Diese Arbeit wurde zum Teil durch die National Science Foundation (CBET 0846453) und die National Institutes of Health (1R01GM089999) unterstützt.

1. Herstellung von Silizium-Master mithilfe von Standard-SU-8 Photolithographie 1 (nicht in diesem Video gezeigt). <ol><li> Verwenden Sie Standard-SU-8 Photolithographie Methoden, um eine SU-8 &quot;Meister&quot; (SU-8 2050 MicroChem, Newton MA) für die Herstellung der verschiedenen Komponenten (PDMS-Membranen mit unterschiedlicher Dicke, Co-Kultur-Kammer) in einem Reinraum. Erstellen Solche Master-Formen können jederzeit Mikrofabrikation Anlage (, z. B. Stanford Mikrofluidik Foundry hergestellt werden <a href="http://thebigone.stanford.edu/foundry/">http://thebigone.stanford.edu/foundry/</a> ). </li><li> Vor PDMS-Replikation, setzen die SU-8 Meister Fluorsilan Dampf in einem Exsikkator an einem Vakuum-Quelle auf einfache Veröffentlichung von PDMS aus dem Master zu erleichtern. Einen Tropfen Fluorsilan auf ein Papiertuch und gelten Vakuum für eine min. Entfernen Sie das Vakuum und damit 30 min für die Abscheidung von Fluorsilan. Halten Sie die SU-8-Master in einem geschlossenen Behälter für die zukünftige Verwendung. </li></ol> 2. Replica Formen von PDMS von SU-8 Master 2 <ol><li> Die fluidische Schicht und die Kontrolle Schicht durch Replikat des PDMS von SU-8 Master gemacht. </li><li> Mix der PDMS-pre-Polymer und Vernetzer bei einem 10:1-wt. Verhältnis und Degas in einem Exsikkator für 1 Std. (oder bis Luftblasen aus der PDMS-Mischung entfernt). </li><li> Legen Sie die SU-8 Master in einer Petrischale und vorsichtig gießen Sie die PDMS-Mischung auf der Oberseite des SU-8-Master. Degas die PDMS wieder. </li><li> Spin der Form bei 1200 rpm für eine Minute, um ein einheitliches PDMS Dicke von etwa 100 um auf die Form zu bekommen. </li><li> Erhitzen Sie die Petrischale mit dem PDMS und der SU-8 Master-Form bei 80 ° C für eine Stunde auf dem PDMS zu heilen. </li><li> Entfernen Sie das gehärtete PDMS-bedeckten SU-8-Meister aus der Heizplatte. </li><li> Schälen Sie die PDMS Form aus dem SU-8-Master. </li><li> Bohren Sie vier Löcher in die PDMS Form mit Hilfe einer 20-Gauge-blunt-end Nadel für zwei Bakterien Buchten, eine Steckdose und eine pneumatische Port. </li></ol> 3. Kleben von Multilayer PDMS-Geräte: Das Gerät ist mit drei Schichten, eine obere Schicht, die den Hauptkanal, einen Mittelweg pneumatische Schicht und einer unteren Schicht bakterielle, dass die Kanäle für die Bakterien enthält enthält montiert. Die drei Schichten zusammengesetzt sind, wie unten beschrieben. <ol><li> Wet die PDMS-Membranen mit Methanol und sorgfältig schälen sie aus ihren SU-8 Master-Form mit einem sauberen Pinzette. Legen Membran auf einem Teflon-Folie. </li><li> Legen Sie die pneumatische Membran und die Fluidik-Schicht auf einer Sauerstoff-Plasma-Kammer. Schalten Sie Plasma für 40 sec (Sauerstoffdruck 10 sccm, HF-Leistung 100 W). </li><li> Nach Exposition gegenüber Plasma, align (innerhalb von 10 Minuten) der Membran auf der Unterseite PDMS-Kanal mit einer Pinzette. Zugabe von Methanol auf der Membran und der PDMS-Schichten hilft Freizügigkeit der Schichten und macht die Ausrichtung zu erleichtern. In diesem Fall ist das Gerät auf ein Stück Gaze gelegt, damit die Methanoldämpfe zu entkommen. </li><li> Cure den ausgerichteten PDMS-Schichten auf einer Heizplatte bei 80 ° C für 8 h </li><li> Nach dem Verkleben der pneumatischen Membran auf die unterste Schicht, wiederholen Sie die Schritte 3,2 bis 3,4 zur Verbindung der oberen Kanal auf die versammelten Composite PDMS-Gerät. </li></ol> 4. Montage der PDMS-Gerät 3,4 <ol><li> Die Bohrungen in den Einlass-und Auslasskanäle für die Aussaat eukaryotischen Zellen und Bakterien eingesetzt. </li><li> Connect Tygon-Schlauch (0,01 Zoll OD) zu den Häfen und den Betrieb der pneumatischen Schicht durch Ziehen mit 20ml Spritze. </li><li> Legen Sie die PDMS-Modell und eine vorgereinigte Glasträger in ein Sauerstoff-Plasma-Kammer. Schalten Sie Plasma für 20 sec (Sauerstoffdruck 20 sccm, RF 300W) ohne Anlegen des Vakuums. </li><li> Um den Kontakt zwischen der Insel Wand (PDMS) Bindung an den Glasträger zu verhindern, sofort eine Verbindung mit einer Spritze, um das Gerät und das Vakuum, indem Sie die Spritze und halten es fest. </li><li> Drücken Sie das PDMS-Modell auf das Glas (innerhalb von 2 Minuten). </li><li> Cure die PDMS-Gerät bei 80 ° C für 10 Minuten. </li></ol> 5. Die Behandlung der Glasoberfläche mit Fibronektin <ol><li> Sterilisieren Gerät unter UV für 15 Minuten nach dem Anschluss Tygon Schlauch an jedem Port. </li><li> Fill und Flow der sterilisierten PBS in den Kanal, um alle Ablagerungen in den Kanal zu entfernen. Bewerben Flow für 10 Minuten, um Luftblasen zu entfernen. Die Luftblasen können leicht entweichen die PDMS wegen seiner Gasdurchlässigkeit. </li><li> Füllen Sie ein Farbstoff-Lösung in den Luftkanal, um die Bildung von Luftblasen in den Kanal zu verhindern. </li><li> Zur Erleichterung der eukaryotischen Zelle Aussaat auf dem Glas, die Einführung einer 1 pg / ml-Lösung von Fibronektin in das Gerät und Inkubation für 40 min bei 37 ° C. </li><li> Waschen Sie die überschüssige Fibronektin mit 1 ml Wachstumsmedium. </li></ol> 6. Bildung und Messung von kommensalen E. coli Biofilm inInseln <ol><li> Verdünnen Sie die symbiotischer Bakterien (zB E. coli BW25113/pCM18) in M9 Minimalmedium bis OD 600 ~ 1 zur Bildung eines Kommensalen Biofilm in den geschlossenen Kammern / Inseln. </li><li> In der Nähe der Insel Wand durch die Anwendung positiver Flüssigkeitsdruck. Eine hausgemachte air-to-Liquid-Druck-Wandler wird verwendet, um Luft Blasenbildung in den Kanal zu verhindern. </li><li> Einführung der kommensalen E. coli in die Inseln mit dem Ventil geschlossen und verhindert, dass Bakterien für 1 Stunde bei 32 ° C legen </li><li> Waschen Sie die losen Bakterien und perfuse einer verdünnten Bakteriensuspension (OD 600 ~ 0,05) bei 0.25mL/hr für 12 Stunden. </li><li> Spülen Sie den Biofilm mit frischem DMEM Nährmedien zu locker angehefteten Bakterien zu entfernen und die Vorbereitungen für eukaryotischen Zelle Anhang. </li><li> Bild des Biofilms mit einem Leica TCS SP5 konfokale Laser Scanning Mikroskop (Bannockburn, IL) und Visualisierung der Biofilm-Architektur mit dem Imaris Software 5. </li></ol> 7. Seeding eukaryotischen Zellen um bakterielle Inseln 6-8 <ol><li> Trypsinize HeLa Zellen aus Zellkulturflasche und resuspendieren in DMEM-Medium für eine Aussaatdichte von 5 x 10 5 Zellen / ml. </li><li> Einführung HeLa-Zellen in das Gerät bei einer Flussrate von 2 ml / min mit einem Picoplus Spritzenpumpe (Harvard Apparattus, MA). Stellen Sie sicher, dass die Insel Wand abgesenkt wird, so dass die bakteriellen Biofilm getrennt von den HeLa-Zell-Region ist. Dieser Schritt sollte auf der Bühne eines Lichtmikroskops durchgeführt werden, so dass die Einheitlichkeit der Aussaat bestimmt werden kann. </li><li> Klemmen Sie die Zelle Steckdose, um die Bewegung der Zellsuspension in den Kanal und Inkubation für 6 h bei 37 ° C zu reduzieren in einer 5% CO 2-Inkubator. </li><li> Perfundieren das Gerät mit Wachstumsmedium bei 0,5 ml / h zu losen Zellen zu entfernen. </li><li> Aktualisieren Sie die DMEM Medium für HeLa und kommensalen E. coli alle 6 Stunden. </li></ol> 8. Einführung von pathogenen Bakterien in die Insel und der Exposition gegenüber eukaryotischen Zellen <ol><li> Bereiten E. coli O157: H7 (EHEC) auf HeLa-Zellen mit einer Multiplizität der Infektion (moi) von 100:1 200:1 (Anzahl der EHEC pro HeLa Zellen) zu infizieren. </li><li> Spülen und waschen sich lose befestigt symbiotischer Bakterien. </li><li> Einführung einer verdünnten EHEC Suspension (OD 600 ~ 0,1) in den Inseln. </li><li> Inkubieren Gerät bei 37 ° C in einer 5% CO 2-Inkubator für 6 Stunden ohne Strom zu ermöglichen EHEC auf Signale im kommensalen Biofilm ausgesetzt werden. </li><li> Heben Sie die PDMS Wand zu EHEC in der kommensalen Inseln aussetzen HeLa-Zellen für 6 Stunden. </li><li> Spülen Sie den Kanal mit PBS und Flecken für lebende und tote Zellen unter Verwendung des Live / Dead-Kit (L3224, Invitrogen, CA). </li><li> Bild mehrere Standorte auf einem Zeiss Axiovert 200M Fluoreszenzmikroskop und schätzen die Zahl der lebenden und toten Zellen. </li></ol> 9. Vertreter Ergebnisse 9 Die Abfolge der Ereignisse in GI-Trakt Infektionen wurde durch die Entwicklung einer Monoschicht von HeLa-Zellen und eine kommensalen E. nachgeahmt coli Biofilm und Freilegen EHEC der kommensalen Biofilm vor der Infektion von HeLa-Zellen. Die Schritte bei der Herstellung der mikrofluidischen Co-Kultur-Modell sind in Abbildung 1 dargestellt. 2A zeigt eine 3-dimensionale Darstellung der Co-Kultur-Gerät. Die beiden Regionen in der Co-Kultur-Gerät der eukaryotischen Zellkulturkammer und die bakterielle Insel sind in Abbildung 2B mit verschiedenen Farbstoffen (lila für den eukaryotischen Regionen und grün für die bakterielle Insel) gezeigt. Die Machbarkeit der Isolierung der Bakterienkultur Regionen aus den umliegenden eukaryotischen Region ist in Abbildung 2C mit Farbstoffen dargestellt. Abbildung 3 zeigt repräsentative Ergebnisse von Co-Kultur von Epithelzellen und Bakterien. 3A zeigt Kolonisierung der kommensalen Biofilm (grün) Insel EHEC (rot). 3B zeigt das Nebeneinander von Epithelzellen und kommensalen E. coli mit EHEC in der bakteriellen Insel. Der RFP Ausdruck EHEC und GFP-exprimierenden symbiotischer Bakterien sind in der Insel, wenn die PDMS Wand abgesenkt lokalisiert ist, welche die Machbarkeit der Isolierung der bakteriellen Insel von Epithelzellen. <img src="/files/ftp_upload/1749/1749fig1.jpg" alt="Abbildung 1" /> Abbildung 1: Cell Seeding Schema in der Co-Kultur-Modell. (1) Die PDMS Wand abgesenkt wird, um eine Insel bilden, sind symbiotischer Bakterien in die Insel eingeführt, und Fibronektin ist rund um die Insel geflossen. (2) HeLa-Zellen sind in den Regionen rund um die Insel ausgesät. (3) Nach HeLa Zellen Konfluenz erreicht und die kommensalen Biofilm entwickelt hat, ist EHEC in der Insel eingeführt. (4) Die PDMS Wand abgehobenbis zu entlarven HeLa-Zellen rund um die Insel zu EHEC. Inset zeigt Details des Ventils. <img src="/files/ftp_upload/1749/1749fig2a.jpg" alt="Abbildung 2a" /> <img src="/files/ftp_upload/1749/1749fig2bc.jpg" alt="Abbildung 2bc" /> Abbildung 2: Microfluidic Modell für Co-Kultur von Epithelzellen und Bakterien. (A) Dreidimensionale Wiedergabe der Co-Kultur-Gerät zeigt pneumatisch betätigte Trapping Regionen zur Bildung bakterieller Inseln unter Epithelzellen. Jede bakterielle Insel (1200 Mikrometer Durchmesser und 1000 um auseinander) hat einen separaten Eingang und Ausgang für die Bereitstellung von Nährmedien und Beseitigung von Abfällen von der Insel. (B) Mikroskopische Aufnahme der Co-Kultur-Gerät mit Farbstoffen, welche die verschiedenen Regionen (epithelialen Zell-Zone, bakterielle Inseln). (C) Die Treue der pneumatischen Trapping-System ist durch eine Senkung der PDMS Wand (links) mit einem pneumatisch aktivierten Kanal (blau), die Einführung Purpur in die geschlossene Insel Inseln und fließenden gelben Farbstoff um es für 48 Stunden gezeigt Wenn die PDMS Wand (rechts) ausgelöst wird, wird die Inselregion in die umliegenden gelben Farbstoff ausgesetzt. Scale-Balken repräsentiert 500 um. <img src="/files/ftp_upload/1749/1749fig3.jpg" alt="Abbildung 3" /> Abbildung 3: Co-Kultur von HeLa-Zellen und Bakterien. (A) Fluoreszenzbild RFP-exprimierenden EHEC und GFP-exprimierenden E. coli BW25113 in Insel. (B) Überlagerung der gesendeten, grünen und roten Fluoreszenz-Bildern in das Gerät. Scale-Balken repräsentiert 200 um.

<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>

Konventionelle Tests für Erreger Befestigung und Besiedlung nutzen eine Monoschicht von eukaryotischen Zellen in Zellkulturplatten, in die Krankheitserreger aufgenommen. Diese Modelle sind nicht physiologisch relevant, da sie nicht integrieren Kommensale bakteriellen Biofilm auf eukaryotischen Zellen entwickelt. Einfache Zugabe eines pre-grown Bakterienkultur zu eukaryotischen Zellen ist unwahrscheinlich, dass diese Konformation führen, wie Biofilmen äußerst Strukturen, die im Laufe der Zeit entwickeln organisiert s...

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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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<tr>
<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

Der menschliche Magen-Darm (GI-Trakt) ist ein einzigartiges Umfeld, in dem intestinalen Epithelzellen und nicht-pathogenen (Kommensalen) Bakterien koexistieren. Es wurde vorgeschlagen, dass die Mikroumgebung, dass der Erreger Begegnungen in der kommensalen Schicht wichtig bei der Bestimmung der Ausdehnung der Besiedlung ist. Aktuelle Kultur Methoden zur Untersuchung von Krankheitserregern Kolonisation sind nicht gut für die Untersuchung dieser Hypothese, da sie nicht aktivieren Co-Kultur von Bakterien und Epithelzellen in einer Weise, dass die GI-Trakt Mikroumgebung imitiert geeignet. Hier beschreiben wir einen mikrofluidischen Co-Kultur-Modell, das unabhängig Kultur von eukaryotischen Zellen und Bakterien ermöglicht, und die Prüfung der Wirkung der kommensalen Mikroumgebung auf Erreger Kolonisation. Die Co-Kultur-Modell ist durch die Entwicklung eines Kommensalen nachgewiesen<em&gt; Escherichia coli</em&gt; Biofilm unter HeLa-Zellen, durch die Einführung von enterohämorrhagischen gefolgt<em&gt; E. coli</em&gt; (EHEC) in die kommensalen Insel, in einer Sequenz, die die Abfolge der Ereignisse in GI-Trakt Infektion imitiert.

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

Dieses Protokoll beschreibt eine mikrofluidische Co-Kultur-Modell für die gleichzeitige und lokalisierte Kultur von Epithelzellen und Bakterien. Dieses Modell kann zur Untersuchung der Rolle der verschiedenen löslichen molekularen Signalen auf Pathogenese sowie Bildschirm die Wirksamkeit der vermeintlichen probiotischen Bakterienstämmen verwendet werden können.

Mikrofluidik-Co-Kultur von Epithelzellen und Bakterien für die Untersuchung Löslich Signal-vermittelte Wechselwirkungen

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 Aufrufe.  Texas A&M University. Dieses Protokoll beschreibt eine mikrofluidische Co-Kultur-Modell für die gleichzeitige und lokalisierte Kultur von Epithelzellen und Bakterien. Dieses Modell kann zur Untersuchung der Rolle der verschiedenen löslichen molekularen Signalen auf Pathogenese sowie Bildschirm die Wirksamkeit der vermeintlichen probiotischen Bakterienstämmen verwendet werden können.

Serie: 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.

Eine Neuronale und Astrocyte Co-Kultur-Assay für High Content Analyse der Neurotoxizität

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

Forschung

Biologie

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.

Ein Multi-Kompartiment-ZNS-Neuron-Glia-Co-Kultur mikrofluidischen Plattform

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.

Isolierung von Brustepithelzellen aus dreidimensionalen Mixed-Zelle Spheroid Co-Kultur

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.

Induktion und Analyse von epithelialen zu mesenchymalen Transition

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.

Untersuchung von Protein-Protein-Interaktionen in lebenden Zellen Mit Biolumineszenz-Resonanz-Energie-Transfer

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.

Cytosolischen Calcium Messungen in Nierenepithelzellen mittels Durchflusszytometrie

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.

Kryoschneiden Verfahren zur Mikrodissektion von Murine Kolonschleimhaut

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.

Mikrofluidik-Geräte mit Measure Lebensdauer und Cellular Phänotypen in Einzel Budding Hefezellen

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.

In Situ-Überwachung von transient gebildeten molekularen Chaperon-Baugruppen in Bakterien, Hefe und menschlichen Zellen

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.

Multizelluläres humanes Alveolarmodell bestehend aus Epithelzellen und primären Immunzellen zur Gefahrenbeurteilung

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