Questo lavoro è stato sostenuto in parte dalla National Science Foundation (cbet 0.846.453) e il National Institutes of Health (1R01GM089999).

1. Fabbricazione di maestri silicio utilizzando le normali SU-8 fotolitografia 1 (non mostrato in questo video). <ol><li> Uso standard SU-8 metodi di fotolitografia per creare un SU-8 &quot;maestri&quot; (SU-8 2050, MicroChem, Newton, MA) per la realizzazione delle diverse componenti (membrane PDMS di diverso spessore, co-coltura da camera) in una camera sterile. Stampi maestro tale può essere fabbricato in qualunque impianto di microfabbricazione (ad esempio, Stanford Microfluidica Fonderia; <a href="http://thebigone.stanford.edu/foundry/">http://thebigone.stanford.edu/foundry/</a> ). </li><li> Prima della replica PDMS, esporre il SU-8 maestri fluorosilane a vapore in un essiccatore collegato a una fonte di vuoto per facilitare la liberazione facile PDMS dal master. Aggiungere una goccia di fluorosilane ad un tovagliolo di carta e si applicano a vuoto per un min. Rimuovere il vuoto e attendere 30 minuti per la deposizione di fluorosilane. Tenere il SU-8 master in un contenitore chiuso per un uso futuro. </li></ol> 2. Stampaggio replica del PDMS da SU-8 master 2 <ol><li> Lo strato fluidico e il livello di controllo sono realizzati per stampaggio replica del PDMS da SU-8 master. </li><li> Miscelare il PDMS pre-polimero e cross-linker ad un peso 10:1. rapporto e Degas in un essiccatore per 1 ora (o fino a quando le bolle d&#39;aria vengono rimossi dalla miscela PDMS). </li><li> Posizionare il SU-8 master in una capsula di Petri e con attenzione versare il composto PDMS sulla parte superiore del SU-8 master. Degassare la PDMS di nuovo. </li><li> Spin lo stampo a 1200 rpm per un minuto per ottenere uno spessore uniforme di circa 100μm PDMS sullo stampo. </li><li> Riscaldare la capsula di Petri contenente il PDMS e il SU-8 stampo maestro a 80 ° C per un&#39;ora per curare il PDMS. </li><li> Rimuovere il curato PDMS coperto SU-8 maestro dalla piastra. </li><li> Sbucciare lo stampo PDMS dal SU-8 master. </li><li> Praticare quattro fori nello stampo PDMS con un calibro 20 ago smussato-end per due ingressi batteri, una presa e una porta pneumatica. </li></ol> 3. Incollaggio di multistrato dispositivi PDMS: Questo dispositivo è assemblato con tre strati, uno strato superiore che contiene il canale principale, uno strato pneumatico medio, e uno strato inferiore di batteri che contiene i canali per i batteri. I tre strati sono assemblati come descritto di seguito. <ol><li> Bagnare le membrane PDMS con metanolo e con attenzione pelarle dalla loro SU-8 stampo master utilizzando una pinza pulita. Membrana posto su un foglio di teflon. </li><li> Posizionare la membrana pneumatica e lo strato di fluido in una camera di plasma di ossigeno. Accendere il plasma per 40 sec (ossigeno pressione 10 SCCM, 100W di potenza RF). </li><li> Dopo l&#39;esposizione al plasma, allineare (entro 10 minuti) la membrana sul fondo PDMS canale utilizzando pinze. L&#39;aggiunta di metanolo sulla membrana e gli strati PDMS aiuta la libera circolazione degli strati, e rende più facile l&#39;allineamento. In questo caso, il dispositivo è posizionato su un pezzo di garza per consentire la fuga di vapori di metanolo. </li><li> Curare gli strati PDMS allineati su una piastra a 80 ° C per 8 ore </li><li> Dopo l&#39;incollaggio della membrana pneumatica sullo strato inferiore, ripetere i passaggi 3,2-3,4 per legare il canale in alto sul dispositivo montato composito PDMS. </li></ol> 4. Montaggio del dispositivo PDMS 3,4 <ol><li> Praticare dei fori nei porti di ingresso e di uscita utilizzati per la semina delle cellule eucariotiche e batteri. </li><li> Collegare il tubo Tygon (0,01 pollici OD) per le porte e far funzionare lo strato pneumatico tirando con una siringa da 20 mL. </li><li> Posizionare il modello PDMS e una lastra di vetro pre-lavaggio in una camera di plasma di ossigeno. Accendere il plasma per 20 secondi (pressione di ossigeno 20 SCCM, RF di potenza 300W) senza applicare il vuoto. </li><li> Al fine di evitare il contatto tra la parete dell&#39;isola (PDMS) l&#39;adesione al vetrino, immediatamente collegare una siringa al dispositivo e applicare vuoto tirando la siringa e tenerlo in posizione. </li><li> Premere delicatamente il modello PDMS sul vetro (entro 2 minuti). </li><li> Curare il dispositivo PDMS a 80 ° C per 10 minuti. </li></ol> 5. Trattare la superficie di vetro con fibronectina <ol><li> Sterilizzare dispositivo ai raggi UV per 15 minuti dopo il collegamento di tubi Tygon ad ogni porta. </li><li> Compilare e il flusso del sterilizzati PBS nel canale per rimuovere i detriti nel canale. Applicare flusso per 10 minuti per rimuovere le bolle d&#39;aria. Le bolle d&#39;aria può facilmente sfuggire al PDMS a causa della sua permeabilità ai gas. </li><li> Riempire un soluzione colorante colore nel canale pneumatico per prevenire la formazione di bolle d&#39;aria nel canale. </li><li> Per facilitare la semina delle cellule eucariotiche sul vetro, introdurre un 1 mg / ml soluzione della fibronectina nel dispositivo e incubare per 40 minuti a 37 ° C. </li><li> Lavare la fibronectina in eccesso con 1 ml di terreni di crescita. </li></ol> 6. Formazione e misura di commensali E. coli in biofilmisole <ol><li> Diluire i batteri commensali (ad esempio, E. coli BW25113/pCM18) nei media M9 minimo diametro esterno 600 ~ 1 per formare un biofilm commensale in camere chiuse / isole. </li><li> Chiudere il muro isola applicando positiva-liquido pressione. Un fatto in casa aria-liquido convertitore di pressione viene utilizzato per prevenire la formazione di bolle d&#39;aria nel canale. </li><li> Introdurre il commensale E. coli nelle isole con la valvola chiusa, e permettere ai batteri di attaccare per 1 ora a 32 ° C. </li><li> Lavare i batteri distaccato e profumato una sospensione batterica diluita (OD 600 ~ 0,05) a 0.25mL/hr per 12 ore. </li><li> Sciacquare il biofilm con freschi terreni di coltura DMEM per rimuovere i batteri vagamente-attached e prepararsi per il fissaggio delle cellule eucariotiche. </li><li> Immagine del biofilm utilizzando una Leica TCS SP5 microscopio confocale a scansione laser (Bannockburn, IL), e visualizzare l&#39;architettura biofilm utilizzando il software IMARIS 5. </li></ol> 7. Semina cellule eucariotiche intorno alle isole batteriche 6-8 <ol><li> Trypsinize cellule HeLa dal fiasco del tessuto culturale e risospendere in DMEM media per una densità di semina di 5 x 10 5 cellule / ml. </li><li> Introdurre cellule HeLa nel dispositivo ad un flusso di 2 mL / min con una pompa a siringa Picoplus (Harvard Apparattus, MA). Assicurarsi che il muro isola è abbassato in modo che il biofilm batterico è separato dalla regione delle cellule HeLa. Questa operazione deve essere eseguita sul palco di un microscopio ottico in modo che l&#39;uniformità di semina può essere determinato. </li><li> Fissare la presa cellula per ridurre il movimento della sospensione cellulare nel canale e incubare per 6 ore a 37 ° C in un 5% di CO 2 incubatore. </li><li> Profumato il dispositivo con mezzo di crescita a 0,5 ml / ora per rimuovere le cellule senza legami. </li><li> Aggiornare i media DMEM per HeLa e commensali E. coli ogni 6 ore. </li></ol> 8. Introduzione di batteri patogeni nell&#39;isola e l&#39;esposizione delle cellule eucariotiche <ol><li> Preparare E. coli O157: H7 (EHEC) per infettare le cellule HeLa in una molteplicità di infezione (moi) di 100:1 200:1 (numero di EHEC per cellula HeLa). </li><li> Sciacquare e lavare i batteri commensali liberamente iscritti. </li><li> Introdurre una sospensione diluita EHEC (OD 600 ~ 0,1) nelle isole. </li><li> Incubare dispositivo a 37 ° C in un 5% di CO 2 incubatore per 6 ore senza flusso per consentire EHEC di essere esposti a segnali presenti nel biofilm commensale. </li><li> Sollevare il muro PDMS per esporre EHEC nelle isole commensale di cellule HeLa per 6 ore. </li><li> Sciacquare il canale con PBS e colorare per le cellule vive e morte con il kit Live / Dead (L3224, Invitrogen, CA). </li><li> Posizioni di immagini su un microscopio a fluorescenza Zeiss Axiovert 200M e stimare il numero di cellule vive e morte. </li></ol> 9. Risultati Rappresentante 9 La sequenza di eventi nelle infezioni del tratto gastrointestinale è stato imitato con lo sviluppo di un monostrato di cellule HeLa e un commensale E. coli biofilm ed esponendo EHEC al biofilm commensale prima infezione di cellule HeLa. I passi necessari per la fabbricazione della microfluidica co-coltura modello sono mostrati in figura 1. Figura 2A mostra un 3-dimensionale prestazione del co-coltura dispositivo. Le due regioni in co-coltura dispositivo della camera di coltura delle cellule eucariotiche e l&#39;isola batterica sono mostrati in figura 2B con coloranti diversi colori (viola per le regioni degli eucarioti e verde per l&#39;isola batterica). La fattibilità di isolare le regioni coltura batterica dalla regione circostante eucariote è mostrato nella figura 2C utilizzando coloranti colori. Figura 3 mostra i risultati rappresentante di co-coltura delle cellule epiteliali e batteri. 3A figura mostra colonizzazione del biofilm commensale (verde) isola EHEC (rosso). Figura 3B mostra la giustapposizione di cellule epiteliali e commensali E. coli con EHEC nell&#39;isola batterica. Il RFP esprimendo EHEC e batteri che esprimono GFP commensali sono localizzati nell&#39;isola quando la parete PDMS si abbassa, che illustrano la fattibilità di isolare l&#39;isola batterica da cellule epiteliali. <img src="/files/ftp_upload/1749/1749fig1.jpg" alt="Figura 1" /> Figura 1: schema di semina cella nel modello di co-cultura. (1) Il muro PDMS si abbassa a formare un&#39;isola, batteri commensali sono introdotti nel l&#39;isola, e fibronectina è scorreva intorno all&#39;isola. (2) le cellule HeLa sono seminate nelle regioni che circondano l&#39;isola. (3) Dopo cellule HeLa raggiungere la confluenza e il biofilm commensale si è sviluppata, EHEC viene introdotto l&#39;isola. (4) Il muro PDMS è sollevatofino a esporre le cellule HeLa che circondano l&#39;isola di EHEC. Inserto mostra i dettagli del funzionamento della valvola. <img src="/files/ftp_upload/1749/1749fig2a.jpg" alt="Figura 2a" /> <img src="/files/ftp_upload/1749/1749fig2bc.jpg" alt="Figura 2BC" /> Figura 2: modello Microfluidic per la co-coltura delle cellule epiteliali e batteri. (A) tridimensionale resa dei co-cultura dispositivo che indica le regioni cattura pneumatico-attuato per la formazione di isole batteriche tra le cellule epiteliali. Ogni isola batterica (1200 micron di diametro e 1000 micron a parte) ha un ingresso separato e uscita per la fornitura di mezzi di comunicazione la crescita e la rimozione dei rifiuti dall&#39;isola. (B) Micrografia della co-coltura dispositivo con coloranti a colori che mostrano le diverse regioni (zona delle cellule epiteliali, isole batterica). (C) La fedeltà del sistema di cattura pneumatico è mostrato abbassando il muro PDMS (pannello di sinistra), utilizzando un pneumatico attivato canale (blu), l&#39;introduzione di porpora nelle isole chiuso dell&#39;isola, e che scorre colorante giallo intorno ad esso per 48 h. Quando il muro si alza PDMS (pannello di destra), la regione insulare è esposto al colorante giallo circostante. Barra di scala rappresenta 500 micron. <img src="/files/ftp_upload/1749/1749fig3.jpg" alt="Figura 3" /> Figura 3: Co-coltura di cellule HeLa e batteri. (A) immagine di fluorescenza di RFP che esprimono EHEC e GFP che esprimono E. coli BW25113 in isola. (B) Sovrapposizione di trasmissione, verde e rosso immagini di fluorescenza nel dispositivo. Barra di scala rappresenta 200 micron.

<ol>
	<li>McDonald, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prototyping+of+microfluidic+devices+in+poly(dimethylsiloxane)+using+solid-object+printing.">Prototyping of microfluidic devices in poly(dimethylsiloxane) using solid-object printing.</a> Anal Chem. 74, 1537-1545 (2002).</li><li>Jeon, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+fabrication+of+integrated+passive+valves+and+pumps+for+flexible+polymer+3-dimensional+microfluidic+systems.">Design and fabrication of integrated passive valves and pumps for flexible polymer 3-dimensional microfluidic systems.</a> Biomed Microdevices. 4, 117-121 (2002).</li><li>Baek, J. Y., Park, J. Y., Ju, J. I., Lee, T. S., Lee, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pneumatically+controllable+flexible+and+polymeric+microfluidic+valve+fabricated+via+in+situ+development.">A pneumatically controllable flexible and polymeric microfluidic valve fabricated via in situ development.</a> J Micromech Microeng. 15, 1015-1020 (2005).</li><li>Grover, W. H., Ivester, R. H., Jensen, E. C., Mathies, R. A., A, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+multiplexed+control+of+latching+pneumatic+valves+using+microfluidic+logical+structures.">Development and multiplexed control of latching pneumatic valves using microfluidic logical structures.</a> Lab Chip. 6, 623-631 (2006).</li><li>Lee, J., Jayaraman, A., Wood, T. K., K, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Indole+is+an+inter-species+biofilm+signal+mediated+by+SdiA.">Indole is an inter-species biofilm signal mediated by SdiA.</a> BMC Microbiol. 7, 42-42 (2007).</li><li>Hsu, C. H., Folch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+devices+with+tunable+topographies.">Microfluidic devices with tunable topographies.</a> Appl Phys Lett. 86, 023508-023508 (2005).</li><li>Hui, E. E., Bhatia, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micromechanical+control+of+cell-cell+interactions.">Micromechanical control of cell-cell interactions.</a> Proc Natl Acad Sci USA. 104, 5722-5726 (2007).</li><li>Lee, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrating+sensing+hydrogel+microstructures+into+micropatterned+hepatocellular+cocultures.">Integrating sensing hydrogel microstructures into micropatterned hepatocellular cocultures.</a> Langmuir. 25, 3880-3886 (2009).</li><li>Kim, J., Hegde, M., Jayaraman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-culture+of+epithelial+cells+and+bacteria+for+investigating+host-pathogen+interactions.">Co-culture of epithelial cells and bacteria for investigating host-pathogen interactions.</a> Lab-on-Chip. , (2009).</li></ol>

Test convenzionale per attaccamento patogeno e la colonizzazione utilizzare un monostrato di cellule eucariote in piastre di coltura dei tessuti in cui vengono aggiunti gli agenti patogeni. Questi modelli non sono fisiologicamente rilevanti in quanto non contengono un commensale biofilm batterici sviluppato su cellule eucariotiche. Semplice aggiunta di un pre-cresciuta coltura batterica alle cellule eucariotiche è improbabile che possa portare a questa conformazione come biofilm sono altamente organizzate strutture che...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>SU-8 2050</td>
<td>&nbsp;</td>
<td>Microchem Corp, MA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>high-resolution (16,256 dpi) photolithography mask</td>
<td>&nbsp;</td>
<td>Fineline-Imaging Inc, CO</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Trichloro(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)silane</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>77279</td>
<td>&nbsp;</td>
<tr>
<td>PDMS</td>
<td>&nbsp;</td>
<td>Dow Corning, WI</td>
<td>184 SIL ELAST KIT 0.5KG</td>
<td>&nbsp;</td>
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>SH30002.02</td>
<td>&nbsp;</td>
<tr>
<td>Programmable spin coater</td>
<td>&nbsp;</td>
<td>Laurell Tech Corp</td>
<td>WS0650S</td>
<td>&nbsp;</td>
<tr>
<td>Mask aligner</td>
<td>&nbsp;</td>
<td>Neutronix-Quintel, PA</td>
<td>Q4000</td>
<td>&nbsp;</td>
<tr>
<td>Oxygen plasma etcher</td>
<td>&nbsp;</td>
<td>March Plasma System, CA</td>
<td>CS-1701</td>
<td>&nbsp;</td>
<tr>
<td>Syringe pump</td>
<td>&nbsp;</td>
<td>Harvard Apparatus, MA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Live/Dead Viability/Cytotoxicity Kit</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>L-3224</td>
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microfluidic co-culture of epithelial cells and bacteria for investigating soluble signal-mediated interactions

L&#39;essere umano gastrointestinale (GI) è un ambiente unico in cui cellule epiteliali intestinali e non patogeni (commensale) batteri coesistono. E &#39;stato proposto che il microambiente che il patogeno incontra nello strato commensali è importante per determinare l&#39;entità della colonizzazione. Metodi di coltura per lo studio attuale colonizzazione dei patogeni non sono adatti per indagare questa ipotesi in quanto non consentono di co-coltura di batteri e cellule epiteliali in un modo che simula il microambiente del tratto gastrointestinale. Qui descriviamo una microfluidica co-coltura modello che consente la cultura indipendente delle cellule eucariotiche e batteri, e testare l&#39;effetto del microambiente commensale sulla colonizzazione dei patogeni. La co-coltura modello è dimostrato attraverso lo sviluppo di un commensale<em&gt; Escherichia coli</emBiofilm&gt; tra le cellule HeLa, seguito da introduzione di enteroemorragica<em&gt; E. coli</em&gt; (EHEC) nell&#39;isola commensali, in una sequenza che riproduce la sequenza degli eventi nelle infezioni del tratto gastrointestinale.

Watch this Scientific Journal Video about Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions at JoVE.com

Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

Questo protocollo descrive un microfluidica co-coltura modello per la cultura contemporanea e localizzato delle cellule epiteliali e batteri. Questo modello può essere utilizzato per indagare il ruolo dei diversi segnali solubili molecolare sulla patogenesi e schermo l&#39;efficacia del putativo ceppi batterici probiotici.

Microfluidica Co-coltura di cellule epiteliali e batteri per le indagini solubile del segnale mediato da Interazioni

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has ...

microfluidic-co-culture-epithelial-cells-bacteria-for-investigating

Research

JoVE Journal

Biology

14.0K Views.  Texas A&M University. Questo protocollo descrive un microfluidica co-coltura modello per la cultura contemporanea e localizzato delle cellule epiteliali e batteri. Questo modello può essere utilizzato per indagare il ruolo dei diversi segnali solubili molecolare sulla patogenesi e schermo l'efficacia del putativo ceppi batterici probiotici.

Video: Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

Un neuronale e astrociti Co-Cultura Assay per l&#39;analisi dei contenuti elevati di neurotossicità

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Ricerca

Biologia

A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to six independent experiments in parallel for higher-throughput. We developed a new fabrication method to create microfluidic devices having both micro and macro scale structures within the same device through a single soft-lithography process, enabling mass fabrication with good repeatability.
The multi-compartment microfluidic co-culture platform is composed of one soma compartment for neurons and six axon/glia compartments for oligodendrocytes (OLs). The soma compartment and axon/glia compartments are connected by arrays of axon-guiding microchannels that function as physical barriers to confine neuronal soma in the soma compartment, while allowing axons to grow into axon/glia compartments. OLs loaded into axon/glia compartments can interact only with axons but not with neuronal soma or dendrites, enabling localized axon-glia interaction studies. The microchannels also enabled fluidic isolation between compartments, allowing six independent experiments to be conducted on a single device for higher throughput.
Soft-lithography using poly(dimethylsiloxane) (PDMS) is a commonly used technique in biomedical microdevices. Reservoirs on these devices are commonly defined by manual punching. Although simple, poor alignment and time consuming nature of the process makes this process not suitable when large numbers of reservoirs have to be repeatedly created. The newly developed method did not require manual punching of reservoirs, overcoming such limitations. First, seven reservoirs (depth: 3.5 mm) were made on a poly(methyl methacrylate) (PMMA) block using a micro-milling machine. Then, arrays of ridge microstructures, fabricated on a glass substrate, were hot-embossed against the PMMA block to define microchannels that connect the soma and axon/glia compartments. This process resulted in macro-scale reservoirs (3.5 mm) and micro-scale channels (2.5 &mu;m) to coincide within a single PMMA master. A PDMS replica that served as a mold master was obtained using soft-lithography and the final PDMS device was replicated from this master.
Primary neurons from E16-18 rats were loaded to the soma compartment and cultured for two weeks. After one week of cell culture, axons crossed microchannels and formed axonal only network layer inside axon/glia compartments. Axons grew uniformly throughout six axon/glia compartments and OLs from P1-2 rats were added to axon/glia compartments at 14 days in vitro for co-culture.

We developed a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research. The platform is capable of conducting up to six independent experiments in parallel and was fabricated using a newly developed macro/micro hybrid fabrication method.

Un multi-vano SNC Neuron-glia Co-cultura Microfluidic piattaforma

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to ...

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

Isolamento di cellule epiteliali mammarie da tridimensionale Mixed-cell Spheroid Co-cultura

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Induzione e Analisi dei epiteliale a Mesenchymal Transizione

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

Indagare interazioni proteina-proteina in cellule vive utilizzando il trasferimento bioluminescenza Resonance Energy

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

Misure calcio citosolico nelle cellule epiteliali renali mediante citometria di flusso

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

Cryosectioning Method for Microdissection of Murine Colonic Mucosa

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The colonic epithelium is dynamically turned over and epithelial cells are generated in the stem cell containing crypts of Lieberk&#252;hn. Progenitor cells produced in the crypt-bases migrate toward the luminal surface, undergoing a process of cellular differentiation before being shed into the gut lumen. In order to study these processes at the molecular level, we have developed a simple method for the microdissection of two spatially distinct regions of the colonic mucosa; the proliferative crypt zone, and the differentiated surface epithelial cells. Our objective is to isolate specific crypt and surface epithelial cell populations from mouse colonic mucosa for the isolation of RNA and protein.

Here we describe a simple method for the isolation of spatially distinct murine colonic epithelial surface cells from the underlying crypt-base cells.

Metodo criosezionamento per Microdissezione di Murine colon mucosa

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The ...

Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects on the lifespan across species. However, the molecular causes of aging and death remain elusive. To gain a systematic understanding of the molecular mechanisms underlying yeast aging, we need high-throughput methods to measure lifespan and to quantify various cellular and molecular phenotypes in single cells. Previously, we developed microfluidic devices to track budding yeast mother cells throughout their lifespan while flushing away newborn daughter cells. This article presents a method for preparing microfluidic chips and for setting up microfluidic experiments. Multiple channels can be used to simultaneously track cells under different conditions or from different yeast strains. A typical setup can track hundreds of cells per channel and allow for high-resolution microscope imaging throughout the lifespan of the cells. Our method also allows detailed characterization of the lifespan, molecular markers, cell morphology, and the cell cycle dynamics of single cells. In addition, our microfluidic device is able to trap a significant amount of fresh mother cells that can be identified by downstream image analysis, making it possible to measure the lifespan with higher accuracy.

This article presents a protocol optimized for the production of microfluidic chips and the setup of microfluidic experiments to measure the lifespan and cellular phenotypes of single yeast cells.

Uso di dispositivi microfluidici misurare la durata della vita e cellulare fenotipi in singole cellule di lievito in erba

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects ...

In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the JDP family could form transient heterocomplexes in eukaryotes to fine-tune substrate selection for the 70 kDa heat shock protein (Hsp70) chaperone-based protein disaggregases. The JDP complexes target acute/chronic stress induced aggregated proteins and presumably help assemble the disaggregases by recruiting multiple Hsp70s to the surface of protein aggregates. The extent of the protein quality control (PQC) network formed by these physically interacting JDPs remains largely uncharacterized in vivo. Here, we describe a microscopy-based in situ protein interaction assay named the proximity ligation assay (PLA), which is able to robustly capture&#160;these transiently formed chaperone complexes in distinct cellular compartments of eukaryotic cells. Our work expands the employment of PLA from human cells to yeast (Saccharomyces cerevisiae) and bacteria (Escherichia coli), thus rendering an important tool to monitor the dynamics of transiently formed protein assemblies in both prokaryotic and eukaryotic cells.

Cognate J-domain proteins cooperate with the Hsp70 chaperone to assist in a myriad of biological processes ranging from protein folding to degradation. Here, we describe an in situ proximity ligation assay, which allows the monitoring of these transiently formed chaperone machineries in bacterial, yeast and human cells.

Monitoraggio in Situ di gruppi di chaperone molecolari formati in farmaci, lieviti e cellule umane

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the ...

Multicellular Human Alveolar Model Composed of Epithelial Cells and Primary Immune Cells for Hazard Assessment

A human alveolar cell coculture model is described here for simulation of the alveolar epithelial tissue barrier composed of alveolar epithelial type II cells and two types of immune cells (i.e., human monocyte-derived macrophages [MDMs] and dendritic cells [MDDCs]). A protocol for assembling the multicellular model is provided. Alveolar epithelial cells (A549 cell line) are grown and differentiated under submerged conditions on permeable inserts in two-chamber wells, then combined with differentiated MDMs and MDDCs. Finally, the cells are exposed to an air-liquid interface for several days. As human primary immune cells need to be isolated from human buffy coats, immune cells differentiated from either fresh or thawed monocytes are compared in order to tailor the method based on experimental needs. The three-dimensional models, composed of alveolar cells with either freshly isolated or thawed monocyte-derived immune cells, show a statistically significant increase in cytokine (interleukins 6 and 8) release upon exposure to proinflammatory stimuli (lipopolysaccharide and tumor necrosis factor &#945;) compared to untreated cells. On the other hand, there is no statistically significant difference between the cytokine release observed in the cocultures. This shows that the presented model is responsive to proinflammatory stimuli in the presence of MDMs and MDDCs differentiated from fresh or thawed peripheral blood monocytes (PBMs). Thus, it is a powerful tool for investigations of acute biological response to different substances, including aerosolized drugs or nanomaterials.

Presented here is a protocol for primary human blood monocyte isolation as well as their differentiation into macrophages and dendritic cells and assembly with epithelial cells into a multicellular human lung model. Biological responses of cocultures composed of immune cells differentiated from either freshly isolated or thawed monocytes, upon exposure to proinflammatory stimuli, are compared.

Modello alveolare umano multicellulare composto da cellule epiteliali e cellule immunitarie primarie per la valutazione del pericolo

A human alveolar cell coculture model is described here for simulation of the alveolar epithelial tissue barrier composed of alveolar epithelial type II ...