Ce travail a été soutenu par AAUW solidarité internationale au Adeola Adewola, NIH / NCRR (U42RR023245) à José Oberholzer, et le projet du diabète de Chicago.

A. microfabrication de 3-couche périphérique périfusion microfluidique Fond du puits Maître Protocole (150 um puits profonds) <ol><li> Nettoyez la galette en utilisant une lame de rasoir si nécessaire. Nettoyez avec de l&#39;acétone, du méthanol, et de l&#39;IAP. Effectuer le traitement au plasma à 50 Watts pendant 30 s. </li><li> Spin-100 SU8 @ 2000 rpm. [Etape 1: 500 min, 10 s, 100 rpm / s, étape 2: 2000 rpm, 30 s, 300 rpm / s]. [NOTE: Ne placez pas la plaquette avec des pincettes après la filature SU8] </li><li> Cuire la galette molle à 65 ° C pendant 20 min et à 95 ° C pendant 50 min. </li><li> La plaquette est exposée à une lumière UV à travers un masque désiré. Dose pour 150 de hauteur est de 650 um mJ / cm 2. </li><li> Post-exposition cuire la galette à 65 ° C pendant 1 min et à 95 ° C pendant 12 min. </li><li> Développer la galette dans SU8 développeur pendant 15 min. </li></ol> Maître Microchannel Protocole (500 um puits profonds) <ol><li> Nettoyez la galette en utilisant une lame de rasoir si nécessaire. Nettoyez avec de l&#39;acétone, du méthanol, et de l&#39;IAP. Effectuer le traitement au plasma à 50 Watts pendant 30 s. </li><li> Spin SU8-2150 @ 1000 tpm. [Etape 1: 500 min, 10 s, 100 rpm / s, étape 2: 1000 rpm, 30 s, 300 rpm / s]. </li><li> Cuire la galette molle à 65 ° C pendant 15 min et à 95 ° C pendant 2 heures et 30 min. </li><li> La plaquette est exposée à une lumière UV à travers un masque désiré. La dose d&#39;exposition pour les 650 um de hauteur est de 685 mJ / cm 2. </li><li> Post-exposition cuire la galette à 65 ° C pendant 5 min et à 95 ° C pendant 35 min. </li><li> Développer la galette dans SU8 développeur pendant 20-30 min. </li></ol> Préparation de la solution PDMS <ol><li> Polydiméthylsiloxane (PDMS) solution est préparée en mélangeant soigneusement 10 parties d&#39;élastomère de silicone avec 1 partie d&#39;agent de durcissement d&#39;un kit standard de Sylgard 184. </li><li> Les bulles générées dans la solution PDMS pendant le processus de mixage sont retirés en utilisant un dessiccateur à vide. </li><li> La bulle sans solution de PDMS est lentement distribué sur la SU8 maîtres et un plat vide de Pétri pour la troisième couche. </li><li> La température de la plaque chauffante est fixée à 75 ° C et le PDMS est guéri à cette température pendant 2 heures </li><li> Entrées, sorties et les puits de change sont perforées à l&#39;aide du perforateur taille appropriée trou. </li><li> Les couches sont collées sur une lame de verre (taille de 0,1 mm) en utilisant un dispositif à plasma de poche. </li></ol> B. Montage expérimental <ol><li> Débit d&#39;éthanol à 70% à travers le dispositif de micro-à stériliser. Débit de l&#39;eau déminéralisée pour laver l&#39;éthanol. Perfuser 50 ml de BSA à 0,5% grâce à l&#39;appareil pour éviter l&#39;adsorption non spécifique de l&#39;insuline sur les parois de microcanaux. </li><li> La pente de la rampe de glucose et d&#39;autres gradients connexes générées par le logiciel LabVIEW qui communique avec la seringue pompes sont testées pour s&#39;assurer les gradients sont stables. </li><li> 25-30 îlots des souris ont été incubés avec 5 Fura-2/AM uM (un indicateur de calcium, Molecular Probes, CA) et de 2,5 uM rhodamine 123 (Rh123, un indicateur potentiel mitochondrial, Sigma, MO) pendant 30 min à 37 ° C dans tampon Krebs Ringer (KRB) contenant 2 mM de glucose </li><li> Les îlots des souris sont soigneusement pipette dans le dispositif microfluidique périfusion travers l&#39;orifice d&#39;entrée. </li><li> Le réseau microfluidique est alors l&#39;installation en reliant l&#39;entrée à l&#39;aide de pompes à seringues tube Tygon et un connecteur en Y et la sortie de collecteur de fraction. L&#39;appareil se trouve sur une étape de chauffage (37 ° C) sur le microscope et la tubulure d&#39;entrée est chauffée sur une plaque chauffante pour maintenir la température de la chambre et une solution à 37 ° C. </li><li> Immédiatement après l&#39;installation, les îlots des souris sont perifused avec KRB contenant 2 mM de glucose pendant 10 min puis une rampe de glucose (2mm 25mm) pendant 25 min. </li><li> Time-lapse images sont recueillies et analysées toutes les 15 s par le logiciel SimplePCI. Le perifusate est également collecté toutes les minutes en utilisant un collecteur de fraction d&#39;analyser la sécrétion d&#39;insuline en utilisant un kit ELISA. </li></ol> Les résultats représentatifs Îlots de souris ont été perifused avec un dégradé linéaire de 2-25 mM de glucose. Comme le montre la figure 1, l&#39;influx de calcium et de la sécrétion d&#39;insuline sont déclenchées après environ 13 minutes de périfusion, correspondant à 6 mM de glucose. Les changements dans le potentiel mitochondrial sont vus auparavant comme prévu, à environ 11 minutes. Ces données démontrent l&#39;avantage d&#39;utiliser ce réseau microfluidique afin de caractériser l&#39;îlot physiologie. <img src="/files/ftp_upload/1649/1649fig1.jpg" alt="Figure 1" /> Figure 1. Les réactions physiologiques à la stimulation des cellules des îlots.

<ol>
	<li>Mohammed, J. S., Wang, Y., Harvat, T. A., Oberholzer, J., Eddington, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+device+for+multimodal+characterization+of+pancreatic+islets.">Microfluidic device for multimodal characterization of pancreatic islets.</a> Lab Chip. 9, 97-106 (2009).</li></ol>

Les systèmes traditionnels de périfusion îlots de Langerhans (macroscopique et microscopique) ont quelques limitations, y compris complexes de configuration et de conception, de hautes exigences techniques, et la difficulté de créer l&#39;utilisateur prescrits gradients chimiques dans le système. Le système microfluidique périfusion décrite ici surmonte ces limitations avec une géométrie simple de conception et de fabrication. Plus important encore, ce système peut être intégré à l&#39;approche d&#39;ima...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>60ml Syringes</td>
<td>&nbsp;</td>
<td>BD</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Fura-2 fluorescence dye</td>
<td>&nbsp;</td>
<td>Molecular Probes Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Rhodamine123 Fluorescence dye</td>
<td>&nbsp;</td>
<td>Molecular Probes Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Glucose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Bovine Serum Albumin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>30" Silicone tubings</td>
<td>&nbsp;</td>
<td>Cole Palmer</td>
<td>&nbsp;</td>
<td>1/16 x 1/8in</td>
</tr>
<tr>
<td>1.5ml Eppendorf tubes</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Y-connectors</td>
<td>&nbsp;</td>
<td>Cole Palmer</td>
<td>&nbsp;</td>
<td>1/16&#x201D; &amp; 4mm</td>
</tr>
<tr>
<td>Syringe connectors</td>
<td>&nbsp;</td>
<td>Cole Palmer</td>
<td>&nbsp;</td>
<td>female luer plug 1/16&#x201D;</td>
</tr>
<tr>
<td>Straight connectors</td>
<td>&nbsp;</td>
<td>Cole Palmer</td>
<td>&nbsp;</td>
<td>1/16&#x201D;</td>
</tr>
<tr>
<td>Elbow connector</td>
<td>&nbsp;</td>
<td>Cole Palmer</td>
<td>&nbsp;</td>
<td>1/16&#x201D;</td>
</tr>
<tr>
<td>Havard syringe pump</td>
<td>&nbsp;</td>
<td>Harvard Apparatus</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Perifusion device</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Hot plate</td>
<td>&nbsp;</td>
<td>PMC</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Thermometer</td>
<td>&nbsp;</td>
<td>Omega Engineering Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Fraction collector</td>
<td>&nbsp;</td>
<td>Gibson</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Pippettor</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Inverted epiflorescence microscope</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>IX71</td>
<td>&nbsp;</td>
<tr>
<td>Charge-coupled device</td>
<td>&nbsp;</td>
<td>QImaging</td>
<td>&nbsp;</td>
<td>Retiga-SRV, Fast 1394</td>
</tr>		</tbody>
		</table>
		</div>

a multi-parametric islet perifusion system within a microfluidic perifusion device

Un dispositif microfluidique périfusion îlot a été développé pour l&#39;évaluation de la sécrétion d&#39;insuline des îlots dynamiques multiples et simultanées imagerie de fluorescence de l&#39;influx de calcium et mitochondrial changements potentiels. Le dispositif se compose de trois couches: la première couche contient un tableau de micro-puits (diamètre de 500 um et 150 um de profondeur) qui aident à immobiliser les îlots tandis exposés à des flux et d&#39;optimiser la surface exposée des îlots; la seconde couche contient une circulaire périfusion chambre (3 mm de profondeur, 7 mm de diamètre), et la troisième couche contient un canal d&#39;amenée de mélange que les fans avant injection dans la chambre périfusion (2 mm de largeur, 19 mm de longueur et de 500 um de hauteur) pour optimiser l&#39;efficacité du mélange avant d&#39;entrer dans la chambre périfusion. La création de gradients de glucose différents dont un linéaire, en forme de cloche, et des formes carrées peuvent également être créés dans le réseau périfusion microfluidique et est démontrée.

Watch this Scientific Journal Video about A Multi-Parametric Islet Perifusion System within a Microfluidic Perifusion Device at JoVE.com

A Multi-Parametric Islet Perifusion System within a Microfluidic Perifusion Device

Un dispositif microfluidique périfusion îlot a été développé pour l&#39;évaluation de la sécrétion d&#39;insuline des îlots dynamiques multiples et simultanées imagerie de fluorescence de l&#39;influx de calcium et mitochondrial changements potentiels.

Un système multi-paramétriques Islet périfusion dans un dispositif microfluidique périfusion

A microfluidic islet perifusion device was developed for the assessment of dynamic insulin secretion of multiple islets and simultaneous fluorescence ...

a-multi-parametric-islet-perifusion-system-within-microfluidic

Research

JoVE Journal

Biology

11.8K vues.  University of Illinois, Chicago. Un dispositif microfluidique périfusion îlot a été développé pour l'évaluation de la sécrétion d'insuline des îlots dynamiques multiples et simultanées imagerie de fluorescence de l'influx de calcium et mitochondrial changements potentiels.

Vidéo: A Multi-Parametric Islet Perifusion System within a Microfluidic Perifusion Device

Murine Pancreatic Islet Isolation

Isolement murin îlots pancréatiques

Recherche

Biologie

Fabrication of a Microfluidic Device for the Compartmentalization of Neuron Soma and Axons

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for culturing neurons. Previously, a silicon wafer was patterned with the design for the neuron microfluidic device using SU-8 and photolithography to create a master mold, or what we simply refer to as a "master". Next, we pour the silicon polymer PDMS on top of the master which is then cured by heating the PDMS to 80&deg;C for 1 hour. The PDMS forms a negative mold of the device. The PDMS is then carefully cut and lifted away from the master. Holes are punched where the reservoirs will be and the excess PDMS trimmed away from the device. Nitrogen is used to blow away any excess debris from the device. At this point the devices are now ready for use and can either bonded to corning No. 1 cover glass with a plasma sterilizer/cleaner or can be reversibly bound to the cover glass by simply placing the device on top of the cover glass. The reversible bonding of the device to glass is covered in a separate video and requires first that the device be sterilized either with 70% ethanol or by autoclaving. Plasma treating sterilizes the devices so no further treatment is necessary. It is, however, important, when plasma-treating the devices, to add liquid to the devices within 10 minutes of the plasma treatment while the surfaces are still hydrophilic. Waiting longer than 10 minutes to add liquid to the device makes it difficult for the liquid to enter the device. The neuron devices are typically plasma-bound to cover glass and 0.5 mg/ml poly-L-lysine (PLL) in pH 8.5 borate buffer is immediately added to the device. After a minimum of 3 hours incubating with PLL, the devices are washed with dH2O water a minimum of 3 times with at least 15 minutes between each wash. Next, the water is removed and fresh media is added to the device. At this point the device is ready for use. It is important to remember at this point to never remove all the media from the device. Always leave media in the main channel.

In this video we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to farbricate a microfluidic device for culturing neurons.

Fabrication d&#39;un dispositif microfluidique pour la compartimentation du Neuron Soma et des axones

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for ...

A Microfluidic Device with Groove Patterns for Studying Cellular Behavior

We describe a microfluidic device with microgrooved patterns for studying cellular behavior. This microfluidic platform consists of a top fluidic channel and a bottom microgrooved substrate. To fabricate the microgrooved channels, a top poly(dimethylsiloxane) (PDMS) mold containing the impression of the microfluidic channels was aligned and bonded to a microgrooved substrate. Using this device, mouse fibroblast cells were immobilized and patterned within microgrooved substrates (25, 50, 75, and 100 &mu;m wide). To study apoptosis in a microfluidic device, media containing hydrogen peroxide, Annexin V, and propidium iodide was perfused into the fluidic channel for 2 hours. We found that cells exposed to the oxidative stress became apoptotic. These apoptotic cells were confirmed by Annexin V that bound to phosphatidylserine at the outer leaflet of the plasma membrane during the apoptosis process. Using this microfluidic device with microgrooved patterns, the apoptosis process was observed in real-time and analyzed by using an inverted microscope containing an incubation chamber (37&deg;C, 5% CO2). Therefore, this microfluidic device incorporated with microgrooved substrates could be useful for studying the cellular behavior and performing high-throughput drug screening.

We describe a protocol for the fabrication of microfluidic devices that can enable cell capture and culture. In this approach patterned microstructures such as grooves within microfluidic channels are used to create low shear stress regions within which cell can dock.

Un dispositif microfluidique avec des modèles Groove pour étudier le comportement cellulaire

We describe a microfluidic device with microgrooved patterns for studying cellular behavior.  This microfluidic platform consists of a top fluidic channel ...

A Gradient-generating Microfluidic Device for Cell Biology

 The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described.  A microfluidic platform is an enabling experimental tool, because it can precisely manipulate fluid flows, enable high-throughput experiments, and generate stable soluble concentration gradients.  Compared to conventional gradient generators, poly(dimethylsiloxane) (PDMS)-based  microfluidic devices can generate stable concentration gradients of growth factors with well-defined profiles.  Here, we developed simple gradient-generating microfluidic devices with three separate inlets.  Three microchannels combined into one microchannel to generate concentration gradients.  The stability and shape of growth factor gradients were confirmed by fluorescein isothyiocyanate (FITC)-dextran with a molecular weight similar to epidermal growth factor (EGF).  Using this microfluidic device, we demonstrated that fibroblasts exposed to concentration gradients of EGF migrated toward higher concentrations.  The directional orientation of cell migration and motility of migrating cells were quantitatively assessed by cell tracking analysis.  Thus, this gradient-generating microfluidic device might be useful for studying and analyzing the behavior of migrating cells.

We describe a protocol for the microfabrication of the gradient-generating microfluidic device that can generate spatial and temporal gradients in well-defined microenvironment. In this approach, the gradient-generating microfluidic device can be used to study directed cell migration, embryogenesis, wound healing, and cancer metastasis.

Un dispositif microfluidique Dégradé génératrices de biologie cellulaire

 The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described.  A microfluidic platform is an ...

Preparing E18 Cortical Rat Neurons for Compartmentalization in a Microfluidic Device

In this video, we demonstrate the preparation of E18 cortical rat neurons. E18 cortical rat neurons are obtained from E18 fetal rat cortex previously dissected and prepared. The E18 cortex is, upon dissection, immediately dissociated into individual neurons. It is possible to store E18 cortex in Hibernate E buffer containing B27 at 4&deg;C for up to a week before the dissociation is performed. However, there will be a drop in cell viability. Typically we obtain our E18 Cortex fresh. It is transported to the lab in ice cold Calcium free Magnesium free dissection buffer (CMFM). Upon arrival, trypsin is added to the cortex to a final concentration of 0.125%. The cortex is then incubated at 37&deg;C for 8 minutes. DMEM containing 10% FBS is added to the cortex to stop the reaction. The cortex is then centrifuged at 2500 rpm for 2 minutes. The supernatant is removed and 2 ml of Neural Basal Media (NBM) containing 2% B27 (vol/vol) and 0.25% Glutamax (vol/vol) is added to the cortex which is then re-suspended by pipetting up and down. Next, the cortex is triturated with previously fire polished glass pipettes, each with a successive smaller opening. After triturating, the cortex is once again centrifuged at 2500 rpm for 2 minutes. The supernatant is then removed and the cortex pellet re-suspended with 2 ml of NBM containing B27 and Glutamax. The cell suspension is then passed through a 40 um nylon cell strainer. Next the cells are counted. The neurons are now ready for loading into the neuron microfluidic device.

In this video we demonstrate the preparation of E18 Cortical Rat Neurons.

Préparation E18 neurones de rat corticale compartimentation dans un dispositif microfluidique

In this video, we demonstrate the preparation of E18 cortical rat neurons. E18 cortical rat neurons are obtained from E18 fetal rat cortex previously ...

Cell Capture Using a Microfluidic Device

Capturez cellule à l&#39;aide d&#39;un dispositif microfluidique

Assessing Neural Stem Cell Motility Using an Agarose Gel-based Microfluidic Device

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is largely due to the difficulty with which one can create a cell-compatible and steady microenvironment using conventional microfabrication techniques and materials. We address this problem via the introduction of a novel microfabrication material, agarose gel, as the base material for the microfluidic device. Agarose gel is highly malleable, and permeable to gas and nutrients necessary for cell survival, and thus an ideal material for cell-based assays. We have shown previously that agarose gel based devices have been successful in studying bacterial and neutrophil cell migration2. In this report, three parallel microfluidic channels are patterned in an agarose gel membrane of about 1mm thickness. Constant flows with media/buffer are maintained in the two side channels using a peristaltic pump. Cells are maintained in the center channel for observation. Since the nutrients and chemicals in the side channels are constantly diffusing from the side to center channel, the chemical environment of the center channel is easily controlled via the flow along the side channels. Using this device, we demonstrate that the movement of neural stem cells can be monitored optically with ease under various chemical conditions, and the experimental results show that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells. Motility of neural stem cells is an important biomarker for assessing cells aggressiveness, thus tumorigenic factor3. Deciphering the mechanism underlying NSC motility will yield insight into both disorders of neural development and into brain cancer stem cell invasion.

We demonstrate that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells(NSCs) using a novel agarose gel based microfluidic device. This technology can be readily adaptable to other mammalian cell systems where cell sources are scarce, such as human neural stem cells, and the turn around time is critical.

Évaluer la motilité Neural Stem Cell utilisant un gel d&#39;agarose à base dispositif microfluidique

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is ...

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 ...

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

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has been proposed that the microenvironment that the pathogen encounters in the commensal layer is important in determining the extent of colonization. Current culture methods for investigating pathogen colonization are not well suited for investigating this hypothesis as they do not enable co-culture of bacteria and epithelial cells in a manner that mimics the GI tract microenvironment. Here we describe a microfluidic co-culture model that enables independent culture of eukaryotic cells and bacteria, and testing the effect of the commensal microenvironment on pathogen colonization. The co-culture model is demonstrated by developing a commensal Escherichia coli biofilm among HeLa cells, followed by introduction of enterohemorrhagic E. coli (EHEC) into the commensal island, in a sequence that mimics the sequence of events in GI tract infection.

This protocol describes a microfluidic co-culture model for simultaneous and localized culture of epithelial cells and bacteria. This model can be used for investigating the role of different soluble molecular signals on pathogenesis as well as screen the effectiveness of putative probiotic bacterial strains.

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 ...

A Microfluidic Device for Quantifying Bacterial Chemotaxis in Stable Concentration Gradients

Chemotaxis allows bacteria to approach sources of attractant chemicals or to avoid sources of repellent chemicals. Bacteria constantly monitor the concentration of specific chemoeffectors by comparing the current concentration to the concentration detected a few seconds earlier. This comparison determines the net direction of movement. Although multiple, competing gradients often coexist in nature, conventional approaches for investigating bacterial chemotaxis are suboptimal for quantifying migration in response to concentration gradients of attractants and repellents. Here, we describe the development of a microfluidic chemotaxis model for presenting precise and stable concentration gradients of chemoeffectors to bacteria and quantitatively investigating their response to the applied gradient. The device is versatile in that concentration gradients of any desired absolute concentration and gradient strength can be easily generated by diffusive mixing. The device is demonstrated using the response of Escherichia coli RP437 to gradients of amino acids and nickel ions.

This protocol describes the development of a microfluidic device for investigating bacterial chemotaxis in stable concentration gradients of chemoeffectors.