Questo lavoro è stato finanziato in parte da finanziamenti della Texas South Management Technology (POCrr 2009,041), l&#39;Istituto per l&#39;integrazione di Medicina e Scienza dal Centro Risorse Nazionale per la Ricerca (UL 1RR025767), e dal National Institute of Dental Research &amp; craniofacciale ( 5R21DE017294).

1. Preparazione dei vetrini funzionalizzati <ol><li> Collocare i vetrini in un rack scorrevole estraibile, e lavare due volte immergendolo in una vaschetta di colorazione contenente 99% di etanolo (grado istologico). Pulire le diapositive pulire con tovaglioli di carta (assicurando di non generare polvere di carta), e asciugare con un getto di gas azoto compresso. </li></ol> NOTA: non utilizzare Kim-Wipes per pulire le diapositive in quanto genererebbe polveri sottili di carta. <ol start="2"><li> Immergere il rack diapositiva contenente i vetrini in una vaschetta di colorazione piena di acido solforico concentrato e incubare a temperatura ambiente per una notte. </li></ol> NOTA: per evitare il contatto con la pelle, usare guanti resistenti agli agenti chimici e occhiali protettivi mentre si lavora con acidi concentrati e prodotti chimici tossici e corrosivi. <ol start="3"><li> Sonicare i vetrini per 30 min e lavare con Milli-Q (18 mQ) acqua per 30 minuti, seguite con un altro lavaggio in unoCetona per 5 min. Questo trattamento espone i gruppi silanolici sulla superficie del vetro. </li><li> Ricoprire i vetrini puliti con 2,5% (vol / vol in acqua) di 3-amminopropiltrietossisilano (APTES) soluzione, immergendo il portavetrini in APTES per 30 minuti, lavando 3 volte in acqua Milli-Q per 15 minuti ogni lavaggio, e cottura i vetrini in un forno a 110 ° C per 15 min. Cottura consente reticolazione delle APTES, con conseguente-NH2-funzionalizzazione della superficie. </li></ol> NOTA: la soluzione APTES in un contenitore di plastica in quanto i depositi APTES preferenzialmente sulle pareti del contenitore in vetro. <ol start="5"><li> Utilizzando un rivestitore a rotazione, rivestimento i vetrini con 1% (peso / volume in toluene) Polistirene-co-anidride maleica (MA-PS) (Sigma) per ottenere un mono-strato di rivestimento idrofobo 6. Aggiungere 2,0 ml di PS-MA su un vetrino pulito montato su un rivestitore a rotazione e mantello a 3.000 rpm per 30 s. Queste condizioni possono variare a seconda dei parametri di spin di rivestimento come rivestimento cosìluzione, substrato, spessore del rivestimento e governata dalla seguente equazione 7 </li></ol><img alt="Equazione 1" src="/files/ftp_upload/3845/3845eq1.jpg" /> dove h è lo spessore della pellicola di rivestimento, e è il tasso di evaporazione, η, C e ρ sono viscosità, la concentrazione e la densità della soluzione di rivestimento, rispettivamente, ed ω è la velocità angolare. NOTA: toluene è nocive per inalazione per lungo tempo e l&#39;uso di una cappa aspirante è raccomandato. <ol start="6"><li> Le slide possono essere memorizzati in un rack scorrevole fino ad un mese secco, privo di polvere condizioni a 2 - 8 ° C. </li></ol> 2. Preparazione degli inoculi Lievito e Encapsulation Collagene <ol><li> Preparare una coltura di una notte di C. albicans ceppo SC5314, in lievito Peptone destrosio [YPD; 10 g / L di estratto di lievito, 20 g / L e peptone 20 g / L destrosio] mezzo liquido inoculando una singola colonia di C. albicans in 10 - 20 mL di YPD 8. Incubare cultura in un agitatore orbitale (circa 150 - 200 rpm) a 30 ° C per una notte. Harvest 1 mL Candida albicans cellule di lievito da overnight colture YPD (per centrifugazione a 5.000 rpm per 5-10 minuti) e si lavano due volte per 10 min in 1 mL di soluzione salina tamponata con fosfato sterile (PBS; tampone fosfato 10 mM, 2,7 mM di cloruro di potassio, cloruro di sodio 137 mM, pH 7,4) (Sigma) per lavaggio. Harvest lavato anche cellule per centrifugazione a 1900 rpm per 10 minuti. Risospendere le cellule lavate in 1 mL di tampone di ricostruzione (0,2 N NaOH con soluzione di bicarbonato di sodio 2,2% (peso / vol) e 4,8% (peso / vol) HEPES, pH 7,2). </li></ol> NOTE: C. albicans è un microrganismo Group Risk 1/BSL1. Ricordate sempre di usare buone tecniche asettiche / sterile per il lavoro con questo microrganismo e seguire le procedure istituzionali per il corretto smaltimento dei materiali a rischio biologico. <ol start="2"><li> Contare le cellule utilizzando un hemocytOmeter su un microscopio a campo chiaro e regolare ad una densità cellulare di 5 x 10 7 cellule / ml. </li><li> Ulteriori diluire la sospensione dieci volte mediante aggiunta di 10 × RPMI-1640 integrato con L-glutammina e tamponata con morpholinepropanesulfonic (MOPS) acido (pH 7,2). </li><li> Incapsulare le cellule di lievito in collagene mescolando la sospensione cellulare in RPMI-1640 con collagene (1,8 mg / mL) (tipo 1 di coda di ratto, BD Biosciences, Bedford, MA), per ottenere una concentrazione finale di 4 x 10 6 cellule / mL. Mantenere il collagene-sospensione cellulare su ghiaccio per impedire la gelificazione del collagene prima della stampa. </li></ol> 3. Preparazione di Ca BChip <ol><li> Pulire e disinfettare tutte le superfici di micro-distributore, tra cui la fonte piastra stazione di lavaggio e la stazione di vuoto, slide piatto vuoto e camera di stampa, pulendo con isopropanolo al 70%. </li><li> Posizionare e tenere sotto vuoto il numero desiderato di PS-MA-rivestite vetrini sul ponte scorrevole del microfonoroarrayer. </li><li> Vortex la sospensione cellulare in collagene vigorosamente e aspirare 100 pl di ben miscelato sospensione in un pozzetto di una piastra a 96 pozzetti, appena prima della stampa. Posizionare questa piastra fonte nella stazione di carico. </li><li> Accendere l&#39;umidificatore per mantenere una umidità relativa del 100% nella camera microarray durante il processo di stampa. </li><li> Stampa 50 nL di sospensione di cellule in una matrice di 48 righe e 16 colonne con punti distanziati di 1,2 mm di distanza utilizzando un microarray spotter (Omnigrid Micro, Inc. Digilab, Holliston, MA) da parte di non-contatto con la deposizione conica coniche 190 consigli orifizio ceramica micron (Digilab). </li><li> Prime, risciacquare e asciugare, aspirare le punte per due volte dopo ogni ciclo di stampa. </li><li> Subito dopo la stampa, posizionare il vetrino in un a tenuta d&#39;aria, camera umidificata (cassette ibridazione, ArrayIt Corporation, Sunnyvale, CA) e incubare a 37 ° C per 24 ore per consentire la formazione di biofilm. </li></ol> 4. Test di sensibilità di PrI biofilm eformed a Ca BChip contro gli agenti antifungini <ol><li> Dalle soluzioni di borsa o polvere di farmaci antifungini, preparare una soluzione di lavoro in RPMI-1640. Tipiche concentrazioni massime della soluzione di lavoro sono 1.024 ug / mL per il fluconazolo e 16 ug / mL per amfotericina B 5. Altre concentrazioni possono essere utilizzati per diversi agenti. </li><li> Preparare otto differenti concentrazioni del composto diluendo la soluzione madre in due diluizioni, coprono una gamma con la stima IC 50 del composto nel mezzo. </li><li> Dopo 24 ore di crescita biofilm, utilizzare il micro-distributore per stampare 50 nL di otto differenti concentrazioni dei farmaci, insieme positivo (cioè senza cellule farmaci nei mezzi solo) e negativo (trattato con ipoclorito di sodio per 20 min) controlli, in almeno sei repliche in cima alle biofilm. </li></ol> NOTA: Mantenere una umidità relativa del 100% per impedire l&#39;essiccamento delle macchie mentre addMOZIONE farmaci. <ol start="4"><li> Subito dopo l&#39;aggiunta dei farmaci, incubare il chip con i farmaci in una camera umidificata a 37 ° C per 24 ore. </li><li> Lavare i farmaci fuori dal dunking il CaBChip 3-5 volte per 2 minuti ogni volta in PBS e macchia incubando il BChip Ca con 0,5 pM FUN1 a 37 ° C per 30 min 9. </li><li> Lavare la macchia da inzuppare il BChip Ca 3-5 volte in PBS per 1-3 min ogni dunk, e asciugare la BChip Ca utilizzando un flusso di azoto. Per saperne di intensità di fluorescenza utilizzando uno scanner microarray (GenePix 4100A, Axon Instruments, CA) ad una lunghezza d&#39;onda di 532 nm con un guadagno di 380 PMT. </li><li> Impostare l&#39;intensità di fluorescenza del controllo positivo (nessun farmaco) e morti (candeggina trattati) macchie biofilm al 100% e 0%, rispettivamente. </li><li> Determinare l&#39;inibizione percentuale di riduzione della intensità di fluorescenza (F) rispetto alla media dei punti di controllo utilizzando la seguente equazione </li></ol><img alt="Equazione 2" src="/files/ftp_upload/3845/3845eq2.jpg" /> dove F, F e F max o sono prime intensità di fluorescenza di farmaco-trattato, no-farmaco di controllo e trattati con macchie di candeggina, rispettivamente. Le impostazioni dello scanner sono stati adeguati per ottenere C max e F o di 30000 e 4000 RFU, rispettivamente. <ol start="9"><li> Calcolare la concentrazione inibitoria del 50% o IC 50 inserendo la variabile dell&#39;equazione Hill pendenza utilizzando software Prism GraphPad (La Jolla, CA). </li></ol> 5. Risultati rappresentativi Un rappresentante Ca BChip, costituito da una matrice 48 × 16 di nano-biofilm di C. albicans, è mostrato in Figura 2. La microscopia in campo chiaro mostra una caratteristica architettonica complessiva del nano-biofilm. La digitalizzazione delle immagini al microscopio elettronico della mostra biofilm che le ife fungine sono incorporati nella matrice difibre collagene, che sono circa 2 um e 100 nm di diametro, rispettivamente. Le FUN1 macchiate di immagini di microarray scanner mostrano il lievito e le forme ife, che sono caratteristiche dei biofilm fungine. Le immagini 2D-e 3D-fluorescenza confocale di FUN1 macchiate biofilm può essere visto nelle eterogeneità spaziale, con regioni di cellule metabolicamente attive intercalati nella matrice extracellulare (composto di collagene come il materiale inglobante e probabilmente anche di materiale exopolymeric prodotto dalla cellule biofilm), che non è macchiate di colorante metabolica. Lo spessore del biofilm è stata stimata essere di circa 50 um. Il BChip Ca è stato utilizzato per stimare la suscettibilità di due farmaci antifungini, fluconazolo e amfotericina B, ed i risultati sono mostrati nella Figura 3. Coerentemente con rapporti pubblicati su standard di settore da 96 saggi e piastra, i biofilm sono resistenti contro il fluconazolo 10 e il calcolato IC 50 per<html> amfotericina B è pari a 0,3 mg / ml 11. <img alt="Figura 1" src="/files/ftp_upload/3845/3845fig1.jpg" /> Figura 1. Diagramma di flusso per la fabbricazione di Ca BChip. <img alt="Figura 2" src="/files/ftp_upload/3845/3845fig2.jpg" /> Figura 2. Un&#39;immagine ad alta thoughput Ca BChip, stampato utilizzando un micro-distributore robotizzato e contenente 768 punti su PS-MA rivestite con diapositive. Ogni spot emisferica è di 50 nL in volume, circa 700 micron di diametro, e con distanza di 1,2 millimetri tra le macchie. Altri prodotti (in senso orario) sono microscopia ottica, scanner microarray, microscopia 2D-e 3D-fluorescenza, e la scansione di immagini al microscopio elettronico dei biofilm individuali sul BChip Ca. La figura SEM ad alto ingrandimento di 25.000 x mostra un filamento ifale larghezza di 2 micron. Figura 3 &quot;src =&quot; / files/ftp_upload/3845/3845fig3.jpg &quot;/&gt; Figura 3. Risultati dei test di sensibilità antifungina e determinazione dei valori di IC 50 per (A) fluconazolo e (B) amfotericina B usando Ca BChip. Le immagini al microscopio a fluorescenza di amfotericina B-trattati biofilm sono mostrati in (C). I risultati sono media ± errore standard significa per due chip separati contenenti 10 repliche per ogni condizione.

<ol>
	<li>Edmond, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nosocomial+bloodstream+infections+in+United+States+hospitals:+a+three-year+analysis.">Nosocomial bloodstream infections in United States hospitals: a three-year analysis.</a> Clin. Infect. Dis. 29, 239-244 (1999).</li><li>Ramage, G., Bachmann, S., Patterson, T. F., Wickes, B. L., Lopez-Ribot, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+multidrug+efflux+pumps+in+relation+to+fluconazole+resistance+in+Candida+albicans+biofilms.">Investigation of multidrug efflux pumps in relation to fluconazole resistance in Candida albicans biofilms.</a> J. Antimicrob. Chemother. 49, 973-980 (2002).</li><li>Srinivasan, A., Uppuluri, P., Lopez-Ribot, J., Ramasubramanian, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+High-Throughput+Candida+albicans+Biofilm+Chip.">Development of a High-Throughput Candida albicans Biofilm Chip.</a> PLoS ONE. 6, 19036-19036 (2011).</li><li>Ramage, G., Vandewalle, K., Wickes, B. L., Lopez-Ribot, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+biofilm+formation+by+Candida+albicans.">Characteristics of biofilm formation by Candida albicans.</a> Rev. Iberoam. Micol. 18, 163-170 (2001).</li><li>Pierce, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+reproducible+96-well+plate-based+method+for+the+formation+of+fungal+biofilms+and+its+application+to+antifungal+susceptibility+testing.">A simple and reproducible 96-well plate-based method for the formation of fungal biofilms and its application to antifungal susceptibility testing.</a> Nat. Protoc. 3, 1494-1500 (2008).</li><li>Lee, M. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+cellular+microarray+for+high-throughput+toxicology+assays.">Three-dimensional cellular microarray for high-throughput toxicology assays.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 59-63 (2008).</li><li>Meyerhofer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+resist+films+produced+by+spinning.">Characteristics of resist films produced by spinning.</a> Journal of Applied Physics. 49, (1978).</li><li>Ramage, G., Vande Walle, K., Wickes, B. L., Lopez-Ribot, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardized+method+for+in+vitro+antifungal+susceptibility+testing+of+Candida+albicans+biofilms.">Standardized method for in vitro antifungal susceptibility testing of Candida albicans biofilms.</a> Antimicrob. Agents Chemother. 45, 2475-2479 (2001).</li><li>Chandra, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilm+formation+by+the+fungal+pathogen+Candida+albicans:+Development,+architecture,+and+drug+resistance.">Biofilm formation by the fungal pathogen Candida albicans: Development, architecture, and drug resistance.</a> Journal of Bacteriology. 183, 5385-5394 (2001).</li><li>Jabra-Rizk, M. A., Falkler, W. A., Meiller, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fungal+biofilms+and+drug+resistance.">Fungal biofilms and drug resistance.</a> Emerg. Infect. Dis. 10, 14-19 (2004).</li><li>Tobudic, S., Lassnigg, A., Kratzer, C., Graninger, W., Presterl, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antifungal+activity+of+amphotericin+B,+caspofungin+and+posaconazole+on+Candida+albicans+biofilms+in+intermediate+and+mature+development+phases.">Antifungal activity of amphotericin B, caspofungin and posaconazole on Candida albicans biofilms in intermediate and mature development phases.</a> Mycoses. 53, 208-214 (2010).</li></ol>

Jose L. Lopez-Ribot e Anand K. equità Ramasubramanian proprio in MicrobeHTS Technologies, Inc., che sta sviluppando agenti antifungini. MicrobeHTS Technologies, Inc. ha fornito alcun sostegno finanziario per questi studi.

Abbiamo sviluppato un cell-based ad alta densità microarray, Ca BChip, costituito da volumi nanolitri biofilm di Candida albicans. Il microarray è stata stampata su substrati di vetro modificato, che ha consentito per il fissaggio solido di spot gel di collagene, fornendo idrofobicità necessaria per un non-diffusione, gel emisferica 3D. Un singolo Ca BChip può sostituire circa otto piastre a 96 pozzetti, e chip diversi possono essere stampati e trattati allo stesso tempo. Il chip utilizza ...

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> 3-amminopropiltrietossisilano (APTES) </td><td> Sigma-Aldrich </td><td> 440140 </td></tr><tr><td> Polistirene-Co-anidride maleica (PS-MA) </td><td> Sigma-Aldrich </td><td> 426946 </td></tr><tr><td> Microscopia diapositive di vetro </td><td> Fisher Scientific </td><td> 12-549-3 </td></tr><tr><td> Coda di tipo Rat collagene I </td><td> BD Biosciences </td><td> 354236 </td></tr><tr><td> Robotic micro-distributore </td><td> Omnigrid Micro </td><td> MICROSYS4000/4100A </td></tr><tr><td> Microarray Scanner </td><td> GenePix Personal 4100A </td><td> GENEPIX4100A </td></tr><tr><td> Ibridazione Cassette </td><td> ArrayIt Corporation </td><td> AHCXD </td></tr><tr><td> FUN1 [2-clororo-4-(2,3-diidro-3-metil-(benzo -1,3-tiazol-2-il)-metilidene)-1-fenil quinoliniumiodide] </td><td> Invitrogen Corp. </td><td> F-7030 </td></tr><tr><td> Fluconazolo </td><td> Sicor Pharmaceuticals, Inc. </td><td> J02AC01 </td></tr><tr><td> Amfotericina B </td><td> Sigma </td><td> A2411 </td></tr><tr><td> RPMI-1640 </td><td> Mediatech, Inc. </td><td> 50-020-PC </td></tr><tr><td> Ceramic Tip 190 micron orifizio </td><td> Digilab </td><td> 60020441-00 </td></tr><tr><td> GraphPad Prism Software </td><td> GraphPad Software, Inc. </td><td></td></tr><tr><td> GenePix Pro V4.1 </td><td> Molecular Devices </td><td></td></tr></tbody></table>

candida albicans biofilm chip (cabchip) for high-throughput antifungal drug screening

Candida albicans rimane il principale agente eziologico della candidosi, che attualmente rappresenta la quarta più comune infezione nosocomiale sangue negli ospedali degli Stati Uniti 1. Queste infezioni opportunistiche rappresentano una crescente minaccia per un numero crescente di individui compromessi, e portare i tassi di mortalità eccessivamente elevati. Ciò è in parte dovuto al limitato arsenale di farmaci antifungini, ma anche alla comparsa di resistenza agli agenti antifungini più comunemente usati. A complicare ulteriormente trattamento è il fatto che una maggioranza di manifestazioni di candidosi sono associati con la formazione di biofilm, e le cellule all&#39;interno di questi biofilm mostrano livelli aumentati di resistenza agli clinicamente più utilizzati agenti antifungini 2. Qui si descrive lo sviluppo di una alta densità microarray che consiste di C. albicans nano-biofilm, che abbiamo chiamato Ca BChip 3. Brevemente, un micro-distributore robotico viene utilizzato to lievito cellule stampa di C. albicans su una substrato solido. Durante la stampa, le cellule di lievito sono racchiusi in una matrice tridimensionale usando un volume basso quanto 50 nL e immobilizzato su un substrato di vetro con un idoneo rivestimento. Dopo la stampa iniziale, le diapositive vengono incubate a 37 ° C per 24 ore per consentire per sviluppo biofilm. Durante questo periodo le macchie crescere in pienamente sviluppati &quot;nano-biofilm&quot; che visualizzare tipici caratteristiche strutturali e fenotipica associati con mature C. albicans biofilm (complessità vale a dire morfologica, tre l&#39;architettura dimensionale e resistenza ai farmaci) 4. Complessivamente, il BChip Ca è composto di ~ 750 biofilm equivalenti e spazialmente distinto; con il vantaggio supplementari che chip multipli può essere stampato e elaborati contemporaneamente. La vitalità cellulare è stimato misurando l&#39;intensità fluorescente di FUN1 metabolica macchia utilizzando un scanner microarray. Questo chip fungina è l&#39;ideale per use in vero high-throughput screening per la scoperta di nuovi farmaci antifungini. Rispetto agli standard attuali (cioè il 96-well modello piastra microtitolo della formazione di biofilm 5), i principali vantaggi del chip biofilm fungine sono automazione, miniaturizzazione, risparmi nei costi e quantità dei reagenti e dei tempi di analisi, nonché l&#39;eliminazione di lavoro operazioni intensive. Noi crediamo che il chip in modo significativo ad accelerare il processo di scoperta di nuovi farmaci antifungini.

Watch this Scientific Journal Video about Candida albicans Biofilm Chip CaBChip for High-throughput Antifungal Drug Screening at JoVE.com

Candida albicans Biofilm Chip CaBChip for High-throughput Antifungal Drug Screening

Abbiamo sviluppato una piattaforma ad alta densità microarray costituito da nano-3D di biofilm<em&gt; C. albicans</em&gt; Chiamato<em&gt; Ca</em&gt; BChip. Il profilo suscettibilità di farmaci testati su una<em&gt; Ca</em&gt; BChip è paragonabile a quello tradizionale a 96 pozzetti modello di piastra, suggerendo che il chip funghi è ideale per una vera high-throughput screening di farmaci antifungini.

Candida albicans Biofilm Chip (Ca BChip) per High-throughput screening farmaco antifungino

Candida  albicans remains the main etiological agent of candidiasis, which  currently represents the fourth most common nosocomial bloodstream infection ...

candida-albicans-biofilm-chip-cabchip-for-high-throughput-antifungal

Research

JoVE Journal

Bioengineering

Candida albicans Biofilm Chip (CaBChip) for High-throughput Antifungal Drug Screening

13.6K Views.  University of Texas at San Antonio. Abbiamo sviluppato una piattaforma ad alta densità microarray costituito da nano-3D di biofilm C. albicans Chiamato Ca BChip. Il profilo suscettibilità di farmaci testati su una Ca BChip è paragonabile a quello tradizionale a 96 pozzetti modello di piastra, suggerendo che il chip funghi è ideale per una vera high-throughput screening di farmaci antifungini.

Video: Candida albicans Biofilm Chip CaBChip for High-throughput Antifungal Drug Screening

Cell-based Calcium Assay for Medium to High Throughput Screening of TRP Channel Functions using FlexStation 3

The Molecular Devices' FlexStation 3 is a benchtop multi-mode microplate reader capable of automated fluorescence measurement in multi-well plates. It is ideal for medium- to high-throughput screens in academic settings. It has an integrated fluid transfer module equipped with a multi-channel pipetter and the machine reads one column at a time to monitor fluorescence changes of a variety of fluorescent reagents. For example, FlexStation 3 has been used to study the function of Ca2+-permeable ion channels and G-protein coupled receptors by measuring the changes of intracellular free Ca2+ levels. Transient receptor potential (TRP) channels are a large family of nonselective cation channels that play important roles in many physiological and pathophysiological functions. Most of the TRP channels are calcium permeable and induce calcium influx upon activation. In this video, we demonstrate the application of FlexStation 3 to study the pharmacological profile of the TRPA1 channel, a molecular sensor for numerous noxious stimuli. HEK293 cells transiently or stably expressing human TRPA1 channels, grown in 96-well plates, are loaded with a Ca2+-sensitive fluorescent dye, Fluo-4, and real-time fluorescence changes in these cells are measured before and during the application of a TRPA1 agonist using the FLEX mode of the FlexStation 3. The effect of a putative TRPA1 antagonist was also examined. Data are transferred from the SoftMax Pro software to construct concentration-response relationships of TRPA1 activators and inhibitors.

This video provides a detailed protocol for studying the pharmacological profile of human TRPA1 channels using FlexStation 3. The protocol covers details of cell preparation, dye loading and operation of the microplate reader, FlexStation 3.

Saggio basato su cellule di calcio per le medie e High Throughput Screening del PRT Funzioni canale utilizzando FlexStation 3

The Molecular Devices' FlexStation 3 is a benchtop multi-mode microplate reader capable of automated fluorescence measurement in multi-well plates.  It is ...

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Bioingegneria

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

Microarrays polimerici per Discovery High Throughput di Biomateriali

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold nanoclusters (Au NCs) within the drug-loaded protein, based on the differential fluorescence signal emitted by the Au NCs. Albumin proteins such as human serum albumin (HSA) and bovine serum albumin (BSA) are selected as the model proteins. Four small molecular drugs (e.g., ibuprofen, warfarin, phenytoin, and sulfanilamide) of different binding affinities to the albumin proteins are tested. It was found that the formation rate of fluorescent Au NCs inside the drug loaded albumin protein under denaturing conditions (i.e., 60 &#176;C or in the presence of urea) is slower than that formed in the pristine protein (without drugs). Moreover, the fluorescent intensity of the as-formed NCs is found to be inversely correlated to the binding affinities of these drugs to the albumin proteins. Particularly, the higher the drug-protein binding affinity, the slower the rate of Au NCs formation, and thus a lower fluorescence intensity of the resultant Au NCs is observed. The fluorescence intensity of the resultant Au NCs therefore provides a simple measure of the relative binding strength of different drugs tested. This method is also extendable to measure the specific drug-protein binding constant (KD) by simply varying the drug content preloaded in the protein at a fixed protein concentration. The measured results match well with the values obtained using other prestige but more complicated methods.

A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

Una rapida e quantitativa metodo fluorimetrico per lo screening di stupefacenti piccole molecole di proteine ​​Targeting

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold ...

Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native context at a molecular level. The technique involves initially preserving the sample in its native state by rapidly freezing the specimen in vitreous ice, then collecting a series of micrographs from different angles at high magnification, and finally computationally reconstructing a 3-D density map. The frozen-hydrated specimen is extremely sensitive to the electron beam and so micrographs are collected at very low electron doses to limit the radiation damage. As a result, the raw cryo-tomogram has a very low signal to noise ratio characterized by an intrinsically noisy image. To better visualize subjects of interest, conventional imaging analysis and sub-tomogram averaging in which sub-tomograms of the subject are extracted from the initial tomogram and aligned and averaged are utilized to improve both contrast and resolution. Large datasets of tilt-series are essential to understanding and resolving the complexes at different states, conditions, or mutations as well as obtaining a large enough collection of sub-tomograms for averaging and classification. Collecting and processing this data can be a major obstacle preventing further analysis. Here we describe a high-throughput cryo-ET protocol based on a computer-controlled 300kV cryo-electron microscope, a direct detection device (DDD) camera and a highly effective, semi-automated image-processing pipeline software wrapper library tomoauto developed in-house. This protocol has been effectively utilized to visualize the intact type III secretion system (T3SS) in Shigella flexneri minicells. It can be applicable to any project suitable for cryo-ET.

We present a protocol on how to utilize high-throughput cryo-electron tomography to determine high resolution in situ structures of molecular machines. The protocol permits large amounts of data to be processed, avoids common bottlenecks and reduces resource downtime, allowing the user to focus on important biological questions.

Usando Tomoauto: un protocollo per High-throughput automatizzato Cryo-elettrone Tomografia

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

Una piattaforma Microfluidic per l&#39;isolamento Single-cell high-throughput e cultura

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

High-throughput Identification of Bacteria Repellent Polymers for Medical Devices

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as contributing to antimicrobial resistance. One possible avenue for the reduction of device-associated infections is the identification of bacteria-repellent polymer coatings for these devices, which would prevent bacterial binding at the initial attachment step. A method for the identification of such repellent polymers, based on the parallel screening of hundreds of polymers using a microarray, is described here. This high-throughput method resulted in the identification of a range of promising polymers that resisted binding of various clinically relevant bacterial species individually and also as multi-species communities. One polymer, PA13 (poly(methylmethacrylate-co-dimethylacrylamide)), demonstrated significant reduction in attachment of a number of hospital isolates when coated onto two commercially available central venous catheters. The method described could be applied to identify polymers for a wide range of applications in which modification of bacterial attachment is important.

A high-throughput microarray method for the identification of polymers which reduce bacterial surface binding on medical devices is described.

High-throughput identificazione di batteri Polimeri repellente per dispositivi medici

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as ...

Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these procedures in basic research and preclinical drug development, it is important to develop protocols for automated generation and analysis under standardized conditions. Here, we present a technique to generate engineered heart tissue (EHT) from cardiomyocytes of different species (rat, mouse, human). The technique relies on the assembly of a fibrin-gel containing dissociated cardiomyocytes between elastic polydimethylsiloxane (PDMS) posts in a 24-well format. Three-dimensional, force-generating EHTs constitute within two weeks after casting. This procedure allows for the generation of several hundred EHTs per week and is technically limited only by the availability of cardiomyocytes (0.4-1.0 x 106/EHT). Evaluation of auxotonic muscle contractions is performed in a modified incubation chamber with a mechanical interlock for 24-well plates and a camera placed on top of this chamber. A software controls a camera moved on an XYZ axis system to each EHT. EHT contractions are detected by an automated figure recognition algorithm, and force is calculated based on shortening of the EHT and the elastic propensity and geometry of the PDMS posts. This procedure allows for automated analysis of high numbers of EHT under standardized and sterile conditions. The reliable detection of drug effects on cardiomyocyte contraction is crucial for cardiac drug development and safety pharmacology. We demonstrate, with the example of the hERG channel inhibitor E-4031, that the human EHT system replicates drug responses on contraction kinetics of the human heart, indicating it to be a promising tool for cardiac drug safety screening.

Here, we show the generation of human engineered heart tissue from induced pluripotent stem cells (hiPSC)-derived cardiomyocytes. We present a method to analyze contraction force and exemplary alteration of contraction pattern by the hERG channel inhibitor E-4031. This method shows high level of robustness and suitability for cardiac drug screening.

Automated Analysis Contrazione di ingegneria dei tessuti umani Cuore per lo screening cardiaco Drug Safety

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

Microtissues 3D per la terapia rigenerativa iniettabile e droga di High-throughput Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

Use of High-Throughput Automated Microbioreactor System for Production of Model IgG1 in CHO Cells

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of experimental conditions while minimizing potential process variability. Applications of this approach include: clone screening, temperature and pH shifts,&#160;media and supplement optimization. Furthermore, the small reactor volumes are conducive to large Design of Experiments that investigate a wide range of conditions. This allows upstream processes to be significantly optimized before scale-up where experimentation is more limited in scope due to time and economic constraints. Automated microscale bioreactor systems offer various advantages over traditional small scale cell culture units, such as shake flasks or spinner flasks. However, during pilot scale process development&#160;significant care must be taken to ensure that these advantages are realized. When run with care, the system can enable high level automation, can be programmed to run DOE&#39;s with a higher number of variables&#160;and can reduce sampling time when integrated with a nutrient analyzer or cell counter. Integration of the expert-derived heuristics presented here, with current automated microscale bioreactor experiments can minimize common pitfalls that hinder meaningful results. In the extreme, failure to adhere to the principles laid out here can lead to equipment damage that requires expensive repairs. Furthermore, the microbioreactor systems have small culture volumes&#160;making characterization of cell culture conditions difficult. The number and amount of samples taken in-process in batch mode culture is limited as operating volumes cannot fall below 10 mL. This method will discuss the benefits and drawbacks of microscale bioreactor systems.

A detailed protocol for the concurrent operation of 48 parallel cell cultures under varied conditions in a microbioreactor system is presented. Cell culture process, harvest and subsequent antibody titer analysis are described.

Uso di High-Throughput Microbioreactor sistema automatizzato per la produzione del modello IgG1 in cellule CHO

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of ...

Applications for Open Source Microplate-Compatible Illumination Panels

Microplates are commonly used in the modern laboratory environment for a wide variety of tasks both in small-scale laboratory benchtop operations as well as large-scale high-throughput screening (HTS) campaigns. Though laboratory automation has greatly increased the utility of microplates there remain instances where automation-based instrumentation is not feasible, cost-effective or compatible with microplate formatting needs. In these cases, microplates must be manually prepared. Problematic to manual microplate manipulations is that a number of difficulties can arise pertaining to the accurate tracking of sample operations, data record keeping and quality control (QC) inspection for well artifacts or formatting errors. As microplate well densities increase (i.e., 96-well, 384-well, 1536-well) the potential for introducing errors also drastically increases.&#160; Moreover, for small bench-top laboratory operations there exists a need to improve the ease and accuracy of sample handling in a cost-effective fashion. Herein, we describe a system that acts as a semi-automated pipetting guide referred to as the Microplate Assistive Pipetting Light Emitter (M.A.P.L.E.). &#160;M.A.P.L.E. has multiple uses for supporting compound hit-picking and microplate preparation for assay development in high-throughput screening or laboratory benchtop operations, as well as QC/quality assurance (QA) diagnostic evaluation of microplate quality or visualizing well formatting errors.

Microplate Assistive Pipetting Light Emitter (M.A.P.L.E.) is a computer-driven device that systematically illuminates microtiter wells to provide guidance for the manual preparation of microplates.&#160; M.A.P.L.E. improves the accuracy of microplate preparation while automating data recordkeeping. &#160;In addition, it can assist with examining microplate quality or aid in the detection of errors.&#160; &#160;

Applicazioni per pannelli di illuminazione compatibili con microplacca open source

Microplates are commonly used in the modern laboratory environment for a wide variety of tasks both in small-scale laboratory benchtop operations as well ...