Questo lavoro è stato sostenuto da AAUW internazionale borsa di studio per Adeola Adewola, NIH / NCRR (U42RR023245) a Jose Oberholzer, e il Progetto Diabete Chicago.

Microfabrication A. di 3-strati dispositivo perifusion microfluidica Bene fondo Maestro protocollo (150 micron pozzi profondi) <ol><li> Pulire le fette che utilizzano una lama di rasoio, se necessario. Pulire con acetone, metanolo, e IPA. Eseguire il trattamento al plasma a 50 Watt per 30 secondi </li><li> Spin SU8-100 @ 2000 rpm. [Fase 1: 500 giri, 10 s, 100 giri / s, Fase 2: 2000 rpm, 30 s, 300 giri / s]. [NOTA: non tenere il wafer con una pinzetta dopo filatura SU8] </li><li> Morbido cuocere il wafer a 65 ° C per 20 min e 95 ° C per 50 min. </li><li> Il wafer viene esposto alla luce UV attraverso una maschera desiderata. Dose per 150 micron altezza è di 650 mJ / cm 2. </li><li> Post-esposizione cuocere il wafer a 65 ° C per 1 minuto e 95 ° C per 12 min. </li><li> Sviluppare il wafer in SU8 sviluppatore per 15 min. </li></ol> Microcanali Maestro protocollo (500 micron pozzi di profondità) <ol><li> Pulire le fette che utilizzano una lama di rasoio, se necessario. Pulire con acetone, metanolo, e IPA. Eseguire il trattamento al plasma a 50 Watt per 30 secondi </li><li> Spin SU8-2150 @ 1000 rpm. [Fase 1: 500 giri, 10 s, 100 giri / s, Fase 2: 1000 rpm, 30 s, 300 giri / s]. </li><li> Morbido cuocere il wafer a 65 ° C per 15 minuti e 95 ° C per 2 ore e 30 min. </li><li> Il wafer viene esposto alla luce UV attraverso una maschera desiderata. Dose di esposizione per 650 micron altezza è 685 mJ / cm 2. </li><li> Post-esposizione cuocere il wafer a 65 ° C per 5 minuti e 95 ° C per 35 min. </li><li> Sviluppare il wafer in SU8 sviluppatore per 20-30 min. </li></ol> PDMS soluzione di preparazione <ol><li> Polidimetilsilossano (PDMS) soluzione viene preparata mescolando accuratamente da 10 parti di elastomeri di silicone con 1 parte di catalizzatore di un kit standard Sylgard 184. </li><li> Le bolle generate nella soluzione PDMS durante il processo di miscelazione vengono rimossi con un essiccatore a vuoto. </li><li> La bolla senza soluzione di PDMS sta lentamente erogato sulla SU8 maestri e un piatto vuoto Petri per il terzo strato. </li><li> La temperatura della piastra è impostato a 75 ° C e il PDMS è curata a questa temperatura per 2 ore </li><li> Insenature, prese e pozzi di cambio sono perforati utilizzando l&#39;apposito perforatrice dimensioni. </li><li> Gli strati sono legati insieme su un vetrino (dimensioni 0,1 mm) utilizzando un dispositivo palmare plasma. </li></ol> B. Apparato Sperimentale <ol><li> Etanolo al 70% del flusso attraverso la micro-dispositivo per la sterilizzazione. DI flusso d&#39;acqua per lavare l&#39;etanolo. Profumato 50 ml di BSA 0,5% attraverso il dispositivo per evitare l&#39;adsorbimento non specifico di insulina alle pareti microcanali. </li><li> La rampa di pendenza di glucosio e di altre pendenze legate generati dal software LabVIEW che comunica con la siringa sono testati pompe per assicurarsi che le pendenze sono stabili. </li><li> 25-30 isolotti topi sono stati incubati con 5 Fura-2/AM mM (un indicatore di calcio, sonde molecolari, CA) e 2,5 mM rodamina 123 (Rh123, un indicatore potenziale mitocondriale, Sigma, MO) per 30 minuti a 37 ° C in Krebs-Ringer buffer (KRB) contenente 2 glucosio mM </li><li> Gli isolotti topi vengono attentamente pipettati nel dispositivo perifusion microfluidi attraverso la porta di entrata. </li><li> La rete è l&#39;installazione della microfluidica collegando l&#39;ingresso alle pompe siringa usando tubi Tygon e di un connettore a Y e la presa di raccoglitore di frazioni. Il dispositivo si siede su una fase di riscaldamento (37 ° C) sul microscopio e il tubo di aspirazione viene riscaldata su una piastra per mantenere la temperatura della camera e la soluzione a 37 ° C. </li><li> Immediatamente dopo l&#39;installazione, gli isolotti topi sono perifused con KRB contenente 2 mM glucosio per 10 minuti e poi una rampa di glucosio (2mm 25mm) per 25 min. </li><li> Time-lapse immagini vengono raccolti e analizzati ogni 15 s da un software SimplePCI. Il perifusate è anche raccolto ogni minuto usando un raccoglitore di frazioni di analizzare alla secrezione insulinica mediante il kit ELISA. </li></ol> Rappresentante Risultati Isolotti del mouse sono stati perifused con una sfumatura lineare di 2-25 mM glucosio. Come mostrato nella Figura 1, afflusso di calcio e la secrezione di insulina sono innescate dopo circa 13 minuti di perifusion, corrispondenti a 6 mM glucosio. Cambiamenti nelle potenzialità mitocondriali sono visto in precedenza come previsto, a circa 11 minuti. Questi dati dimostrano il vantaggio di utilizzare questa rete microfluidica per caratterizzare isolotto fisiologia. <img src="/files/ftp_upload/1649/1649fig1.jpg" alt="Figura 1" /> Figura 1. Reazioni fisiologiche alla stimolazione delle cellule insulari.

<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>

I sistemi tradizionali di perifusion isolotto (macroscala e microscala) hanno alcune limitazioni tra cui complesso di configurazione e design, elevate esigenze tecniche, e la difficoltà di creare utenti prescritto gradienti chimici nel sistema. Il sistema perifusion microfluidica descritto qui di superare queste limitazioni con geometria semplice di progettazione e fabbricazione. Più importante, questo sistema può essere integrato con l&#39;approccio di imaging a fluorescenza che forniscono come uno strumento unico p...

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		<th>Material Name</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<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>
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<td>Rhodamine123 Fluorescence dye</td>
<td>&nbsp;</td>
<td>Molecular Probes Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Glucose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Bovine Serum Albumin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>30" Silicone tubings</td>
<td>&nbsp;</td>
<td>Cole Palmer</td>
<td>&nbsp;</td>
<td>1/16 x 1/8in</td>
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<tr>
<td>1.5ml Eppendorf tubes</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Y-connectors</td>
<td>&nbsp;</td>
<td>Cole Palmer</td>
<td>&nbsp;</td>
<td>1/16&#x201D; &amp; 4mm</td>
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<td>Syringe connectors</td>
<td>&nbsp;</td>
<td>Cole Palmer</td>
<td>&nbsp;</td>
<td>female luer plug 1/16&#x201D;</td>
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<td>Straight connectors</td>
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<td>Cole Palmer</td>
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<td>Elbow connector</td>
<td>&nbsp;</td>
<td>Cole Palmer</td>
<td>&nbsp;</td>
<td>1/16&#x201D;</td>
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<td>Havard syringe pump</td>
<td>&nbsp;</td>
<td>Harvard Apparatus</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Perifusion device</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Hot plate</td>
<td>&nbsp;</td>
<td>PMC</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Thermometer</td>
<td>&nbsp;</td>
<td>Omega Engineering Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<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>
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a multi-parametric islet perifusion system within a microfluidic perifusion device

Un dispositivo microfluidic perifusion isolotto è stato sviluppato per la valutazione della secrezione insulinica dinamica di isole multiple e simultanee imaging di fluorescenza di afflusso di calcio e mitocondriale potenziali cambiamenti. Il dispositivo è costituito da tre strati: primo strato contiene un array di microscala pozzi (500 micron di diametro e 150 micron di profondità) che aiutano a immobilizzare le isolette, mentre esposto al flusso e massimizzare la superficie esposta delle isole, il secondo strato contiene una circolare perifusion camera (3 mm di profondità, 7 mm di diametro) e il terzo strato contiene un ingresso di miscelazione dei canali che i fan prima dell&#39;iniezione nella camera perifusion (2 mm in larghezza, 19 mm di lunghezza e 500 micron di altezza) per l&#39;ottimizzazione l&#39;efficienza di miscelazione prima di entrare nella camera di perifusion. La creazione di gradienti di glucosio diverse tra cui una lineare, forma a campana, e forme squadrate può anche essere creato nella rete perifusion microfluidica ed è dimostrato.

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 dispositivo microfluidic perifusion isolotto è stato sviluppato per la valutazione della secrezione insulinica dinamica di isole multiple e simultanee imaging di fluorescenza di afflusso di calcio e mitocondriale potenziali cambiamenti.

Un multi-parametrica Perifusion Sistema isolotto all&#39;interno di un dispositivo Perifusion Microfluidic

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 Views.  University of Illinois, Chicago. Un dispositivo microfluidic perifusion isolotto è stato sviluppato per la valutazione della secrezione insulinica dinamica di isole multiple e simultanee imaging di fluorescenza di afflusso di calcio e mitocondriale potenziali cambiamenti.

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

Murine Pancreatic Islet Isolation

Murino pancreatica isolamento Islet

Ricerca

Biologia

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.

Fabbricazione di un dispositivo a microfluidi per la compartimentazione dei Neuron Soma e assoni

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 dispositivo a microfluidi con pattern Groove per studiare il comportamento cellulare

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 gradiente di generazione del dispositivo Microfluidic di Biologia Cellulare

 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.

Preparazione E18 neuroni corticali di ratto per compartimentazione in un dispositivo a microfluidi

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

Cattura cellula Utilizzando un dispositivo a microfluidi

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.

Valutare motilità delle cellule staminali neurali L&#39;utilizzo di un gel a base di Agarose dispositivo a microfluidi

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

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

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.

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

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

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.