="width:128px;">Brain Bits LLC</td>
			<td dir="LTR" style="width:108px;">NB4-500</td>
			<td dir="LTR" style="width:156px;">N/A</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;"></td>
			<td dir="LTR" style="width:128px;"></td>
			<td dir="LTR" style="width:108px;"></td>
			<td dir="LTR" style="width:156px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Class 2 red diode laser</td>
			<td>Newport</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Photo-detector</td>
			<td>Noah Industries</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Model 2100 Pulse stimulator</td>
			<td>A-M systems</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Multiclamp 700B Digitizer</td>
			<td>Axon Instruments</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Patch clamp microscope and stage</td>
			<td>Olympus</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Delta T4 culture dish controller</td>
			<td>Bioptechs</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Axoscope software</td>
			<td>Molecular Devices</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">LabVIEW software</td>
			<td>National Instruments</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">37oC, 5% CO2 incubator</td>
			<td>NAPCO</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Class 2 microbiological flow hood</td>
			<td>Labconco</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Pipettes and tips</td>
			<td>Eppendorf</td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

utilization of microscale silicon cantilevers to assess cellular contractile function in vitro

Lo sviluppo di test più predittivi e biologicamente rilevanti in vitro si basa sulla promozione dei sistemi di coltura di cellule versatili che facilitano la valutazione funzionale delle cellule teste di serie. A tal fine, la tecnologia cantilever microscala offre una piattaforma con cui misurare la funzionalità contrattile di una gamma di tipi cellulari, incluse scheletrico, cardiaco e cellule muscolari lisce, attraverso la valutazione della contrazione indotta substrato piegatura. Applicazione di matrici a sbalzo multiplex fornisce i mezzi per sviluppare moderata a protocolli di high-throughput per valutare l&#39;efficacia dei farmaci e la tossicità, fenotipo della malattia e la progressione, così come le interazioni neuromuscolari e altri cellula-cellula. Questo manoscritto fornisce i dettagli per la fabbricazione di matrici a sbalzo affidabili per questo scopo, ed i metodi necessari al successo le cellule di coltura su queste superfici. Ulteriore descrizione è fornita sui passi necessari per eseguire anale funzionaleYsis di tipi di cellule contrattili mantenuta su tali array utilizzando un nuovo sistema laser e foto-rilevatore. I dati rappresentativi forniti evidenzia la precisione e la natura riproducibile dell&#39;analisi della funzione contrattile possibile utilizzare questo sistema, così come la vasta gamma di studi cui tale tecnologia può essere applicata. Adozione diffusa di successo di questo sistema potrebbe fornire gli investigatori i mezzi per eseguire rapidi, studi funzionali a basso costo in vitro, portando a previsioni più accurate delle prestazioni dei tessuti, lo sviluppo della malattia e la risposta al trattamento terapeutico.

Cultura di successo di cellule contrattili su cantilever è una procedura relativamente semplice, utilizzando tecniche standard di coltura cellulare (Figura 5). La percentuale di cellule di supporto cantilever amministrazioni varierà a seconda del tipo di cellula in esame e specifica cultura tecnica impiegata. Utilizzo di cellule embrionali primarie derivate da ratto arti posteriori, l&#39;attività contrattile è stata rilevata il 12% di cantilever esaminati (n = 4). Analisi della funzione contrattile...

Watch this Scientific Journal Video about Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro at JoVE.com

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

Questo protocollo descrive l&#39;uso di cantilever silicio microscala come superfici di coltura flessibili per misurare la contrattilità delle cellule muscolari in vitro. Contrazione cellulare provoca cantilever flessione, che può essere misurata, registrata e convertita in letture di forza, fornendo un sistema non invasivo e scalabile per misurare funzione contrattile in vitro.

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

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

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

10.1K Views.  University of Central Florida.  Questo protocollo descrive l'uso di cantilever silicio microscala come superfici di coltura flessibili per misurare la contrattilità delle cellule muscolari in vitro. Contrazione cellulare provoca cantilever flessione, che può essere misurata, registrata e convertita in letture di forza, fornendo un sistema non invasivo e scalabile per misurare funzione contrattile in vitro. 

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

Utilization of Plasmonic and Photonic Crystal Nanostructures for Enhanced Micro- and Nanoparticle Manipulation

A method to manipulate the position and orientation of submicron particles nondestructively would be an incredibly useful tool for basic biological research. Perhaps the most widely used physical force to achieve noninvasive manipulation of small particles has been dielectrophoresis(DEP).1 However, DEP on its own lacks the versatility and precision that are desired when manipulating cells since it is traditionally done with stationary electrodes. Optical tweezers, which utilize a three dimensional electromagnetic field gradient to exert forces on small particles, achieve this desired versatility and precision.2 However, a major drawback of this approach is the high radiation intensity required to achieve the necessary force to trap a particle which can damage biological samples.3 A solution that allows trapping and sorting with lower optical intensities are optoelectronic tweezers (OET) but OET's have limitations with fine manipulation of small particles; being DEP-based technology also puts constraint on the property of the solution.4,5
This video article will describe two methods that decrease the intensity of the radiation needed for optical manipulation of living cells and also describe a method for orientation control. The first method is plasmonic tweezers which use a random gold nanoparticle (AuNP) array as a substrate for the sample as shown in Figure 1. The AuNP array converts the incident photons into localized surface plasmons (LSP) which consist of resonant dipole moments that radiate and generate a patterned radiation field with a large gradient in the cell solution. Initial work on surface plasmon enhanced trapping by Righini et al and our own modeling have shown the fields generated by the plasmonic substrate reduce the initial intensity required by enhancing the gradient field that traps the particle.6,7,8 The plasmonic approach allows for fine orientation control of ellipsoidal particles and cells with low optical intensities because of more efficient optical energy conversion into mechanical energy and a dipole-dependent radiation field. These fields are shown in figure 2 and the low trapping intensities are detailed in figures 4 and 5. The main problems with plasmonic tweezers are that the LSP's generate a considerable amount of heat and the trapping is only two dimensional. This heat generates convective flows and thermophoresis which can be powerful enough to expel submicron particles from the trap.9,10 The second approach that we will describe is utilizing periodic dielectric nanostructures to scatter incident light very efficiently into diffraction modes, as shown in figure 6.11 Ideally, one would make this structure out of a dielectric material to avoid the same heating problems experienced with the plasmonic tweezers but in our approach an aluminum-coated diffraction grating is used as a one-dimensional periodic dielectric nanostructure. Although it is not a semiconductor, it did not experience significant heating and effectively trapped small particles with low trapping intensities, as shown in figure 7. Alignment of particles with the grating substrate conceptually validates the proposition that a 2-D photonic crystal could allow precise rotation of non-spherical micron sized particles.10 The efficiencies of these optical traps are increased due to the enhanced fields produced by the nanostructures described in this paper.

Plasmonic tweezers and photonic crystal nanostructures are shown to produce useful enhancements in the efficiency and orientation control of optically trapping micro- and nano-particles.

L&#39;utilizzo di nanostrutture di cristallo plasmoniche e fotonici per Enhanced manipolazione micro e nanoparticelle

A method to manipulate the position and orientation of submicron particles nondestructively would be an incredibly useful tool for basic biological ...

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Bioingegneria

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs 1-6. Yet the development of artificial molecular machines centers on devices capable of directional motion, i.e. molecular motors, and on their scaled-down mechanical parts (wheels, axels, pendants etc) 7-9. This imitates the macro-machines, even though the physical properties essential for these devices, such as inertia and momentum conservation, are not usable in the nanoworld environments 10. Alternative designs, which do not follow the mechanical macromachines schemes and use mechanisms developed in the evolution of biological molecules, can take advantage of the specific conditions of the nanoworld. Besides, adapting actual biological molecules for the purposes of nano-design reduces potential dangers the nanotechnology products may pose. Here we demonstrate the assembly and application of one such bio-enabled construct, a semi-artificial molecular device which combines a naturally-occurring molecular machine with artificial components. From the enzymology point of view, our construct is a designer fluorescent enzyme-substrate complex put together to perform a specific useful function. This assembly is by definition a molecular machine, as it contains one 12. Yet, its integration with the engineered part - fluorescent dual hairpin - re-directs it to a new task of labeling DNA damage12.
Our construct assembles out of a 32-mer DNA and an enzyme vaccinia topoisomerase I (VACC TOPO). The machine then uses its own material to fabricate two fluorescently labeled detector units (Figure 1). One of the units (green fluorescence) carries VACC TOPO covalently attached to its 3'end and another unit (red fluorescence) is a free hairpin with a terminal 3'OH. The units are short-lived and quickly reassemble back into the original construct, which subsequently recleaves. In the absence of DNA breaks these two units continuously separate and religate in a cyclic manner. In tissue sections with DNA damage, the topoisomerase-carrying detector unit selectively attaches to blunt-ended DNA breaks with 5'OH (DNase II-type breaks)11,12, fluorescently labeling them. The second, enzyme-free hairpin formed after oligonucleotide cleavage, will ligate to a 5'PO4 blunt-ended break (DNase I-type breaks)11,12, if T4 DNA ligase is present in the solution 13,14 . When T4 DNA ligase is added to a tissue section or a solution containing DNA with 5'PO4 blunt-ended breaks, the ligase reacts with 5'PO4 DNA ends, forming semi-stable enzyme-DNA complexes. The blunt ended hairpins will interact with these complexes releasing ligase and covalently linking hairpins to DNA, thus labeling 5'PO4 blunt-ended DNA breaks.
This development exemplifies a new practical approach to the design of molecular machines and provides a useful sensor for detection of apoptosis and DNA damage in fixed cells and tissues.

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

We demonstrate the assembly and application of a molecular-scale device powered by a topoisomerase protein. The construct is a bio-molecular sensor which labels two major types of DNA breaks in tissue sections by attaching two different fluorophores to their ends.

In Assemblea in vitro di semi-artificiale della macchina molecolare e il suo utilizzo per il rilevamento del danno al DNA

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs 1-6. Yet the development of artificial molecular ...

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised the only means available, the development of noninvasive imaging modalities (echocardiography, MRI, CT) having high temporal and spatial resolution provide a new window for quantitative diastolic function assessment. Echocardiography is the agreed upon standard for diastolic function assessment, but indexes in current clinical use merely utilize selected features of chamber dimension (M-mode) or blood/tissue motion (Doppler) waveforms without incorporating the physiologic causal determinants of the motion itself.&#160;The recognition that all left ventricles (LV) initiate filling by serving as mechanical suction pumps allows global diastolic function to be assessed based on laws of motion that apply to all chambers. What differentiates one heart from another are the parameters of the equation of motion that governs filling. Accordingly, development of the Parametrized Diastolic Filling (PDF) formalism&#160;has shown that the entire range of clinically observed early transmitral flow (Doppler E-wave) patterns are extremely well fit by the laws of damped oscillatory motion.&#160;This permits analysis of individual E-waves in accordance with a causal mechanism (recoil-initiated suction) that yields three (numerically) unique lumped parameters whose physiologic analogues are chamber stiffness (k), viscoelasticity/relaxation (c), and load (xo).&#160;The recording of transmitral flow (Doppler E-waves) is standard practice in clinical cardiology and, therefore, the echocardiographic recording method is only briefly reviewed. Our focus is on determination of the PDF parameters from routinely recorded E-wave data.&#160;As the highlighted results indicate, once the PDF parameters have been obtained from a suitable number of load varying E-waves, the investigator is free to use the parameters or construct indexes from the parameters (such as stored energy 1/2kxo2, maximum A-V pressure gradient kxo, load independent index of diastolic function, etc.) and select the aspect of physiology or pathophysiology to be quantified.

Accurate, causality-based quantification of global diastolic function has been achieved by kinematic modeling-based analysis of transmitral flow via the Parametrized Diastolic Filling (PDF) formalism. PDF generates unique stiffness, relaxation,&#160;and load parameters and elucidates &#39;new&#39; physiology while providing sensitive and specific indexes of dysfunction.

Quantificazione di Global Funzione diastolica da Analisi basata su modellazione cinematica del flusso transmitralico tramite il formalismo di riempimento parametrizzate diastolica

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

Analyzing Mixing Inhomogeneity in a Microfluidic Device by Microscale Schlieren Technique

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

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

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

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

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

Oculare Test (EIT) per identificazione del pericolo di sostanze chimiche irritanti oculari utilizzando Ricostruito umana Cornea come epiteliale (RHCE) Tessuto Modello

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Techniques for the Evolution of Robust Pentose-fermenting Yeast for Bioconversion of Lignocellulose to Ethanol

Lignocellulosic biomass is an abundant, renewable feedstock useful for production of fuel-grade ethanol and other bio-products. Pretreatment and enzyme saccharification processes release sugars that can be fermented by yeast. Traditional industrial yeasts do not ferment xylose (comprising up to 40% of plant sugars) and are not able to function in concentrated hydrolyzates. Concentrated hydrolyzates are needed to support economical ethanol recovery, but they are laden with toxic byproducts generated during pretreatment. While detoxification methods can render hydrolyzates fermentable, they are costly and generate waste disposal liabilities. Here, adaptive evolution and isolation techniques are described and demonstrated to yield derivatives of the native Scheffersomyces stipitis strain NRRL Y-7124 that are able to efficiently convert hydrolyzates to economically recoverable ethanol despite adverse culture conditions. Improved individuals are enriched in an evolving population using multiple selection pressures reliant on natural genetic diversity of the S. stipitis population and mutations induced by exposures to two diverse hydrolyzates, ethanol or UV radiation. Final evolution cultures are dilution plated to harvest predominant isolates, while intermediate populations, frozen in glycerol at various stages of evolution, are enriched on selective media using appropriate stress gradients to recover most promising isolates through dilution plating. Isolates are screened on various hydrolyzate types and ranked using a novel procedure involving dimensionless relative performance index (RPI) transformations of the xylose uptake rate and ethanol yield data. Using the RPI statistical parameter, an overall relative performance average is calculated to rank isolates based on multiple factors, including culture conditions (varying in nutrients and inhibitors) and kinetic characteristics. Through application of these techniques, derivatives of the parent strain had the following improved features in enzyme saccharified hydrolyzates at pH 5-6: reduced initial lag phase preceding growth, reduced diauxic lag during glucose-xylose transition, significantly enhanced fermentation rates, improved ethanol tolerance and accumulation to 40 g/L.

Adaptive evolution and isolation techniques are described and demonstrated to yield derivatives of Scheffersomyces stipitis strain NRRL Y-7124 that are able to rapidly consume hexose and pentose mixed sugars in enzyme saccharified undetoxified hydrolyzates and to accumulate over 40 g/L ethanol.

Tecniche per l&#39;evoluzione della robusta lievito Pentoso-fermentazione per bioconversione della lignocellulosa a etanolo

Lignocellulosic biomass is an abundant, renewable feedstock useful for production of fuel-grade ethanol and other bio-products. Pretreatment and enzyme ...

Measuring and Modeling Contractile Drying in Human Stratum Corneum

Stratum corneum (SC) is the most superficial skin layer. Its contact with the external environment means that this tissue layer is subjected to both cleansing agents and daily variations in ambient moisture; both of which can alter the water content of the tissue. Reductions in water content from severe barrier dysfunction or low humidity environments can alter SC stiffness and cause a build-up of drying stresses. In extreme conditions, these factors can cause mechanical rupture of the tissue. We have established a high throughput method of quantifying dynamic changes in the mechanical properties of SC upon drying. This technique can be employed to quantify changes in the drying behavior and mechanical properties of SC with cosmetic cleanser and moisturizer treatments. This is achieved by measuring dynamic variations in spatially resolved in-plane drying displacements of circular tissue samples adhered to an elastomer substrate. In-plane radial displacements acquired during drying are azimuthally averaged and fitted with a profile based on a linear elastic contractility model. Dynamic changes in drying stress and SC elastic modulus can then be extracted from the fitted model profiles.

This article describes a method of quantifying the dynamic drying behavior and mechanical properties of stratum corneum by measuring spatially resolved in-plane drying displacements of circular tissue samples adhered to an elastomer substrate. This technique can be used to measure how different chemical treatments alter drying and tissue mechanical properties.

Misurazione e modellazione contrattile asciugatura in Human strato corneo

Stratum corneum (SC) is the most superficial skin layer. Its contact with the external environment means that this tissue layer is subjected to both ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

Un<em&gt; In Vitro</em&gt; Organ Culture Modello del disco intervertebrale Murine

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (&#8804;14 days). These spheroid models (&#8805;500 &#181;m in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the &#8216;gold standard&#8217; micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

This procedure was established to be used for developing advanced 3D hepatic cultures in vitro, which can provide a more physiologically relevant assessment of the genotoxic hazards associated with nanomaterial exposures over both an acute or long-term, repeated dose regimes.

Modelli avanzati di fegato 3D per test di genotossicità in vitro a seguito dell'esposizione a lungo termine ai nanomateriali

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...

An In vitro System to Gauge the Thrombolytic Efficacy of Histotripsy and a Lytic Drug

Deep vein thrombosis (DVT) is a global health concern. The primary approach to achieve vessel recanalization for critical obstructions is catheter-directed thrombolytics (CDT). To mitigate caustic side effects and the long treatment time associated with CDT, adjuvant and alternative approaches are under development. One such approach is histotripsy, a focused ultrasound therapy to ablate tissue via bubble cloud nucleation. Pre-clinical studies have demonstrated strong synergy between histotripsy and thrombolytics for clot degradation. This report outlines a benchtop method to assess the efficacy of histotripsy-aided thrombolytic therapy, or lysotripsy.
Clots manufactured from fresh human venous blood were introduced into a flow channel whose dimensions and acousto-mechanical properties mimic an iliofemoral vein. The channel was perfused with plasma and the lytic&#160;recombinant tissue-type plasminogen activator. Bubble clouds were generated in the clot with a focused ultrasound source designed for the treatment of femoral venous clots. Motorized positioners were used to translate the source focus along the clot length. At each insonation location, acoustic emissions from the bubble cloud were passively recorded, and beamformed to generate passive cavitation images. Metrics to gauge treatment efficacy included clot mass loss (overall treatment efficacy), and the concentrations of D-dimer (fibrinolysis) and hemoglobin (hemolysis) in the perfusate. There are limitations to this in vitro design, including lack of means to assess in vivo side effects or dynamic changes in flow rate as the clot lyses. Overall, the setup provides an effective method to assess the efficacy of histotripsy-based strategies to treat DVT.

Histotripsy-aided lytic delivery or lysotripsy is under development for the treatment of deep vein thrombosis. An in vitro procedure is presented here to assess the efficacy of this combination therapy. Key protocols for the clot model, image guidance, and assessment of treatment efficacy are discussed.