وأيد هذا العمل من قبل الجمعية الأميركية للجامعيات الزمالة الدولية لAdewola اديولا ، NIH / NCRR (U42RR023245) لخوسيه أوبرهولزر ، ومشروع السكري شيكاغو.

ألف التصنيع الدقيق من 3 طبقات الجهاز perifusion ميكروفلويديك حسنا أسفل ماجستير البروتوكول (150 ميكرون الآبار العميقة) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تنظيف الرقاقة باستخدام شفرة حلاقة اذا لزم الامر. نظيفة مع الأسيتون والميثانول ، ومعهد الإدارة العامة. تنفيذ المعاملة البلازما في 50 واط لمدة 30 ثانية. </li><li style=";text-align:right;direction:rtl"> تدور SU8 - 100 @ 2000 دورة في الدقيقة. [الخطوة 1 : 500 دورة في الدقيقة ، 10 ثانية و 100 دورة في الدقيقة / ثانية ، الخطوة 2 : 2000 دورة في الدقيقة ، 30 ثانية ، و 300 دورة في الدقيقة / ق]. [ملاحظة : لا تحمل الرقاقة مع ملاقط بعد الغزل SU8] </li><li style=";text-align:right;direction:rtl"> خبز لينة الرقاقة عند 65 درجة مئوية لمدة 20 دقيقة وعند 95 درجة مئوية لمدة 50 دقيقة. </li><li style=";text-align:right;direction:rtl"> يتعرض الرقاقة للأشعة فوق البنفسجية من خلال قناع المطلوب. الجرعة عن 150 ميكرومتر ارتفاع 650 ميغا جول / سم 2. </li><li style=";text-align:right;direction:rtl"> بعد التعرض خبز الرقاقة عند 65 درجة مئوية لمدة 1 دقيقة وعلى 95 درجة مئوية لمدة 12 دقيقة. </li><li style=";text-align:right;direction:rtl"> تطوير رقاقة في SU8 المطور لمدة 15 دقيقة. </li></ol> بروتوكول متناهية ماجستير (500 ميكرون الآبار العميقة) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تنظيف الرقاقة باستخدام شفرة حلاقة اذا لزم الامر. نظيفة مع الأسيتون والميثانول ، ومعهد الإدارة العامة. تنفيذ المعاملة البلازما في 50 واط لمدة 30 ثانية. </li><li style=";text-align:right;direction:rtl"> تدور SU8 - 2150 @ 1000 دورة في الدقيقة. [الخطوة 1 : 500 دورة في الدقيقة ، 10 ثانية و 100 دورة في الدقيقة / ثانية ، الخطوة 2 : 1000 دورة في الدقيقة ، 30 ثانية ، و 300 دورة في الدقيقة / ق]. </li><li style=";text-align:right;direction:rtl"> لينة خبز الرقاقة عند 65 درجة مئوية لمدة 15 دقيقة وعند 95 درجة. مئوية لمدة 2 ساعة ودقيقة و 30 </li><li style=";text-align:right;direction:rtl"> يتعرض الرقاقة للأشعة فوق البنفسجية من خلال قناع المطلوب. جرعة التعرض لارتفاع 650 ميكرون هو 685 ميغا جول / سم 2. </li><li style=";text-align:right;direction:rtl"> بعد التعرض خبز الرقاقة عند 65 درجة مئوية لمدة 5 دقائق وعلى 95 درجة مئوية لمدة 35 دقيقة. </li><li style=";text-align:right;direction:rtl"> تطوير رقاقة في SU8 المطور لمدة 20-30 دقيقة. </li></ol> PDMS حل إعداد <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> مستعدة Polydimethylsiloxane (PDMS) حل عن طريق خلط دقيق 10 أجزاء من المطاط الصناعي السيليكون مع 1 جزء من وكيل علاج لعدة Sylgard القياسية 184. </li><li style=";text-align:right;direction:rtl"> تتم إزالة فقاعات ولدت في حل PDMS أثناء عملية الخلط باستخدام مجفف فراغ. </li><li style=";text-align:right;direction:rtl"> هو الاستغناء ببطء فقاعة خالية حل PDMS على وسادة SU8 طبق بيتري فارغة للطبقة ثالثة. </li><li style=";text-align:right;direction:rtl"> يتم تعيين درجة حرارة لوحة الساخن الى 75 درجة مئوية غير مملح وPDMS وعند هذه الدرجة لمدة 2 ساعة </li><li style=";text-align:right;direction:rtl"> واللكم مداخل ومنافذ الصرف والآبار خارج باستخدام ثقب الناخس الحجم المناسب. </li><li style=";text-align:right;direction:rtl"> مستعبدين طبقات معا على شريحة زجاجية (حجم 0.1 ملم) باستخدام جهاز البلازما المحمولة. </li></ol> باء الإعداد التجريبي <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تدفق الايثانول 70 ٪ من خلال الجهاز الصغير لتعقيم. DI تدفق المياه لغسل والايثانول. يروي BSA 50 مل من 0.5 ٪ من خلال جهاز لمنع امتصاص الأنسولين غير محددة على الجدران متناهية. </li><li style=";text-align:right;direction:rtl"> التدرج المنحدر الجلوكوز والتدرجات الأخرى ذات الصلة التي تم إنشاؤها بواسطة برنامج LABVIEW الذي يتصل مع المحقنة يتم اختبار المضخات للتأكد من التدرجات مستقرة. </li><li style=";text-align:right;direction:rtl"> وحضنت 25-30 الفئران الجزر مع Fura-2/AM ميكرومتر 5 (مؤشر الكالسيوم وتحقيقات الجزيئية ، كاليفورنيا) ، و 2.5 ميكرومتر رودامين 123 (Rh123 ، وهو مؤشر إمكانات الميتوكوندريا ، سيغما ، MO) لمدة 30 دقيقة عند 37 درجة مئوية في كريبس ، رينغر العازلة (KRB) التي تحتوي على السكر في 2 ملي </li><li style=";text-align:right;direction:rtl"> وpipetted بعناية الجزر الفئران في الجهاز perifusion ميكروفلويديك من خلال مدخل الميناء. </li><li style=";text-align:right;direction:rtl"> شبكة ميكروفلويديك ثم يتم الإعداد من خلال ربط مدخل إلى مضخات الحقن باستخدام أنابيب Tygon وY - موصل ومنفذا لجمع الكسور. الجهاز يجلس على مرحلة التسخين (37 درجة مئوية) على المجهر ويتم تسخين الأنابيب مدخل على موقد للحفاظ على درجة حرارة الغرفة والحل عند 37 درجة مئوية. </li><li style=";text-align:right;direction:rtl"> مباشرة بعد الإعداد ، وperifused الجزر تحتوي على الفئران مع KRB 2 ملي الجلوكوز لمدة 10 دقيقة ثم منحدر الجلوكوز (2mm و25mM) لمدة 25 دقيقة. </li><li style=";text-align:right;direction:rtl"> يتم جمع الوقت الفاصل بين الصور وحلل كل ليالي 15 بواسطة برنامج SimplePCI. وتجمع أيضا perifusate كل دقيقة باستخدام جزء من هواة جمع وتحليل افراز الانسولين باستخدام ELISA عدة. </li></ol> ممثل النتائج وكانت الجزر perifused الفأر مع الانحدار الخطي للجلوكوز ملي 25/02. كما هو مبين في الشكل 1 ، وأثار تدفق الكالسيوم وإفراز الأنسولين بعد حوالي 13 دقيقة من perifusion الموافق الجلوكوز 6 ملم. وتعتبر التغيرات في إمكانات الميتوكوندريا في وقت سابق كما هو متوقع ، في حوالي 11 دقيقة. هذه البيانات يدل على الاستفادة من استخدام هذه الشبكة لتوصيف ميكروفلويديك جزيرة الفيزيولوجيا. <img src="/files/ftp_upload/1649/1649fig1.jpg" alt="الشكل 1" /> الشكل 1. الاستجابات الفسيولوجية للجزيرة تحفيز الخلية.

<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>

النظم التقليدية perifusion جزيرة (macroscale والميكروسكيل) بعض القيود المعقدة بما في ذلك الإعداد والتصميم ، والمتطلبات التقنية العالية ، وصعوبة إنشاء المستخدم التدرجات الكيميائية المنصوص عليها في النظام. نظام perifusion ميكروفلويديك الموصوفة هنا يتغلب على هذه القيود مع هندسة بسيطة للتصميم والتصنيع. أكثر أهمية ، ويمكن أن تكون متكاملة مع نهج هذا النظام التصوير مضان التي توفر أداة فريدة لدراسة جزيرة الفيزيولوجيا. تظاهر النظام مع نسبة الإشارة ضوضاء عالية والمكانية والزمانية القرار من هذه الإشارات مضان. النموذج الأولي للجهاز الحالي كما يمكن أن تعقد الاجهزة perifusion متعددة في رقاقة واحدة ، وتقديم أنواع مختلفة من microenvironments لأغراض تطبيق على نطاق واسع.

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		<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>
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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>
<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>
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<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>
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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>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>
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<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

ووضعت جزيرة perifusion ميكروفلويديك الجهاز لتقييم افراز الانسولين من الجزر الحيوية متعددة ومضان التصوير المتزامن لتدفق الكالسيوم والتغيرات المحتملة الميتوكوندريا. الجهاز يتكون من ثلاث طبقات : الطبقة الأولى تحتوي على مجموعة من الآبار الميكروسكيل (500 و 150 ميكرومتر قطرها ميكرون العمق) التي تساعد على شل الجزر في حين تتعرض لتدفق وزيادة المساحة المكشوفة من الجزر ، والثاني يحتوي على طبقة دائرية perifusion غرفة (3 ملم العميق ، 7 مم) ، وطبقة ثالثة تحتوي على مدخل القناة لخلط أن المشجعين من قبل حقنها في غرفة perifusion (2 ملم في العرض ، 19 ملم في الطول ، و 500 ميكرومتر في الطول) لتحسين كفاءة خلط قبل دخول غرفة perifusion. إنشاء تدرجات مختلفة بما في ذلك الجلوكوز على شكل جرس الخطية ، والأشكال المربعة ويمكن أيضا أن تنشأ في شبكة perifusion ميكروفلويديك وتظاهر هو.

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

ووضعت جزيرة perifusion ميكروفلويديك الجهاز لتقييم افراز الانسولين من الجزر الحيوية متعددة ومضان التصوير المتزامن لتدفق الكالسيوم والتغيرات المحتملة الميتوكوندريا.

A Perifusion جزيرة متعدد حدودي النظام داخل جهاز ميكروفلويديك Perifusion

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

Nanyang Technological University

Research

JoVE Journal

Biology

11.8K Views.  University of Illinois, Chicago. ووضعت جزيرة perifusion ميكروفلويديك الجهاز لتقييم افراز الانسولين من الجزر الحيوية متعددة ومضان التصوير المتزامن لتدفق الكالسيوم والتغيرات المحتملة الميتوكوندريا.

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

Murine Pancreatic Islet Isolation

فأري عزل جزيرة البنكرياس

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.

تصنيع جهاز ميكروفلويديك للتجزئة سوما الخلية العصبية والمحاوير

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.

جهاز ميكروفلويديك مع أنماط Groove لدراسة السلوك الخليوي

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.

جهاز لتوليد التدرج ميكروفلويديك لبيولوجيا الخلية

 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.

إعداد E18 العصبونات القشرية للتجزئة الجرذ في جهاز ميكروفلويديك

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

القبض على خلية باستخدام جهاز ميكروفلويديك

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.

تقييم العصبية على الحركة باستخدام الخلايا الجذعية جل المستندة Agarose الأجهزة ميكروفلويديك

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.

حجرة متعددة الخلية العصبية الجهاز العصبي المركزي ، الدبقية منهاج ميكروفلويديك المشارك الثقافة

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

The Microfluidic Probe: Operation and Use for Localized Surface Processing

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. Microfluidics is commonly associated with closed microchannels which limit their use to samples that can be introduced, and cultured in the case of cells, within a confined volume. On the other hand, micropipetting system have been used to locally perfuse cells and surfaces, notably using push-pull setups where one pipette acts as source and the other one as sink, but the confinement of the flow is difficult in three dimensions. Furthermore, pipettes are fragile and difficult to position and hence are used in static configuration only. 
The microfluidic probe (MFP) circumvents the constraints imposed by the construction of closed microfluidic channels and instead of enclosing the sample into the microfluidic system, the microfluidic flow can be directly delivered onto the sample, and scanned across the sample, using the MFP. . The injection and aspiration openings are located within a few tens of micrometers of one another so that a microjet injected into the gap is confined by the hydrodynamic forces of the surrounding liquid and entirely aspirated back into the other opening. The microjet can be flushed across the substrate surface and provides a precise tool for localized deposition/delivery of reagents which can be used over large areas by scanning the probe across the surface. 
In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer.

In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer solution.

ودقق ميكروفلويديك : تشغيل واستخدام لتصنيع السطحي المترجمة

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. ...

Platelet Adhesion and Aggregation Under Flow using Microfluidic Flow Cells

Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium has been exposed. The platelet adhesion cascade takes place in the presence of shear flow, a factor not accounted for in conventional (static) well-plate assays. This article reports on a platelet-aggregation assay utilizing a microfluidic well-plate format to emulate physiological shear flow conditions. Extracellular proteins, collagen I or von Willebrand factor are deposited within the microfluidic channel using active perfusion with a pneumatic pump. The matrix proteins are then washed with buffer and blocked to prepare the microfluidic channel for platelet interactions. Whole blood labeled with fluorescent dye is perfused through the channel at various flow rates in order to achieve platelet activation and aggregation. Inhibitors of platelet aggregation can be added prior to the flow cell experiment to generate IC50 dose response data.

The platelet adhesion cascade takes place in the presence of shear flow, a factor not accounted for in conventional (static) well-plate assays. This article reports on a platelet-aggregation assay utilizing a microfluidic well-plate format to emulate physiological shear flow conditions.

التصاق وتكدس الصفيحات تحت التدفق باستخدام خلايا تدفق ميكروفلويديك

Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium has been exposed. The platelet adhesion ...