Ce travail a été soutenu en partie par la National Science Foundation (EFAC 0.846.453) et le National Institutes of Health (1R01GM089999).

1. La fabrication des maîtres de silicium en utilisant la norme SU-8 photolithographie 1 (non représenté dans cette vidéo). <ol><li> Utiliser des méthodes standard de SU-8 photolithographie pour créer un SU-8 &quot;maîtres&quot; (SU-8 2050, MicroChem, Newton, MA) pour fabriquer les différents composants (PDMS de différentes épaisseurs, de co-culture de chambre) dans une salle blanche. Ces moules maître peut être fabriqué dans toute installation de microfabrication (par exemple, Stanford Microfluidique Fonderie; <a href="http://thebigone.stanford.edu/foundry/">http://thebigone.stanford.edu/foundry/</a> ). </li><li> Avant de réplication PDMS, exposer le SU-8 maîtres d&#39;fluorosilane la vapeur dans un dessiccateur attaché à une source de vide pour faciliter la libération facile de PDMS par le maître. Ajouter une goutte de fluorosilane d&#39;une serviette en papier et appliquer le vide pendant une minute. Retirez le vide et laissez 30 min pour le dépôt de fluorosilane. Gardez le SU-8 de maître dans un récipient fermé pour une utilisation future. </li></ol> 2. Réplique de moulage de PDMS de SU-8 master 2 <ol><li> La couche fluidique et la couche de contrôle sont réalisés par moulage réplique de PDMS de SU-8 maître. </li><li> Mélanger le PDMS pré-polymère et cross-linker à un poids de 10h01. Ratio et dégazer dans un dessiccateur pendant 1 heure (ou jusqu&#39;à ce que les bulles d&#39;air sont éliminés du mélange PDMS). </li><li> Placez le SU-8 maître dans une boîte de Pétri et verser délicatement le mélange PDMS sur le dessus de la SU-8 maître. Degas le PDMS à nouveau. </li><li> Spin le moule à 1200 rpm pendant une minute pour obtenir une épaisseur uniforme d&#39;environ 100 microns de PDMS sur le moule. </li><li> Chauffer la boîte de Pétri contenant le PDMS et le SU-8 moule maître à 80 ° C pendant une heure pour guérir le PDMS. </li><li> Retirez le guérir PDMS-couverte SU-8 maîtres de la plaque chauffante. </li><li> Peler le moule PDMS du maître SU-8. </li><li> Percez quatre trous dans le moule PDMS en utilisant un calibre 20 à bout émoussé l&#39;aiguille pour les entrées bactéries deux, une sortie et un port pneumatique. </li></ol> 3. Collage de multicouches appareils PDMS: Cet appareil est assemblé avec trois couches, une couche supérieure qui contient le canal principal, une couche de pneumatiques milieu, et une couche inférieure bactérienne qui contient les canaux pour les bactéries. Les trois couches sont assemblés comme décrit ci-dessous. <ol><li> Mouiller les membranes en PDMS avec du méthanol et soigneusement les peler de leur SU-8 du moule maître à l&#39;aide d&#39;une pince propre. Membranes Déposer sur une plaque en téflon. </li><li> Placez la membrane pneumatique et la couche fluidique sur une chambre à plasma d&#39;oxygène. Tourner sur le plasma pendant 40 secondes (10 sccm pression d&#39;oxygène, de puissance RF 100W). </li><li> Après une exposition au plasma, d&#39;aligner (dans les 10 minutes) de la membrane sur le fond du canal PDMS l&#39;aide de pinces. Addition de méthanol sur la membrane et les couches PDMS permet la libre circulation des couches, et rend l&#39;alignement facile. Dans ce cas, le dispositif est placé sur un morceau de gaze pour permettre aux vapeurs de méthanol à s&#39;échapper. </li><li> Guérissez les couches alignées PDMS sur une plaque chauffante à 80 ° C pendant 8 h. </li><li> Après collage de la membrane pneumatique sur la couche inférieure, répétez les étapes 3.2 à 3.4 pour coller le canal supérieur sur le dispositif de PDMS assemblés composites. </li></ol> 4. L&#39;assemblage du dispositif de PDMS 3,4 <ol><li> Percez des trous dans les ports d&#39;entrée et de sortie utilisés pour l&#39;ensemencement des cellules eucaryotes et les bactéries. </li><li> Relier les tubes Tygon (0,01 pouces OD) aux ports et à exploiter la couche pneumatiques en tirant avec une seringue de 20ml. </li><li> Placez le modèle PDMS et d&#39;une lame de verre pré-nettoyés dans une chambre à plasma d&#39;oxygène. Tourner sur le plasma pendant 20 sec (pression d&#39;oxygène 20 sccm, RF 300W) sans appliquer le vide. </li><li> Afin d&#39;éviter tout contact entre l&#39;île mur (PDMS) collage à la lame de verre, se connecter immédiatement une seringue à l&#39;appareil et appliquer le vide en tirant la seringue et le maintenant en place. </li><li> Appuyez doucement sur le modèle de PDMS sur le verre (dans les 2 minutes). </li><li> Cure le dispositif de PDMS à 80 ° C pendant 10 minutes. </li></ol> 5. Traiter la surface du verre avec de la fibronectine <ol><li> Stériliser appareil sous UV pendant 15 minutes après le raccordement de tuyaux Tygon à chaque port. </li><li> Remplissez et le débit du PBS stérile dans le canal pour enlever tous les débris dans le canal. Appliquer des flux pendant 10 minutes pour enlever les bulles d&#39;air. Les bulles d&#39;air peuvent facilement échapper à la PDMS en raison de sa perméabilité aux gaz. </li><li> Remplir une solution de teinture de couleur dans le canal pneumatique pour éviter la formation de bulles d&#39;air dans le canal. </li><li> Afin de faciliter l&#39;ensemencement des cellules eucaryotes sur le verre, introduire un 1 ug / ml solution de fibronectine dans l&#39;appareil et laisser incuber pendant 40 min à 37 ° C. </li><li> Laver la fibronectine excédent avec 1 ml de milieu de croissance. </li></ol> 6. Formation et évaluation des commensaux E. coli dans les biofilmsîles <ol><li> Diluer les bactéries commensales (par exemple, E. coli BW25113/pCM18) dans les médias M9 minimale à OD 600 ~ 1 pour former un biofilm commensales dans les chambres fermées / îles. </li><li> Fermer le mur île par l&#39;application positive liquide à haute pression. Un convertisseur de pression artisanales air-liquide est utilisé pour prévenir la formation de bulles d&#39;air dans le canal. </li><li> Introduire le commensal E. coli dans les îles avec la vanne fermée, et permettent aux bactéries d&#39;attacher pendant 1 heure à 32 ° C. </li><li> Laver les bactéries seules et perfuser une suspension diluée bactérienne (DO 600 ~ 0,05) à 0.25mL/hr pendant 12 heures. </li><li> Rincer le biofilm avec des produits frais milieux de croissance DMEM pour enlever les bactéries faiblement attachés et se préparer pour la fixation des cellules eucaryotes. </li><li> Image du biofilm en utilisant un Leica TCS SP5 microscope confocal à balayage laser (Bannockburn, IL), et de visualiser l&#39;architecture du biofilm en utilisant le logiciel Imaris 5. </li></ol> 7. Ensemencement des cellules eucaryotes autour des îles bactérienne 6-8 <ol><li> Trypsiniser cellules HeLa par flacon de culture tissulaire et remettre en suspension dans des milieux DMEM pour une densité de semis de 5 x 10 5 cellules / ml. </li><li> Présentez les cellules HeLa dans l&#39;appareil à un débit de 2 mL / min en utilisant une pompe seringue Picoplus (Harvard Apparattus, MA). Assurez-vous que le mur île est abaissé pour que le biofilm bactérien est séparée de la région de cellules HeLa. Cette étape doit être effectuée sur la scène d&#39;un microscope optique afin que l&#39;uniformité de l&#39;ensemencement peut être déterminée. </li><li> Serrer la sortie de la cellule afin de réduire le mouvement de la suspension cellulaire dans le canal et incuber pendant 6 h à 37 ° C dans un 5% de CO 2 incubateur. </li><li> Perfuser l&#39;appareil avec un milieu de croissance à 0,5 ml / h pour enlever les cellules seules. </li><li> Actualiser les médias DMEM pour HeLa et commensaux E. coli toutes les 6 heures. </li></ol> 8. Introduction de bactéries pathogènes dans l&#39;île et l&#39;exposition aux cellules eucaryotes <ol><li> Préparer E. coli O157: H7 (EHEC) pour infecter les cellules HeLa à une multiplicité d&#39;infection (MOI) de 100:1 200:1 (nombre de cellules HeLa par EHEC). </li><li> Rincer et laver faiblement attachés bactéries commensales. </li><li> Introduire une suspension diluée EHEC (DO600 ~ 0,1) dans les îles. </li><li> Incuber appareil à 37 ° C dans un 5% de CO 2 incubateur pendant 6 heures sans écoulement pour permettre à EHEC pour être exposé à des signaux présents dans le biofilm commensales. </li><li> Soulevez le mur de PDMS pour exposer EHEC dans les îles commensales de cellules HeLa pendant 6 heures. </li><li> Rincer le canal avec du PBS et les taches pour les cellules vivantes et mortes en utilisant le kit Live / Dead (L3224, Invitrogen, CA). </li><li> L&#39;emplacement des images multiples sur un microscope Zeiss Axiovert 200M fluorescence et estimer le nombre de cellules vivantes et mortes. </li></ol> 9. Résultats Représentant 9 La séquence des événements dans les infections des voies gastro-intestinal a été imité par le développement d&#39;une monocouche de cellules HeLa et un commensal E. biofilm coli et d&#39;exposer à l&#39;EHEC biofilm commensales avant l&#39;infection des cellules HeLa. Les étapes impliquées dans la fabrication de la microfluidique de co-culture du modèle sont présentés dans la figure 1. Figure 2A montre un rendu en 3 dimensions de l&#39;appareil de co-culture. Les deux régions dans le dispositif de co-culture de la chambre de culture de cellules eucaryotes et les bactéries de l&#39;île sont montrés dans la figure 2B avec des colorants de couleurs différentes (violet pour les régions eucaryotes et vert pour l&#39;île de bactéries). La faisabilité d&#39;isoler les régions de culture bactérienne de la région environnante eucaryotes est montré dans la figure 2C utilisant des colorants de couleurs. La figure 3 montre des résultats représentatifs de la co-culture de cellules épithéliales et des bactéries. Figure 3A montre la colonisation du biofilm commensales (vert) île par EHEC (rouge). La figure 3B montre la juxtaposition de cellules épithéliales et commensaux E. coli avec des EHEC dans l&#39;île bactérienne. La DP exprimant la GFP EHEC et exprimer les bactéries commensales sont localisés dans l&#39;île quand le mur de PDMS est abaissé, ce qui illustre la faisabilité d&#39;isoler l&#39;île bactérienne de cellules épithéliales. <img src="/files/ftp_upload/1749/1749fig1.jpg" alt="Figure 1" /> Figure 1: Schéma de l&#39;ensemencement des cellules dans le modèle de co-culture. (1) Le mur de PDMS est abaissé pour former une île, les bactéries commensales sont introduits dans l&#39;île, et la fibronectine est coulé autour de l&#39;île. (2) des cellules HeLa sont ensemencées dans les régions qui entourent l&#39;île. (3) Après les cellules HeLa atteignent la confluence et le biofilm commensales a développé, à EHEC est introduit dans l&#39;île. (4) Le mur de PDMS est levépour exposer les cellules HeLa qui entoure l&#39;île d&#39;EHEC. En médaillon montre les détails du fonctionnement de la vanne. <img src="/files/ftp_upload/1749/1749fig2a.jpg" alt="Figure 2a" /> <img src="/files/ftp_upload/1749/1749fig2bc.jpg" alt="Figure 2bc" /> Figure 2: Modèle microfluidiques pour la co-culture de cellules épithéliales et des bactéries. (A) en trois dimensions restitution de l&#39;appareil de co-culture montrant pneumatique actionné régions piégeage pour former des îlots bactérienne chez les cellules épithéliales. Chaque île bactérienne (1200 um de diamètre et 1000 um d&#39;intervalle) a une entrée et une sortie pour fournir un milieu de croissance et d&#39;élimination des déchets de l&#39;île. (B) Micrographie de l&#39;appareil de co-culture avec des colorants de couleurs montrant les différentes régions (zone de cellules épithéliales, les îles bactérienne). (C) La fidélité du système de piégeage pneumatique est démontré par l&#39;abaissement du mur PDMS (panneau de gauche) en utilisant un canal pneumatique activé (en bleu), l&#39;introduction de teinture pourpre dans les iles fermée, et qui coule colorant jaune autour d&#39;elle pendant 48 h. Lorsque le mur de PDMS est soulevée (à droite), la région île est exposée à la teinture jaune entoure. La barre d&#39;échelle représente 500 um. <img src="/files/ftp_upload/1749/1749fig3.jpg" alt="Figure 3" /> Figure 3: Co-culture de cellules HeLa et les bactéries. (A) image de fluorescence de la DP-exprimant EHEC et exprimant la GFP E. coli dans les BW25113 île. (B) Superposition d&#39;transmise, vert, rouge et des images de fluorescence dans l&#39;appareil. La barre d&#39;échelle représente 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>

Essais classiques pour l&#39;attachement des agents pathogènes et la colonisation d&#39;utiliser une monocouche de cellules eucaryotes dans des plaques de culture de tissu dans lequel des agents pathogènes sont ajoutés. Ces modèles ne sont pas physiologiquement pertinents car ils n&#39;intègrent pas un biofilm bactérien commensal développés sur les cellules eucaryotes. La simple addition d&#39;une culture pré-cultivé des bactéries aux cellules eucaryotes est peu probable d&#39;aboutir à cette conformation qu...

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		<th>Material Name</th>
		<th>Type</th>
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		<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

La gastro-intestinal humain intestinal (GI) est un environnement unique dans lequel les cellules épithéliales intestinales et non pathogènes (commensaux) les bactéries coexistent. Il a été proposé que le microenvironnement des rencontres que l&#39;agent pathogène dans la couche commensale est importante pour déterminer l&#39;ampleur de la colonisation. Les méthodes de culture actuelles pour enquêter sur la colonisation des pathogènes ne sont pas bien adaptés pour enquêter sur cette hypothèse car ils ne permettent pas de co-culture de bactéries et de cellules épithéliales d&#39;une manière qui imite le microenvironnement tractus gastro-intestinal. Nous décrivons ici un microfluidique de co-culture modèle qui permet la culture indépendante des cellules eucaryotes et les bactéries, et de tester l&#39;effet du microenvironnement commensale sur la colonisation d&#39;agents pathogènes. Le modèle de co-culture se manifeste par l&#39;élaboration d&#39;un commensal<em&gt; Escherichia coli</emBiofilm&gt; parmi les cellules HeLa, suivie par l&#39;introduction de entérohémorragique<em&gt; E. coli</em&gt; (EHEC) dans l&#39;île commensaux, dans une séquence qui imite la séquence des événements dans les infections des voies gastro-intestinales.

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

Ce protocole décrit une microfluidique de co-culture de modèle pour la culture simultanée et localisée des cellules épithéliales et des bactéries. Ce modèle peut être utilisé pour étudier le rôle des différents signaux moléculaires solubles sur la pathogenèse ainsi que l&#39;écran de l&#39;efficacité des souches bactériennes probiotiques putatifs.

Microfluidiques Co-culture de cellules épithéliales et des bactéries d&#39;enquête sur les Soluble signal médiée par des interactions

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 vues.  Texas A&M University. Ce protocole décrit une microfluidique de co-culture de modèle pour la culture simultanée et localisée des cellules épithéliales et des bactéries. Ce modèle peut être utilisé pour étudier le rôle des différents signaux moléculaires solubles sur la pathogenèse ainsi que l'écran de l'efficacité des souches bactériennes probiotiques putatifs.

Vidéo: 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 et astrocytes de co-culture de dosage pour l&#39;analyse du contenu haute de la neurotoxicité

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

Recherche

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.

Un compartiment Multi-SNC neurone-glie co-culture de la plate-forme microfluidique

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.

Isolement des cellules épithéliales mammaires à partir de trois dimensions de cellules mixtes Sphéroïde 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 ...

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.

Induction et analyse des épithéliale à mésenchymateuses 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.

Étudier les interactions protéine-protéine dans les cellules vivantes en utilisant le transfert d&#39;énergie de résonance de bioluminescence

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.

Mesures de calcium cytosolique dans les cellules épithéliales rénales par cytométrie en flux

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.

Cryosectioning Méthode de microdissection de murin côlon Muqueuse

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.

L&#39;utilisation des dispositifs microfluidiques pour mesurer et cellulaire Lifespan dans Phenotypes simples cellules de levure de bourgeonnement

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.

Surveillance in Situ des assemblages de chaperon moléculaire s'est formé de façon transitoire dans les bactéries, les levures et les cellules humaines

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.

Modèle alvéolaire humain multicellulaire composé de cellules épithéliales et de cellules immunitaires primaires pour l’évaluation des risques

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