Diese Arbeit wurde von AAUW internationalen Gemeinschaft zu Adeola Adewola, NIH / NCRR (U42RR023245) zu Jose Oberholzer, und die Chicago Diabetes-Projekt unterstützt.

A. Microfabrication der 3-Schicht-mikrofluidischen perifusion Gerät Bottom Well-Master Protokoll (150 um Tiefbrunnen) <ol><li> Reinigen Sie die Scheibe mit einer Rasierklinge, wenn nötig. Reinigung mit Aceton, Methanol und IPA. Führen Plasmabehandlung bei 50 Watt für 30 s. </li><li> Spin SU8-100 @ 2000 rpm. [Schritt 1: 500 min, 10 s, 100 rpm / s, Schritt 2: 2000 rpm, 30 s, 300 rpm / s]. [Hinweis: Halten Sie nicht die Wafer mit einer Pinzette nach dem Schleudern SU8] </li><li> Softbake der Wafer bei 65 ° C für 20 min und bei 95 ° C für 50 min. </li><li> Der Wafer wird mit UV-Licht durch eine gewünschte Maske belichtet. Dose für 150 pm beträgt 650 mJ / cm 2. </li><li> Post Exposure Bake der Wafer bei 65 ° C für 1 min und bei 95 ° C für 12 min. </li><li> Entwickeln Sie die Wafer in SU8 Entwickler für 15 min. </li></ol> Microchannel Master Protokoll (500 um Tiefbrunnen) <ol><li> Reinigen Sie die Scheibe mit einer Rasierklinge, wenn nötig. Reinigung mit Aceton, Methanol und IPA. Führen Plasmabehandlung bei 50 Watt für 30 s. </li><li> Spin SU8-2150 @ 1000 Umdrehungen pro Minute. [Schritt 1: 500 min, 10 s, 100 rpm / s, Schritt 2: 1000 rpm, 30 s, 300 rpm / s]. </li><li> Softbake der Wafer bei 65 ° C für 15 min und bei 95 ° C für 2 Stunden und 30 min. </li><li> Der Wafer wird mit UV-Licht durch eine gewünschte Maske belichtet. Belichtungsdosis für 650 um Höhe beträgt 685 mJ / cm 2. </li><li> Post Exposure Bake der Wafer bei 65 ° C für 5 min und bei 95 ° C für 35 min. </li><li> Entwickeln Sie die Wafer in SU8 Entwickler für 20-30 min. </li></ol> PDMS-Aufbereitung <ol><li> Polydimethylsiloxan (PDMS)-Lösung wird durch gründliches Mischen 10 Teile aus Silikon-Elastomer mit 1 Teil Härter eines Standard Sylgard 184 Kit vorbereitet. </li><li> Die Blasen in der PDMS-Lösung beim Mischen erzeugt werden entfernt mit einem Vakuum-Exsikkator. </li><li> Die blasenfreie PDMS Lösung wird langsam auf die SU8 Meister und eine leere Petrischale für die dritte Schicht verzichtet. </li><li> Die Temperatur der Heizplatte auf 75 ° C eingestellt ist und der PDMS ist bei dieser Temperatur für 2 Stunden ausgehärtet </li><li> Ein-und Auslässen und den Austausch Brunnen werden mit der entsprechenden Größe Locher gestanzt. </li><li> Die Schichten werden auf einem Glasträger (Größe 0,1 mm) mit einem Handheld-Plasma-Gerät verbunden. </li></ol> B. Versuchsaufbau <ol><li> Durchfluss 70% igem Ethanol durch die Mikro-Gerät zu sterilisieren. Flow-DI-Wasser zum Auswaschen der Ethanol. Perfundieren 50 ml von 0,5% BSA durch das Gerät, um unspezifische Adsorption von Insulin an den Mikrokanal Wänden zu verhindern. </li><li> Die Glucose Rampensteilheit und andere damit zusammenhängende Gradienten von LabView-Software, mit der Spritze Pumpen getestet, um sicherzustellen, dass die Gradienten sind stabil sind kommuniziert. </li><li> 25-30 Mäusen Inseln wurden mit 5 uM Fura-2/AM (ein Kalzium-Indikator, Molecular Probes, CA) und 2,5 uM Rhodamin 123 (Rh123, eine mitochondriale Potenziale Indikator, Sigma, MO) für 30 min bei 37 ° C in inkubiert Krebs-Ringer-Puffer (KRB), enthaltend 2 mM Glucose </li><li> Die Mäuse Inseln sind sorgfältig in mikrofluidischen perifusion Gerät durch die Einlassöffnung pipettiert. </li><li> Die Mikrofluidik-Netzwerk wird dann durch Anschließen der Einmündung in den Spritzenpumpen mit Tygon-Schlauch und ein Y-Stecker und die Steckdose zu Fraktionssammler. Das Gerät sitzt auf einem Heiztisch (37 ° C) auf dem Mikroskop und den Zulaufschlauch ist auf einer Heizplatte erhitzt, um die Temperatur der Kammer und die Lösung bei 37 ° C zu halten </li><li> Unmittelbar nach der Installation werden die Mäuse Inselchen mit KRB mit 2 mM Glukose für 10 min und dann eine Glucose-Rampe (2mm 25mm) für 25 min perifused. </li><li> Zeitraffer-Bilder werden gesammelt und alle 15 s analysiert SimplePCI Software. Die perifusate ist auch jede Minute mit einem Fraktionssammler der Insulin-Sekretion mittels ELISA-Kit analysieren gesammelt. </li></ol> Repräsentative Ergebnisse Maus Inseln wurden mit einem linearen Gradienten von 2-25 mM Glucose perifused. Wie in Abbildung 1 gezeigt, sind Calcium-Einstrom und die Insulinsekretion nach etwa 13 Minuten von perifusion, entsprechend 6 mM Glukose ausgelöst. Änderungen in der mitochondrialen Potentiale sind früher als erwartet, etwa 11 Minuten gesehen. Diese Daten zeigen, der Vorteil der Verwendung dieser mikrofluidischen Netzwerk Insel Physiologie zu charakterisieren. <img src="/files/ftp_upload/1649/1649fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Physiologische Reaktionen auf Zell-Stimulation Insel.

<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>

Traditionelle Insel perifusion Systeme (makroskopischen und mikro-) einige Einschränkungen, einschließlich komplexer der Einrichtung und Design, hohe technische Anforderungen und Schwierigkeiten, benutzerdefinierte vorgeschriebenen chemischen Gradienten in das System zu erstellen. Die mikrofluidischen perifusion hier beschriebene System überwindet diese Einschränkungen mit einfachen Geometrie der Konstruktion und Fertigung. Noch wichtiger ist, kann dieses System mit Fluoreszenz-Imaging-Ansatz, die als ein einzigarti...

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		<thead>
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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>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>
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<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>
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a multi-parametric islet perifusion system within a microfluidic perifusion device

Ein mikrofluidischen Insel perifusion Gerät wurde für die Beurteilung der Dynamik der Insulinsekretion von mehreren Inselchen und gleichzeitige Fluoreszenz-Imaging von Calcium-Einstrom und die mitochondriale möglichen Veränderungen entwickelt. Das Gerät besteht aus drei Schichten: erste Schicht enthält eine Reihe von mikro-Brunnen (500 Mikrometer Durchmesser und 150 um Tiefe), die zu den Inseln zu immobilisieren, während ausgesetzt zu fließen und maximieren die exponierte Fläche der Inseln zu helfen, die zweite Schicht enthält eine kreisförmige perifusion Kammer (3 mm tief, 7 mm Durchmesser), und die dritte Schicht enthält einen Einlass-Mischkanal, dass die Fans vor der Injektion in die perifusion Kammer (2 mm in der Breite, 19 mm in der Länge und 500 pm in der Höhe) zur Optimierung der die Mischwirkung vor dem Eintritt in die perifusion Kammer. Die Schaffung verschiedener Glukose Gradienten mit einem linear, Glockenform, und quadratischen Formen können auch in der mikrofluidischen perifusion Netzwerk geschaffen werden, und demonstriert.

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

Ein mikrofluidischen Insel perifusion Gerät wurde für die Beurteilung der Dynamik der Insulinsekretion von mehreren Inselchen und gleichzeitige Fluoreszenz-Imaging von Calcium-Einstrom und die mitochondriale möglichen Veränderungen entwickelt.

Ein Multi-Parametric Islet Perifusion System innerhalb eines mikrofluidischen Perifusion Geräte

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 Aufrufe.  University of Illinois, Chicago. Ein mikrofluidischen Insel perifusion Gerät wurde für die Beurteilung der Dynamik der Insulinsekretion von mehreren Inselchen und gleichzeitige Fluoreszenz-Imaging von Calcium-Einstrom und die mitochondriale möglichen Veränderungen entwickelt.

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

Murine Pancreatic Islet Isolation

Murine Inselzellen der Bauchspeicheldrüse Isolation

Forschung

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.

Herstellung eines mikrofluidischen Vorrichtung für die Abschottung der Neuron Soma und Axonen

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.

Einer mikrofluidischen Vorrichtung mit Groove Patterns für ein Studium Cellular Behavior

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.

Ein Gradient-Erzeugung Mikrofluidikvorrichtung für Zellbiologie

 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.

Vorbereitung E18 Kortikale Rattenneuronen für Kompartimentierung in einer mikrofluidischen Vorrichtung

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

Zell-Capture mit einer mikrofluidischen Vorrichtung

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.

Die Beurteilung Neural Stem Cell Motilität Mit einem Agarosegel-basierte Mikrofluidikvorrichtung

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.

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

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

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.

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

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

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.