Bu çalışma, Adeola Adewola, NIH / NCRR (U42RR023245) Jose Oberholzer ve Chicago Diyabet Proje AAUW uluslararası burs tarafından desteklenmiştir.

3-katmanlı mikroakışkan perifusion cihazın A. mikroimalat Alt Eh Master Protokolü (150 mm derin kuyu) <ol><li> Gofret gerekirse bir jilet kullanarak temizleyin. Aseton, Metanol ve IPA ile temizleyin. 30 saniye için 50 Watt plazma işlemi gerçekleştirin </li><li> Spin SU8-100 @ 2000 rpm. Adım 1: 500 dak, 10 sn, 100 dev / s, Adım 2: 2000 rpm, 30 s, 300 devir / s]. [NOT: SU8 iplik sonra cımbız ile gofret tutmayın] </li><li> Yumuşak fırında 65 gofret ° C 20 dakika ve 95 ° C de 50 dk. </li><li> Gofret, istenilen bir maske aracılığıyla UV ışınlarına maruz kalmaktadır. Doz 150 mikron yükseklik için 650 mJ / cm 2. </li><li> Mesaj maruz gofret 65 ° C 1 dakika ve 95 ° C de 12 dakika pişirin. </li><li> 15 dakika SU8 geliştirici gofret geliştirin. </li></ol> Mikrokanal Yüksek Lisans Protokolü (500 mm derin kuyu) <ol><li> Gofret gerekirse bir jilet kullanarak temizleyin. Aseton, Metanol ve IPA ile temizleyin. 30 saniye için 50 Watt plazma işlemi gerçekleştirin </li><li> Spin SU8-2150 @ 1000 rpm. Adım 1: 500 dak, 10 sn, 100 dev / s, Adım 2: 1000 rpm, 30 s, 300 devir / s]. </li><li> Yumuşak fırında 65 gofret ° C&#39;de 15 dakika ve 95 ° C 2 saat ve 30 dakika. </li><li> Gofret, istenilen bir maske aracılığıyla UV ışınlarına maruz kalmaktadır. 650 mikron yükseklik için maruz kalma dozu 685 mJ / cm 2 . </li><li> Mesaj maruz gofret 65 ° C 5 dakika ve 95 ° C de 35 dakika pişirin. </li><li> 20-30 dakika süreyle SU8 geliştirici gofret geliştirin. </li></ol> PDMS çözüm hazırlığı <ol><li> Polidimetilsiloksan (PDMS) çözümü 10 parça silikon elastomer standart bir Sylgard 184 kiti 1 kısım sertleştirici iyice karıştırılarak hazırlanır. </li><li> PDMS çözüm karıştırma işlemi sırasında oluşturulan kabarcıklar, bir vakum desikatörde kullanarak kaldırılır. </li><li> Kabarcık PDMS çözüm yavaş SU8 yüksek lisans ve üçüncü katman için boş bir Petri kabı üzerine sürülür. </li><li> Sıcak plaka sıcaklığı 75 ° C ve PDMS 2 saat bu sıcaklıkta kür </li><li> Uygun boyutta delgeç kullanılarak girişleri, çıkışları ve döviz kuyu açılmıştır. </li><li> Katmanları, bir el plazma cihazı kullanılarak bir cam slayt (büyüklüğü 0.1 mm) bir araya yapıştırılır. </li></ol> B. deney düzeneği <ol><li> Mikro cihaz üzerinden akış% 70 etanol sterilize etmek. Akış DI su etanol yıkayın. Mikrokanallı duvarları olmayan insülin özel adsorpsiyon önlemek için cihaz üzerinden 50 ml% 0.5 BSA serpmek. </li><li> Glukoz rampa degrade ve pompaları gradyanlar kararlı olduğundan emin olmak için test şırınga ile iletişim LabView yazılımı tarafından oluşturulan diğer ilgili gradiyent. </li><li> 37 ° C de 30 dakika boyunca 5 mcM Fura-2/AM (bir kalsiyum göstergesi, Moleküler probları, CA) ve 2.5 mcM rodamin 123 (Rh123, mitokondriyal bir potansiyelleri göstergesi, Sigma, MO) 25-30 fareler adacıklar ile inkübe edildi. 2 mM glukoz içeren Krebs-Ringer tampon (KRB) </li><li> Fareler adacıklar dikkatle giriş bağlantı noktası üzerinden mikroakışkan perifusion cihazı pipetlenir. </li><li> Tygon tüp ve bir Y-konektör ve fraksiyon toplayıcı çıkış kullanarak şırınga pompaları için giriş bağlayarak mikroakışkan ağ kurulumu. Cihaz mikroskop (37 ° C) ısıtma aşamasında oturur ve giriş tüp, 37 oda ve çözüm sıcaklığı tutmak için bir ocak ısıtmalı ° C </li><li> Hemen kurulumdan sonra, farelerin adacıklar KRB sonra, 10 dakika ve 25 dakika glukoz rampası (2mm 25mm) için 2 mM glukoz içeren perifused. </li><li> Zaman atlamalı görüntüleri her 15 sn SimplePCI yazılım tarafından toplanır ve analiz edilir. Perifusate da insülin salgılanması ELISA kiti kullanılarak analiz etmek için bir fraksiyon toplayıcı kullanarak her dakika toplanır. </li></ol> Temsilcisi Sonuçlar Fare adacıklar 2-25 mM glukoz doğrusal bir degrade ile perifused. Şekil 1&#39;de gösterildiği gibi, kalsiyum akını ve insülin salgılanması 6 mM glukoz ilgili perifusion yaklaşık 13 dakika sonra tetiklenir. Mitokondrial potansiyelleri değişiklikler, yaklaşık 11 dakika beklendiği gibi daha erken görülür. Bu veri, adacık fizyoloji karakterize etmek için bu mikroakışkan ağını kullanarak avantaj göstermektedir. <img src="/files/ftp_upload/1649/1649fig1.jpg" alt="Şekil 1" /> Şekil 1: Adacık hücre stimülasyonu için fizyolojik tepkiler .

<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>

Geleneksel adacık perifusion sistemleri (macroscale ve microscale), kurulum ve tasarım, yüksek teknik gereklilikler ve sistem kullanıcı öngörülen kimyasal degradeler oluşturmak için zorluk karmaşık dahil olmak üzere, bazı kısıtlamalar var. Burada açıklanan mikroakışkan perifusion sistemi basit tasarım ve imalat geometri ile bu sınırlamaların üstesinden gelir. Daha da önemlisi, bu sistem adacık fizyoloji çalışmak için benzersiz bir araç olarak sağlayan floresan görüntüleme yaklaşımı...

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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>
<tr>
<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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<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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<tr>
<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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<tr>
<td>Straight connectors</td>
<td>&nbsp;</td>
<td>Cole Palmer</td>
<td>&nbsp;</td>
<td>1/16&#x201D;</td>
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<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>
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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>
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<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

Mikroakışkan bir adacık perifusion cihazı, çok sayıda adacık ve kalsiyum girişini ve mitokondriyal potansiyel değişiklikleri eş zamanlı olarak floresan görüntüleme dinamik insülin salgılanması değerlendirilmesi için geliştirilmiştir. Cihaz üç tabakadan oluşur: İlk katman maruz kaldığında, maruz kalan yüzey alanını adacıklar akışı ve en üst düzeye çıkarmak için adacıklar hareketsiz yardımcı microscale kuyular (500 mikron çapında ve 150 mikron derinliğinde) bir dizi içerir; ikinci kat bir genelge içerir perifusion odası (3 mm derinliğinde, 7 mm çapında) ve üçüncü katman bir giriş karıştırma kanal içerdiğini fanlar önce optimize etmek için perifusion odasına enjeksiyon (genişliği 2 mm, 19 mm uzunluğunda, ve yüksekliği 500 mm) karıştırma verimlilik perifusion odasına girmeden önce. Doğrusal bir çan şekli de dahil olmak üzere çeşitli glukoz degradeler, ve kare şekiller oluşturma mikroakışkan perifusion ağı oluşturulacak ve gösterilmiştir.

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

Mikroakışkan bir adacık perifusion cihazı, çok sayıda adacık ve kalsiyum girişini ve mitokondriyal potansiyel değişiklikleri eş zamanlı olarak floresan görüntüleme dinamik insülin salgılanması değerlendirilmesi için geliştirilmiştir.

Mikroakışkan Perifusion Cihaz içindeki Multi-Parametrik Islet Perifusion Sistemi

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 Görüntüleme.  University of Illinois, Chicago. Mikroakışkan bir adacık perifusion cihazı, çok sayıda adacık ve kalsiyum girişini ve mitokondriyal potansiyel değişiklikleri eş zamanlı olarak floresan görüntüleme dinamik insülin salgılanması değerlendirilmesi için geliştirilmiştir.

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

Murine Pancreatic Islet Isolation

Murin Pankreas Adacık İzolasyonu

Biyoloji

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.

Nöron Soma ve Aksonlar compartmentalization mikroakışkan Aygıt Fabrikasyon

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.

Hücresel Davranış incelenmesi için Groove Desenler mikroakışkan Aygıt

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.

Hücre Biyolojisi Degrade üreten mikroakışkan Aygıt

 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.

Mikroakışkan Aygıt compartmentalization E18 Kortikal Rat Nöronlar hazırlanması

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

Hücre Yakalama mikroakışkan Cihaz Kullanımı

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.

Nöral Kök Hücre Motilite değerlendirilmesi Agaroz Jel tabanlı mikroakışkan Cihaz Kullanımı

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.

Çok bölmesi MSS Nöron glia Co-kültür mikroakışkan Platformu

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.

Çözünür Sinyal aracılı Etkileşimleri Araştırılması için Epitel Hücreleri ve Bakteriler mikroakışkan Co-kültür

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.