1. MEA Preparazione <ol><li> MEA per l&#39;uso negli esperimenti di elettroporazione possono essere fabbricati usando la tecnica standard di fotolitografia come descritto in precedenza 18 o acquistati direttamente da fornitori di produttori MEA come i sistemi multicanale <a href="http://www.multichannelsystems.com" target="_blank">(http:/www.multichannelsystems.com/)</a> e Alpha MED Scientific, Inc. <a href="http://www.med64.com/" target="_blank">( http://www.MED64.com)</a> . Le MEA utilizzati nei nostri esperimenti sono stati realizzati in-house presso l&#39;impianto camera pulita in Arizona State University, che è gestito dal Centro per la Ricerca Elettronica dello Stato Solido (CSSER). I MEA utilizzati nei nostri esperimenti avuto 32 elettrodi indio ossido di stagno in un 8 del 4 array con dimensioni elettrodi diametro 50-200 micron all&#39;interno di un vetro 1 cm di diametro interno bene. Bene il vetro è stato incollato sul usando MEA polidimetilsilossano (PDMS). </li><li> Sterilizzare il MEA in autoclave prima cella seeding. Altri metodi di sterilizzazione come un ammollo in 70% etanolo, trattamento UV e trattamento con acqua calda come descritto altrove può anche essere utilizzato fintanto che non compromettere l&#39;integrità del MEA. </li><li> Trasferire il autoclavato MEA di una cappa a flusso laminare e metterla in un piatto polistirene standard sterile Petri. Prima della semina di cellule sulla MEA, sterilizzare la cappa a flusso laminare per accendere la luce UV per 20 min. Se la MEA è sensibile alla luce UV coprire la piastra Petri contenente la MEA con foglio di alluminio sterile mentre la cappa a flusso è sotto illuminazione UV. </li></ol> 2. Cellule semina sul MEA <ol><li> Cellule raccolto da una linea cellulare di esecuzione aderenti cellule di mammifero usando Tripsina / EDTA e alzarle una concentrazione di 100-200 cellule / microlitro in media ml 1 cella per placcatura. Nei nostri esperimenti abbiamo avuto successo nella coltura delle cellule NIH 3T3 fibroblasti linea, linea di cellule HeLa e primari neuroni corticali e ippocampali sulla MEA. </li><li&gt; Mettere a 20-30 ml di goccia media con celle del MEA. Trasferire la piastra di Petri con la MEA per l&#39;incubatore. È importante garantire che la dimensione della goccia cella è sufficiente a coprire tutti gli elettrodi sulla MEA. </li><li> 2 ore dopo la semina delle cellule trasferire la capsula di Petri con la MEA dall&#39;incubatore a cappa a flusso laminare e aggiungere delicatamente 400-500 microlitri di pre supporti riscaldati (37 ° C) al MEA bene. Controllare al microscopio per assicurare che le cellule siano ancora attaccato e distribuito uniformemente sulla superficie MEA. Trasferire il MEA per l&#39;incubatrice. Il periodo di attesa 2 ore prima dell&#39;aggiunta di supporto assicura tempo sufficiente per le cellule di aderire alla superficie MEA. Aggiunta del supporto delle cellule troppo presto dopo la semina le cellule possono lavare via le cellule. Se le cellule non aderiscono sulla MEA, la sua superficie può essere trattata con fattori di adesione cellulare quali aspolylysine, fibronectina e laminina per migliorare l&#39;adesione delle cellule. Nei nostri esperimenti sulle cellule HeLa che di solito trattare la superficie con MEA fibronectin (10 pg / ml) per 1-2 ore. </li><li> 8-12 ore dopo la semina delle cellule, eseguire 1-2 lavaggi con PBS per rimuovere le cellule morte. Medie cellulare può sostituire ogni 24-48 ore, se necessario. In un tipico esperimento, le cellule sono pronte per la trasfezione di solito 24-48 ore dopo la semina. Per mantenere la coerenza in termini di efficienza elettroporazione si consiglia di attendere fino a quando la cultura è almeno l&#39;80% confluenti prima di elettroporazione. </li></ol> 3. Specifici per un sito trasfezione di siRNA <ol><li> Preparare una soluzione di acido nucleico. Per la trasfezione siRNA, preparare una soluzione di 1-2 siRNA pM elettroporazione in tampone ghiacciato elettroporazione per ottenere un volume finale di 200-400 microlitri. In questo articolo il video che dimostrano la trasfezione di cellule HeLa con Alexa 488 coniugato strapazzate sequenza di siRNA. </li><li> Trasferire il MEA con le cellule dal termostato a cappa sterile a flusso laminare. Rimuovere il supporto delle cellule del MEA ed eseguire un lavaggio con PBS. Successivamente, aggiungere 200-400 microlitri della ice freddo 1 pM elettroporazione soluzione siRNA al pozzo. </li><li> Applicare anodici impulsi quadri elettrici agli elettrodi mirati (Fare riferimento al punto 4 per la determinazione dei parametri ottimali di impulso elettrico). Nei nostri esperimenti abbiamo utilizzato un Instruments pragmatiche 2414A generatore di forme d&#39;onda per generare impulsi elettrici e un sistema dual-inline-package (DIP) 16 clip passante di entrare in contatto con i rilievi dei titoli di MEA. Il generatore di forme d&#39;onda può essere connesso alla clip DIP demultiplexor tramite una scheda a circuito stampato per fornire selettività dei singoli elettrodi. Per l&#39;elettrodo di riferimento, posizionare un catodo di acciaio nei media cella. Riducendo l&#39;elettrodo di riferimento acciaio nei media è estremamente importante assicurare che non fa contatto con la superficie MEA. Al contatto, può causare danni alle cellule e il MEA. Si raccomanda di ridurre l&#39;elettrodo di riferimento a una posizione di 1 mm sopra le celle. Un distanziatore con un&#39;altezza di 1 mm può essere posizionato nel pozzo per assistere nel posizionamento del refrimento elettrodo. Un approccio alternativo utilizzando un elettrodo di riferimento esterno è utilizzare elettrodi adiacenti come catodo-anodo coppie. </li><li> Subito dopo l&#39;applicazione degli impulsi elettrici agli elettrodi mirati sulla MEA, trasferire il ritorno alla MEA l&#39;incubatrice. Dopo un periodo di incubazione di 5 minuti, delicatamente sostituire il 75% del tampone di elettroporazione con pre-riscaldato (37 ° C) supporti cellulari. Successivamente, l&#39;imaging fluorescente può essere fatto per confermare la consegna di siRNA fluorescenti etichettato alle cellule. </li></ol> 4. Elettroporazione dei parametri di ottimizzazione <ol><li> Per determinare i parametri ottimali di impulsi elettrici per esperimenti di design elettroporazione, elettroporazione per un intervallo di valori di ampiezza dell&#39;impulso, durata dell&#39;impulso, e il numero di impulsi. Nella nostra esperienza con cellule HeLa, abbiamo osservato che un singolo impulso di tensione di ampiezza da 3 V a 9 V, e la durata di 1 msec a 10 msec per una dimensione di 100 pm elettrodo portato a elettroporazione successo con mila morte delle cellule Nimal. È nostra raccomandazione che un parametro variabile a un tempo e altri sono mantenute costanti. Per quantificare l&#39;efficienza di elettroporazione per ciascuno dei parametri di impulso, usiamo ioduro di propidio (PI), un colorante impermeant cella macchie materiale nucleico, e la metodologia basata assay vivo, come descritto nelle fasi 4,2-4,5. </li><li> Stabilire una coltura cellulare sulla MEA seguendo le indicazioni riportate ai punti 1 e 2. </li><li> Prima di preparare una elettroporazione 300-500 microlitri di 30 ug / ml soluzione PI in tampone ghiacciato elettroporazione e 400 pl di 4 um calcien AM soluzione in PBS. </li><li> Trasferire la MEA dall&#39;incubatore alla cappa a flusso laminare. Rimuovere il supporto delle cellule del MEA ed eseguire un lavaggio con PBS. Successivamente, aggiungere 300-500 microlitri del tampone freddo elettroporazione ghiaccio con PI. </li><li> Applicare impulsi elettrici con ampiezza di pulsazione desiderata e la durata agli elettrodi mirati (Le attrezzature utilizzate nei nostri esperimenti per la generazione e l&#39;applicazione del eletimpulso tric al MEA è descritto nel passaggio 3.3). Per l&#39;elettrodo di riferimento o abbassare un elettrodo di acciaio nel buffer elettroporazione nel MEA pozzo o utilizzare l&#39;elettrodo vicino come riferimento. Selettivamente applicando impulsi di ampiezza variabile o durate di elettrodi diversi esperimenti multipli può essere eseguita sul dispositivo stesso. Per esempio, su un elettrodo MEA 32, 8 esperimenti possono essere eseguiti suddividendo i 32 elettrodi in 8 gruppi, ognuno composto da 4 elettrodi. Ciascuno degli 8 gruppi possono essere stimolato da un singolo impulso rettangolare di durata tensione 5 msec con ampiezze diverse in 3 V-6 gamma V in incrementi di 0,5 V e un gruppo può essere utilizzato come controllo. </li><li> Immediatamente dopo l&#39;applicazione di impulsi di tensione trasferire il MEA per l&#39;incubatore per 5 min. Successivamente, rimuovere delicatamente il 75% del tampone di elettroporazione dal MEA pozzo e sostituirlo con supporti cellulari caldi. Trasferire il MEA per l&#39;incubatrice. </li><li> Dopo un periodo di incubazione di 2-4 hrsostituzione del supporto di cella nella MEA bene con 300-500 ml di 4 mM calceina AM soluzione in PBS. Trasferire il MEA di incubatrice per altri 30 min. </li><li> Dopo l&#39;incubazione delle cellule con calceina AM, sostituire la calceina AM soluzione nel MEA bene con PBS dopo due lavaggi con PBS. Ottenere immagini fluorescenti di cellule utilizzando con un microscopio ottico con filtri per la PI (eccitazione / emissione - 535 nm/617 nm) e calceina AM (eccitazione / emissione - 490 nm/520 nm). L&#39;assorbimento di PI, a causa di elettroporazione, induce le cellule a fluorescenza rossa e la AM Calcein induce le cellule che sono vivi a fluorescenza verde. Tre possibili scenari che si possono osservare sono: 1) cellule elettroporate e vivo (si fluorescenza sia rosso e verde), 2) cellule vive, ma non elettroporate (sarà solo fluorescenza verde) e 3) le cellule morte a causa di elettroporazione (sarà solo fluorescenza rossa) . La vitalità cellulare è stata calcolata come percentuale del numero totale di cellule vive sull&#39;elettrodo e l&#39;eff elettroporazioneiciency è stata calcolata come percentuale del numero totale di cellule caricate con PI / siRNA. </li></ol> 5. Risultati rappresentativi La figura 1 mostra il sito-specifica caricamento di cellule HeLa con PI. Si può osservare che le cellule solo sull&#39;elettrodo dimostrano un assorbimento di PI (Figura 1B). Un test dal vivo eseguita elettroporazione messaggio è stato utilizzato per valutare la vitalità delle cellule (Figura 1A). L&#39;efficienza elettroporazione e la vitalità cellulare per l&#39;esempio mostrato nella figura 1 sono 81,8% e 96,1% rispettivamente. Vitalità cellulare ed efficienza elettroporazione può essere ottenuto ottimizzando i parametri di impulso di elettroporazione. Sito-specifica trasfezione di cellule HeLa con siRNA fluorescente etichettato per un impulso di elettroporazione ottimizzato (8 V, 1 msec) è mostrato in Figura 2. L&#39;efficienza elettroporazione per il siRNA a 8 V, 1 msec è risultato essere 74,4 ± 16,0% evitalità cellulare è risultato essere 87,9 ± 8,4% (tutti i dati rappresentati come media ± deviazione standard, N = 5). <img alt="Figura 1" src="/files/ftp_upload/4415/4415fig1.jpg" /> Figura 1. Sito-specifica consegna di PI in cellule HeLa su un elettrodo 200 micron (6 V, 5 msec). A) Calcien saggio basato vivo eseguita due ore dopo l&#39;elettroporazione. Cellule vive sono indicati con fluorescenza verde. B) Red fluorescenza dimostra l&#39;assorbimento da parte delle cellule del PI sull&#39;elettrodo. C) Una immagine sovrapposta di A e B. Le frecce bianche indicano cellule che sono elettroporata e vivo. Le frecce nere indicano cellule che sono morti. Il cerchio bianco in A, B e C indica la sagoma dell&#39;elettrodo. <img alt="Figura 2" src="/files/ftp_upload/4415/4415fig2.jpg" /> Figura 2. Trasfezione di cellule HeLa con scrambl fluorescente etichettatoLa sequenza di siRNA. A) Immagine di cellule HeLa su un elettrodo di 100 um trasfettate con Alexa 488 siRNA tag (8 V, 1 msec). B) Immagine di nuclei di cellule colorate con DAPI. Il cerchio bianco segna il bordo dell&#39;elettrodo.

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	<li>Fire, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potent+and+specific+genetic+interference+by+double-stranded+RNA+in+Caenorhabditis+elegans.">Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.</a> Nature. 391, 806-811 (1998).</li><li>Caplen, N. J., Parrish, S., Imani, F., Fire, A., Morgan, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+inhibition+of+gene+expression+by+small+double-stranded+RNAs+in+invertebrate+and+vertebrate+systems.">Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems.</a> Proceedings of the National Academy of Sciences. 98, 9742 (2001).</li><li>Elbashir, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Duplexes+of+21-nucleotide+RNAs+mediate+RNA+interference+in+cultured+mammalian+cells.">Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells.</a> Nature. 411, 494-498 (2001).</li><li>Brummelkamp, T. R., Bernards, R., Agami, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+system+for+stable+expression+of+short+interfering+RNAs+in+mammalian+cells.">A system for stable expression of short interfering RNAs in mammalian cells.</a> Science. 296, 550 (2002).</li><li>Lee, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+small+interfering+RNAs+targeted+against+HIV-1+rev+transcripts+in+human+cells.">Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells.</a> Nat. Biotechnol. 20, 500-505 (2002).</li><li>Miyagishi, M., Taira, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=U6+promoter-driven+siRNAs+with+four+uridine+3'+overhangs+efficiently+suppress+targeted+gene+expression+in+mammalian+cells.">U6 promoter-driven siRNAs with four uridine 3' overhangs efficiently suppress targeted gene expression in mammalian cells.</a> Nat. Biotechnol. 20, 497-500 (2002).</li><li>Paul, C. P., Good, P. D., Winer, I., Engelke, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+expression+of+small+interfering+RNA+in+human+cells.">Effective expression of small interfering RNA in human cells.</a> Nat. Biotechnol. 20, 505-508 (2002).</li><li>Xia, H., Mao, Q., Paulson, H. L., Davidson, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=siRNA-mediated+gene+silencing+in+vitro+and+in+vivo.">siRNA-mediated gene silencing in vitro and in vivo.</a> Nat. Biotechnol. 20, 1006-1010 (2002).</li><li>Chu, G., Hayakawa, H., Berg, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+for+the+efficient+transfection+of+mammalian+cells+with+DNA.">Electroporation for the efficient transfection of mammalian cells with DNA.</a> Nucleic Acids Res. 15, 1311 (1987).</li><li>Felgner, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipofection:+a+highly+efficient,+lipid-mediated+DNA-transfection+procedure.">Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure.</a> Proceedings of the National Academy of Sciences. 84, 7413 (1987).</li><li>Raptis, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=applications+of+electroporation+of+adherent+cells+in+situ,+on+a+partly+conductive+slide.">applications of electroporation of adherent cells in situ, on a partly conductive slide.</a> Mol. Biotechnol. 4, 129-138 (1995).</li><li>Jordan, M., Schallhorn, A., Wurm, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfecting+mammalian+cells:+optimization+of+critical+parameters+affecting+calcium-phosphate+precipitate+formation.">Transfecting mammalian cells: optimization of critical parameters affecting calcium-phosphate precipitate formation.</a> Nucleic Acids Res. 24, 596-601 (1996).</li><li>Gao, Y., Liu, X. L., Li, X. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Research+progress+on+siRNA+delivery+with+nonviral+carriers.">Research progress on siRNA delivery with nonviral carriers.</a> International Journal of Nanomedicine. 6, 1017 (2011).</li><li>Ziauddin, J., Sabatini, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarrays+of+cells+expressing+defined+cDNAs.">Microarrays of cells expressing defined cDNAs.</a> Nature. 411, 107-110 (2001).</li><li>Yamauchi, F., Kato, K., Iwata, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatially+and+temporally+controlled+gene+transfer+by+electroporation+into+adherent+cells+on+plasmid+DNA-loaded+electrodes.">Spatially and temporally controlled gene transfer by electroporation into adherent cells on plasmid DNA-loaded electrodes.</a> Nucleic Acids Res. 32, e187 (2004).</li><li>Yamauchi, F., Kato, K., Iwata, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Layer-by-Layer+Assembly+of+Poly+(ethyleneimine)+and+Plasmid+DNA+onto+Transparent+Indium?+Tin+Oxide+Electrodes+for+Temporally+and+Spatially+Specific+Gene+Transfer.">Layer-by-Layer Assembly of Poly (ethyleneimine) and Plasmid DNA onto Transparent Indium? Tin Oxide Electrodes for Temporally and Spatially Specific Gene Transfer.</a> Langmuir. 21, 8360-8367 (2005).</li><li>Jain, T., Muthuswamy, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microsystem+for+transfection+of+exogenous+molecules+with+spatio-temporal+control+into+adherent+cells.">Microsystem for transfection of exogenous molecules with spatio-temporal control into adherent cells.</a> Biosensors and Bioelectronics. 22, 863-870 (2007).</li><li>Jain, T., Muthuswamy, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bio-chip+for+spatially+controlled+transfection+of+nucleic+acid+payloads+into+cells+in+a+culture.">Bio-chip for spatially controlled transfection of nucleic acid payloads into cells in a culture.</a> Lab on a Chip. 7, 1004-1011 (2007).</li><li>Jain, T., Muthuswamy, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microelectrode+array+(MEA)+platform+for+targeted+neuronal+transfection+and+recording.">Microelectrode array (MEA) platform for targeted neuronal transfection and recording.</a> Biomedical Engineering, IEEE Transactions on. 55, 827-832 (2008).</li><li>Jain, T., McBride, R., Head, S., Saez, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+parallel+introduction+of+nucleic+acids+into+mammalian+cells+grown+in+microwell+arrays.">Highly parallel introduction of nucleic acids into mammalian cells grown in microwell arrays.</a> Lab on a Chip. 9, 3557-3566 (2009).</li><li>Huang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+and+high-throughput+electroporation+microchip+applicable+for+siRNA+delivery.">An efficient and high-throughput electroporation microchip applicable for siRNA delivery.</a> Lab Chip. 11, 163-172 (2010).</li><li>Li, Z., Wei, Z., Li, X., Du, Q., Liang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+and+high-throughput+electroporation+chips.">Efficient and high-throughput electroporation chips.</a> , (2011).</li><li>Jain, T., Papas, A., Jadhav, A., McBride, R., Saez, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+electroporation+of+surface-bound+siRNAs+in+microwell+arrays.">In situ electroporation of surface-bound siRNAs in microwell arrays.</a> Lab Chip. 12, 939-947 (2012).</li><li>Haas, K., Sin, W. C., Javaherian, A., Li, Z., Cline, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-Cell+Electroporationfor+Gene+Transfer+In+Vivo.">Single-Cell Electroporationfor Gene Transfer In Vivo.</a> Neuron. 29, 583-591 (2001).</li><li>Akaneya, Y., Jiang, B., Tsumoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAi-induced+gene+silencing+by+local+electroporation+in+targeting+brain+region.">RNAi-induced gene silencing by local electroporation in targeting brain region.</a> J. Neurophysiol. 93, 594 (2005).</li><li>Lee, W. G., Demirci, U., Khademhosseini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscale+electroporation:+challenges+and+perspectives+for+clinical+applications.">Microscale electroporation: challenges and perspectives for clinical applications.</a> Integr. Biol. 1, 242-251 (2009).</li><li>Boukany, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanochannel+electroporation+delivers+precise+amounts+of+biomolecules+into+living+cells.">Nanochannel electroporation delivers precise amounts of biomolecules into living cells.</a> Nature Nanotechnology. 6, 747-754 (2011).</li></ol>

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In questo articolo il video che dimostrano l&#39;uso di MEA per il sito-specifica trasfezione di cellule HeLa con strapazzate sequenza di siRNA. Uno dei vantaggi di questa tecnologia è la sua applicabilità a diverse linee cellulari incluse linee cellulari primarie. Il nostro laboratorio ha già dimostrato l&#39;uso di questa tecnologia per il sito-specifica trasfezione di colture primarie neuronali dell&#39;ippocampo di ratto E18 dal giorno di vita e NIH-3T3 strapazzate con sequenze siRNA e GFP plasmide 18, 19.</...

<table border="1"><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti (opzionale) </td></tr><tr><td> Cellulari media: Avanzata MEM L-Glutammina 200 mM Penicillina / streptomicina Siero fetale bovino </td><td> Gibco / Invitrogen Himedia Laboratori / VWR Lonza Group Ltd. Gibco / Invitrogen </td><td> 12492-013 95057-448 09-757F 16000-044 </td><td> Composizione cella media: 2% FBS, Pennstrep 2% L-glutammina e il 2% in Advance MEM </td></tr><tr><td> Tripsina EDTA </td><td> Mediatech, Inc. </td><td> 25-053-CL </td><td></td></tr><tr><td> PBS </td><td> Mediatech, Inc. </td><td> 21-040-CV </td><td></td></tr><tr><td> Alexa 488 e rodamina tagged strapazzate sequrienza siRNA </td><td> Qiagen, Inc. </td><td> 1027292 </td><td></td></tr><tr><td> Elettroporazione tampone </td><td> Biorad Laboratories </td><td> 165-2677 </td><td></td></tr><tr><td> Generatore di forme d&#39;onda </td><td> Pragmatico </td><td> 2414A </td><td> Qualsiasi forma d&#39;onda / generatore di impulsi che può fornire gli impulsi desiderati possono essere utilizzati. </td></tr></table>

high efficiency, site-specific transfection of adherent cells with sirna using microelectrode arrays (mea)

La scoperta della via di RNAi in eucarioti e il conseguente sviluppo di agenti RNAi, come siRNA e shRNA, hanno raggiunto un metodo efficace per mettere a tacere i geni specifici 1-8 per la genomica funzionale e terapie. Una sfida importante coinvolto in studi basati su RNAi è la consegna di agenti RNAi alle cellule bersaglio. Tradizionali non virali tecniche di parto, come l&#39;elettroporazione di massa e metodi di trasfezione chimici spesso non hanno il controllo spaziale necessaria su consegna e permettere l&#39;efficienza di trasfezione poveri 9-12. I recenti progressi nei metodi di trasfezione chimici come lipidi cationici, polimeri cationici e nanoparticelle hanno portato ad efficienze di trasfezione altamente avanzate 13. Tuttavia, queste tecniche ancora non riescono a offrire un controllo preciso dello spazio sulla consegna che possono beneficiare immensamente miniaturizzata tecnologie high-throughput, studi cella singola e le indagini di interazioni cellula-cellula. nt &quot;&gt; I recenti progressi tecnologici nella consegna del gene hanno permesso alta produttività trasfezione di cellule aderenti 14-23, la maggioranza dei quali utilizzano elettroporazione microscala. elettroporazione Microscale offre spazio-temporale preciso controllo di consegna (fino a singole cellule) ed è stato dimostrato per ottenere alte efficienze 19, 24-26. Inoltre, approcci basati elettroporazione non richiedono un lungo periodo di incubazione (tipicamente 4 ore) con complessi siRNA e DNA come necessario in metodi di trasfezione base chimica e causare l&#39;immissione diretta di siRNA nudo e DNA molecole nel citoplasma cellulare. Di conseguenza espressione genica può essere ottenuto fino a sei ore dopo la trasfezione 27. nostro laboratorio ha precedentemente dimostrato l&#39;utilizzo di matrici di microelettrodi (MEA) per sito-specifica trasfezione aderenti in colture cellulari di mammifero 17-19. Nell&#39;approccio basato MEA, la consegna del carico utile genetica avviene tramite localizzato micro-scala electroporatisu di cellule. Una applicazione di impulsi elettrici agli elettrodi selezionati genera locale campo elettrico che conduce alla elettroporazione di cellule presenti nella regione degli elettrodi stimolati. Il controllo indipendente delle micro-elettrodi fornisce controllo spaziale e temporale sopra trasfezione e permette anche esperimenti di trasfezione multipli basati da eseguire sulla stessa cultura aumentando la produttività e riducendo sperimentale cultura a cultura variabilità. Qui si descrive l&#39;apparato sperimentale e il protocollo per la trasfezione mirata di aderenti cellule HeLa con una fluorescente tag criptato siRNA sequenza usando elettroporazione. Lo stesso protocollo può essere utilizzato anche per la trasfezione di vettori plasmidici. Inoltre, il protocollo qui descritto può essere facilmente estesa ad una varietà di linee cellulari di mammifero con modifiche secondarie. Disponibilità commerciale degli accordi ambientali multilaterali con i modelli di elettrodi sia predefiniti e personalizzati rendono questa t accessibile tecnicala maggior parte dei laboratori di ricerca o con attrezzature di base di coltura cellulare.

Watch this Scientific Journal Video about High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays MEA at JoVE.com

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays MEA

L&#39;articolo descrive il protocollo per sito-specifica di transfezione criptato sequenza di siRNA in una coltura cellulare di mammifero aderente utilizzando una matrice di microelettrodi (MEA).

Alta efficienza, sito-specifica trasfezione di cellule aderenti con siRNA mediante matrici microelettrodi (MEA)

The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for ...

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Research

JoVE Journal

Bioengineering

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays (MEA)

13.4K Views.  Arizona State University. L'articolo descrive il protocollo per sito-specifica di transfezione criptato sequenza di siRNA in una coltura cellulare di mammifero aderente utilizzando una matrice di microelettrodi (MEA).

Video: High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays MEA

Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. Urgent need exists to develop novel clinically-translatable therapeutic strategies that can improve on the dismal survival statistics of pancreatic cancer patients. Although gene therapy in cancer has shown a tremendous promise, the major challenge is in the development of safe and effective delivery system, which can lead to sustained transgene expression. 
Gelatin is one of the most versatile natural biopolymer, widely used in food and pharmaceutical products. Previous studies from our laboratory have shown that type B gelatin could physical encapsulate DNA, which preserved the supercoiled structure of the plasmid and improved transfection efficiency upon intracellular delivery. By thiolation of gelatin, the sulfhydryl groups could be introduced into the polymer and would form disulfide bond within nanoparticles, which stabilizes the whole complex and once disulfide bond is broken due to the presence of glutathione in cytosol, payload would be released 2-5. Poly(ethylene glycol) (PEG)-modified GENS, when administered into the systemic circulation, provides long-circulation times and preferentially targets to the tumor mass due to the hyper-permeability of the neovasculature by the enhanced permeability and retention effect 6. Studies have shown over-expression of the epidermal growth factor receptor (EGFR) on Panc-1 human pancreatic adenocarcinoma cells 7. In order to actively target pancreatic cancer cell line, EGFR specific peptide was conjugated on the particle surface through a PEG spacer.8
Most anti-tumor gene therapies are focused on administration of the tumor suppressor genes, such as wild-type p53 (wt-p53), to restore the pro-apoptotic function in the cells 9. The p53 mechanism functions as a critical signaling pathway in cell growth, which regulates apoptosis, cell cycle arrest, metabolism and other processes 10. In pancreatic cancer, most cells have mutations in p53 protein, causing the loss of apoptotic activity. With the introduction of wt-p53, the apoptosis could be repaired and further triggers cell death in cancer cells 11. 
Based on the above rationale, we have designed EGFR targeting peptide-modified thiolated gelatin nanoparticles for wt-p53 gene delivery and evaluated delivery efficiency and transfection in Panc-1 cells.

Type B gelatin-based engineered nanovectors system (GENS) was developed for systemic gene delivery and transfection in the treatment of pancreatic cancer. By modification with epidermal growth factor receptor (EGFR) specific peptide on the surface of nanparticles, they could target on EGFR receptor and release plasmid under reducing environment, such as high intracellular glutathione concentrations.

Consegna dei geni e trasfezione in cellule umane di cancro al pancreas con Epidermal Growth Factor Receptor mirati Nanovectors Progettato a base di gelatina

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. ...

Ricerca

Bioingegneria

Site-specific Bacterial Chromosome Engineering: &#934;C31 Integrase Mediated Cassette Exchange (IMCE)

The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range1. Integration into the chromosome circumvents issues such as plasmid replication, plasmid stability, plasmid incompatibility, and plasmid copy number variance. This method uses the site-specific integrase from the Streptomyces phage (&Phi;) C312,3. The &Phi;C31 integrase catalyzes a direct recombination between two specific DNA sites: attB and attP (34 and 39 bp, respectively)4. This recombination is stable and does not revert5. A "landing pad" (LP) sequence consisting of a spectinomycin- resistance gene, aadA (SpR), and the E. coli &szlig;-glucuronidase gene (uidA) flanked by attP sites has been integrated into the chromosomes of Sinorhizobium meliloti, Ochrobactrum anthropi, and Agrobacterium tumefaciens in an intergenic region, the ampC locus, and the tetA locus, respectively. S. meliloti is used in this protocol. Mobilizable donor vectors containing attB sites flanking a stuffer red fluorescent protein (rfp) gene and an antibiotic resistance gene have also been constructed. In this example the gentamicin resistant plasmid pJH110 is used. The rfp gene6 may be replaced with a desired construct using SphI and PstI. Alternatively a synthetic construct flanked by attB sites may be sub-cloned into a mobilizable vector such as pK19mob7. The expression of the &Phi;C31 integrase gene (cloned from pHS628) is driven by the lac promoter, on a mobilizable broad host range plasmid pRK78139.
A tetraparental mating protocol is used to transfer the donor cassette into the LP strain thereby replacing the markers in the LP sequence with the donor cassette. These cells are trans-integrants. Trans-integrants are formed with a typical efficiency of 0.5%. Trans-integrants are typically found within the first 500-1,000 colonies screened by antibiotic sensitivity or blue-white screening using 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid (X-gluc). This protocol contains the mating and selection procedures for creating and isolating trans-integrants.

Site-specific Bacterial Chromosome Engineering: &amp;#934;C31 Integrase Mediated Cassette Exchange (IMCE)

A quick and efficient method to integrate foreign DNA of interest into pre-made acceptor strains, termed landing pad strains, is described. The method allows site-specific integration of a DNA cassette into the engineered landing pad locus of a given strain, through conjugation and expression of the &#934;C31 integrase.

Site-specific Ingegneria batterica Cromosoma: ΦC31 integrasi Mediated Cassette Exchange (IMCE)

The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range1. Integration into the chromosome circumvents issues such as ...

Engineering Adherent Bacteria by Creating a Single Synthetic Curli Operon

The method described here consists in redesigning E. coli adherence properties by assembling the minimum number of curli genes under the control of a strong and metal-overinducible promoter, and in visualizing and quantifying the resulting gain of bacterial adherence. This method applies appropriate engineering principles of abstraction and standardization of synthetic biology, and results in the BBa_K540000 Biobrick (Best new Biobrick device, engineered, iGEM 2011).
The first step consists in the design of the synthetic operon devoted to curli overproduction in response to metal, and therefore in increasing the adherence abilities of the wild type strain. The original curli operon was modified in silico in order to optimize transcriptional and translational signals and escape the "natural" regulation of curli. This approach allowed to test with success our current understanding of curli production. Moreover, simplifying the curli regulation by switching the endogenous complex promoter (more than 10 transcriptional regulators identified) to a simple metal-regulated promoter makes adherence much easier to control.
The second step includes qualitative and quantitative assessment of adherence abilities by implementation of simple methods. These methods are applicable to a large range of adherent bacteria regardless of biological structures involved in biofilm formation. Adherence test in 24-well polystyrene plates provides a quick preliminary visualization of the bacterial biofilm after crystal violet staining. This qualitative test can be sharpened by the quantification of the percentage of adherence. Such a method is very simple but more accurate than only crystal violet staining as described previously 1 with both a good repeatability and reproducibility. Visualization of GFP-tagged bacteria on glass slides by fluorescence or laser confocal microscopy allows to strengthen the results obtained with the 24-well plate test by direct observation of the phenomenon.

The design of a synthetic operon encoding both the secretory apparatus and the structural monomers of curli fibers is described. Overproduction of these amyloids and adherent polymers allows a measurable gain of adherence of the E. coli chassis1. Easy ways to visualize and quantify adherence are explained.

Ingegneria batteri aderenti mediante la creazione di un unico sintetico curli Operon

The  method described here consists in redesigning E. coli adherence properties by assembling the minimum number of  curli genes under the control of a ...

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening based on cells in such environments have therefore serious limitations: cell organelles show extreme phenotypes, cell morphologies and sizes are heterogeneous and/or specific cell organelles cannot be properly visualized. In addition, cells in vivo are located in a 3D environment; in this situation, cells show different phenotypes mainly because of their interaction with the surrounding extracellular matrix of the tissue. In order to standardize and generate order of single cells in a physiologically-relevant 3D environment for cell-based assays, we report here the microfabrication and applications of a device for in vitro 3D cell culture. This device consists of a 2D array of microcavities (typically 105 cavities/cm2), each filled with single cells or embryos. Cell position, shape, polarity and internal cell organization become then normalized showing a 3D architecture. We used replica molding to pattern an array of microcavities, &#8216;eggcups&#8217;, onto a thin polydimethylsiloxane (PDMS) layer adhered on a coverslip. Cavities were covered with fibronectin to facilitate adhesion. Cells were inserted by centrifugation. Filling percentage was optimized for each system allowing up to 80%. Cells and embryos viability was confirmed. We applied this methodology for the visualization of cellular organelles, such as nucleus and Golgi apparatus, and to study active processes, such as the closure of the cytokinetic ring during cell mitosis. This device allowed the identification of new features, such as periodic accumulations and inhomogeneities of myosin and actin during the cytokinetic ring closure and compacted phenotypes for Golgi and nucleus alignment. We characterized the method for mammalian cells, fission yeast, budding yeast, C. elegans with specific adaptation in each case. Finally, the characteristics of this device make it particularly interesting for drug screening assays and personalized medicine.

We report a device and a new method to study cells and embryos. Single cells are precisely ordered in microcavity arrays. Their 3D confinement is a step towards 3D environments encountered in physiological conditions and allows organelle orientation. By controlling cell shape, this setup minimizes variability reported in standard assays.

Ordinazione di singole cellule e singola embrioni in 3D confinamento: Un nuovo dispositivo per l&#39;alta Screening contenuti

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening ...

Porous Silicon Microparticles for Delivery of siRNA Therapeutics

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

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

Poroso Silicon microparticelle per consegna di siRNA Therapeutics

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

Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes. The emergence of prion diseases in wildlife populations and their increasing threat to human health has led to increased efforts to find a treatment for these diseases. Recent studies have found numerous anti-prion compounds that can either inhibit the infectious PrPRes isomer or down regulate the normal cellular prion protein. However, most of these compounds do not cross the blood brain barrier to effectively inhibit PrPRes formation in brain tissue, do not specifically target neuronal PrPC, and are often too toxic to use in animal or human subjects. 
We investigated whether siRNA delivered intravascularly and targeted towards neuronal PrPC is a safer and more effective anti-prion compound. This report outlines a protocol to produce two siRNA liposomal delivery vehicles, and to package and deliver PrP siRNA to neuronal cells. The two liposomal delivery vehicles are 1) complexed-siRNA liposome formulation using cationic liposomes (LSPCs), and 2) encapsulated-siRNA liposome formulation using cationic or anionic liposomes (PALETS). For the LSPCs, negatively charged siRNA is electrostatically bound to the cationic liposome. A positively charged peptide (RVG-9r [rabies virus glycoprotein]) is added to the complex, which specifically targets the liposome-siRNA-peptide complexes (LSPCs) across the blood brain barrier (BBB) to acetylcholine expressing neurons in the central nervous system (CNS). For the PALETS (peptide addressed liposome encapsulated therapeutic siRNA), the cationic and anionic lipids were rehydrated by the PrP siRNA. This procedure results in encapsulation of the siRNA within the cationic or anionic liposomes. Again, the RVG-9r neuropeptide was bound to the liposomes to target the siRNA/liposome complexes to the CNS. Using these formulations, we have successfully delivered PrP siRNA to AchR-expressing neurons, and decreased the PrPC expression of neurons in the CNS.

The goal of this protocol is to use cationic/anionic liposomes with a neuro-targeting peptide as a CNS delivery system to enable siRNA to cross the BBB. The optimization of a delivery system for treatments, like siRNA, would allow for more treatment options for prion and other neurodegenerative diseases.

La consegna dei terapeutica siRNA al SNC Utilizzando cationici e anionici liposomi

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes.  The ...

Comprehensive Evaluation of the Effectiveness and Safety of Placenta-Targeted Drug Delivery Using Three Complementary Methods

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to the placenta while minimizing fetal and maternal side effects remains challenging. Targeted nanoparticle carriers provide new opportunities to treat placental disorders. We recently demonstrated that a synthetic placental chondroitin sulfate A binding peptide (plCSA-BP) could be used to guide nanoparticles to deliver drugs to the placenta. In this protocol, we describe in detail a system for assessing the efficiency of drug delivery to the placenta by plCSA-BP that employs three separate methods used in combination: in vivo imaging, high-frequency ultrasound (HFUS), and high-performance liquid chromatography (HPLC). Using in vivo imaging, plCSA-BP-guided nanoparticles were visualized in the placentas of live animals, while HFUS and HPLC demonstrated that plCSA-BP-conjugated nanoparticles efficiently and specifically delivered methotrexate to the placenta. Thus, a combination of these methods can be used as an effective tool for the targeted delivery of drugs to the placenta and development of new treatment strategies for several pregnancy complications.

We describe a system that utilizes three methods to evaluate the safety and effectiveness of placenta-targeted drug delivery: in vivo imaging to monitor nanoparticle accumulation, high-frequency ultrasound to monitor placental and fetal development, and HPLC to quantify drug delivery to tissue.

Valutazione globale dell'efficacia e della sicurezza di Placenta-Targeted Drug Delivery utilizzando tre metodi complementari

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to ...

Interfacing Microfluidics with Microelectrode Arrays for Studying Neuronal Communication and Axonal Signal Propagation

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of electrophysiological activity for long periods. However, the property of sensing signals from all sources around every microelectrode can become unfavorable when trying to understand communication and signal propagation in neuronal circuits. In a neuronal network, several neurons can be simultaneously activated and can generate overlapping action potentials, making it difficult to discriminate and track signal propagation. Considering this limitation, we have established an in vitro setup focused on assessing electrophysiological communication, which is able to isolate and amplify axonal signals with high spatial and temporal resolution. By interfacing microfluidic devices and MEAs, we are able to compartmentalize neuronal cultures with a well-controlled alignment of the axons and microelectrodes. This setup allows recordings of spike propagation with a high signal-to-noise ratio over the course of several weeks. Combined with specialized data analysis algorithms, it provides detailed quantification of several communication related properties such as propagation velocity, conduction failure, firing rate, anterograde spikes, and coding mechanisms.
This protocol demonstrates how to create a compartmentalized neuronal culture setup over substrate-integrated MEAs, how to culture neurons in this setup, and how to successfully record, analyze and interpret the results from such experiments. Here, we show how the established setup simplifies the understanding of neuronal communication and axonal signal propagation. These platforms pave the way for new in vitro models with engineered and controllable neuronal network topographies. They can be used in the context of homogeneous neuronal cultures, or with co-culture configurations where, for example, communication between sensory neurons and other cell types is monitored and assessed. This setup provides very interesting conditions to study, for example, neurodevelopment, neuronal circuits, information coding, neurodegeneration and neuroregeneration approaches.

This protocol aims to demonstrate how to combine in vitro microelectrode arrays with microfluidic devices for studying action potential transmission in neuronal cultures. Data analysis, namely the detection and characterization of propagating action potentials, is performed using a new advanced, yet user-friendly and freely available, computational tool.

Microfluidica di interfacciamento con microelettrodi per studiare comunicazione neuronale e propagazione del segnale assonale

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of ...

Fabrication of Ti3C2 MXene Microelectrode Arrays for In Vivo Neural Recording

Implantable microelectrode technologies have been widely used to elucidate neural dynamics at the microscale to gain a deeper understanding of the neural underpinnings of brain disease and injury. As electrodes are miniaturized to the scale of individual cells, a corresponding rise in the interface impedance limits the quality of recorded signals. Additionally, conventional electrode materials are stiff, resulting in a significant mechanical mismatch between the electrode and the surrounding brain tissue, which elicits an inflammatory response that eventually leads to a degradation of the device performance. To address these challenges, we have developed a process to fabricate flexible microelectrodes based on Ti3C2 MXene, a recently discovered nanomaterial that possesses remarkably high volumetric capacitance, electrical conductivity, surface functionality, and processability in aqueous dispersions. Flexible arrays of Ti3C2 MXene microelectrodes have remarkably low impedance due to the high conductivity and high specific surface area of the Ti3C2 MXene films, and they have proven to be exquisitely sensitive for recording neuronal activity. In this protocol, we describe a novel method for micropatterning Ti3C2 MXene into microelectrode arrays on flexible polymeric substrates and outline their use for in vivo micro-electrocorticography recording. This method can easily be extended to create MXene electrode arrays of arbitrary size or geometry for a range of other applications in bioelectronics and it can also be adapted for use with other conductive inks besides Ti3C2 MXene. This protocol enables simple and scalable fabrication of microelectrodes from solution-based conductive inks, and specifically allows harnessing the unique properties of hydrophilic Ti3C2 MXene to overcome many of the barriers that have long hindered the widespread adoption of carbon-based nanomaterials for high-fidelity neural microelectrodes.

Fabrication of Ti3C2 MXene Microelectrode Arrays for In Vivo Neural Recording

We describe here a method for fabricating Ti3C2 MXene microelectrode arrays and utilizing them for in vivo neural recording.

Fabbricazione di array di microelettrodi Ti3C2 MXene per la registrazione neurale In Vivo

Implantable microelectrode technologies have been widely used to elucidate neural dynamics at the microscale to gain a deeper understanding of the neural ...

Fabrication of 3D Cardiac Microtissue Arrays using Human iPSC-Derived Cardiomyocytes, Cardiac Fibroblasts, and Endothelial Cells

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided a unique opportunity to study the complex interplay among different cardiovascular cell types that drives tissue development and disease. In the area of cardiac tissue models, several sophisticated three-dimensional (3D) approaches use induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to mimic physiological relevance and native tissue environment with a combination of extracellular matrices and crosslinkers. However, these systems are complex to fabricate without microfabrication expertise and require several weeks to self-assemble. Most importantly, many of these systems lack vascular cells and cardiac fibroblasts that make up over 60% of the nonmyocytes in the human heart. Here we describe the derivation of all three cardiac cell types from iPSCs to fabricate cardiac microtissues. This facile replica molding technique allows cardiac microtissue culture in standard multi-well cell culture plates for several weeks. The platform allows user-defined control over microtissue sizes based on initial seeding density and requires less than 3 days for self-assembly to achieve observable cardiac microtissue contractions. Furthermore, the cardiac microtissues can be easily digested while maintaining high cell viability for single-cell interrogation with the use of flow cytometry and single-cell RNA sequencing (scRNA-seq). We envision that this in vitro model of cardiac microtissues will help accelerate validation studies in drug discovery and disease modeling.

Here, we describe an easy-to-use methodology to generate 3D self-assembled cardiac microtissue arrays composed of pre-differentiated human-induced pluripotent stem cell-derived cardiomyocytes, cardiac fibroblasts, and endothelial cells. This user-friendly and low cell requiring technique to generate cardiac microtissues can be implemented for disease modeling and early stages of drug development.

Fabbricazione di array di microtissue cardiaci 3D utilizzando cardiomiociti derivati da iPSC umani, fibroblasti cardiaci e cellule endoteliali

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided ...