Bölüm 1: mikroakışkan nöron cihaz hazırlanması <ol><li> 3 &quot;SU-8 fotolitografi tarafından yapılmış bir model silikon gofret polidimetilsiloksan (PDMS) mikroakışkan kalıp döküm için kullanılır. Master kalıpları, ya da gibi SU-8 desenli silikon gofret bakın&quot; efendileri &quot;. </li><li> PDMS 10:1 w / w oranında sertleştirici ile karıştırılır, PDMS 10 gram, 1 gram sertleştirici eklenir. </li><li> PDMS sonra iyice karıştırılır ve silikon yonga üzerine dökülür. Silikon gofret 10 cm polistren Petri kabı içinde yer alıyor. Yaklaşık 4 mm kalınlığı ile ana kapsayacak şekilde PDMS, yaklaşık 12 g 15 g alır. </li><li> PDMS ile ana ardından Petri kabı kapağı kapalı bir vakum desikatöre yerleştirilir ve vakum uygulanır. Bu sertleştirici karıştırma sırasında tanıtıldı PDMS hava kabarcıkları yardımcı olmak için yapılır. Bu hava kabarcıkları için yaklaşık 15 dakika sürer. </li><li> PDMS ile ana desikatöre kaldırılır. 0.45 um filtresi ile bir hava tabancası kalan tüm kabarcıklarını çıkarmak için kullanılır. </li><li> Usta daha sonra tedavisi için 1 saat için 80 ° C sıcak plaka üzerine yerleştirilir. </li></ol> Not: bir vakum desikatöre mevcut değilse, ana birkaç saat oda sıcaklığında bırakılmalıdır. Bu hava kabarcıkları sonunda kendi kaybolacaktır. Sıcak bir levha mevcut değilse, PDMS oda sıcaklığında 24 saat sonra tedavi olacak. Not: ana kür işlemi sırasında düzey olduğundan emin olmak da önemlidir. Bölüm 2: Nöron mikroakışkan cihaz Kesme <ol><li> PDMS ana tedavi edildikten sonra, temiz bir kaput alınır. </li><li> Cerrahi bıçak kullanarak, bir daire çevresinde cihazların kesilir. Bir PDMS içine bıçak ekler, silikon yonga ile temas kurmaya ama gofret çok fazla baskı önemlidir, başka ana çatlar. </li><li> Cihazlar (her 3 &quot;Silikon Yüksek Lisans başına 4 cihazları vardır) etrafında bir daire şekilde kesilmiş, PDMS ana dikkatlice kaldırdı. Önceden kesilmiş eklenir Bu cerrahi bıçak hafifçe bükerek başlamış olabilir . </li><li> Sonra, rezervuarlar 8 mm doku biyopsisi yumruk kullanarak cihazın içine yumrukladı. </li><li> Cihazlar, sonra dörde ve aşırı PDMS cihaz Corning Kapak Cam (No: 1) 24 mm x 40 mm uyacak böylece uzakta kesilmiş. Azot havanın herhangi bir aşırı enkaz darbe için kullanılır. İnatçı enkaz 3M Scotch Marka 471 vinil bant kullanarak silinebilir. </li></ol> Bölüm 3: Plazma cam kapak fişleri cihazlar yapıştırma. <ol><li> Plazma Cleaner kapak fişleri harrick nöron cihazlar bağ kullanılır. Kısacası, plazma temizleyici cihazlar yerleştirilir ve bir vakum pompası odasından havayı tahliye etmek için kullanılır. </li><li> Vakum basıncı 300 milliTorr ulaşıldıktan sonra, güç elektrik bobini açık ve yüksek ayarlanır. </li><li> - Elektrik bobini &quot;elektronlar, iyonlar ve nötr atomlar veya moleküller oluşan kısmen iyonize gaz, plazma oluşturur <a href="http://www.harrickplasma.com/plasma_physics.php">http://www.harrickplasma.com/plasma_physics.php</a> odasında kalan hava moleküllerini sol. Plazma reaktif türler, cam ve kalıcı bir bağ oluşturur birlikte yerleştirildiği zaman, cihaz, yüzeyleri oluşturur. Plazma da, sıvıların yanı sıra kolaylaştırmak yardımcı hidrofilik yüzeyleri, döner. Ayrıca, plazma temizleyici cihazlar sterilize eder. Mikroakışkan aygıtı içeren PDMS cihazın yüzey plazma temizleyici, cam slaytlar ile birlikte ön yüzü yukarıya yerleştirilir. Cam ve mikroakışkan cihaz plazma tedavi yüzeyler daha sonra steril 60 mm yemekler yerleştirilir, temiz bir tezgah birbirleri ile temas halinde monte edilir. Tedavi yüzeyleri sıkı bir geri dönüşü olmayan bir bağ oluşturur. </li><li> 10 dakika içinde plazma cihazları cam yapıştırma, Poli-L-Lizin (PLL) cihaza eklenir. 10 dakika sonra, cihazın hydrophilicity PLL cihaz girmek için zor hale düşecektir. Not: PLL eklenmesinden sonra cihaz hava kabarcıkları kontrol etmek için önemlidir. Hücreler okside olduğu gibi ana kanal içinde hava kabarcıkları, istenmeyen. Varolan herhangi bir hava kabarcıklarını çıkarmak için kolay bir yöntem, 150 ul (bu durumda PLL) sıvı ile dolu bir P200 pipetlemeyin kullanmak için, ana kanal açılışında ucu doğrudan yerleştirin ve zorla P200 bastırın. Bu birkaç kez tekrar edilmesi gerekebilir ama sonunda kuvvet pipetlemeyin kabarcıkları sürücü olacaktır. </li><li> PLL içeren cihazlar, 37 standart doku kültürü inkübatör geceleme inkübe izin ° C ile% 5 CO 2 . </li><li> Ertesi gün de,kötü alışkanlıklardan otoklavlanmış dH2O iki kez hızlı bir şekilde durulanır. Bu işlem sırasında, sıvı tamamen sadece rezervuarları ana kanallar kaldırıldı asla. Ana kanal sıvı kaldırma kabarcıkları tanıtacak. 2 hızlı durular sonra, cihazlar otoklavlanmış dH2O ile doldurulur ve en az 3 saat bir inkübatör içine geri yerleştirilir ya da gecede vardır. </li><li> Ertesi gün, cihazlar yine otoklavlanmış dH2O ile 2 kat hızlı durulanır. Ardından, gerekli takviyeleri B27 ve glutamax kültür birincil sıçan nöron içeren sinir bazal medya, cihazlar eklenir. Cihazlar, ileride kullanmak üzere inkübatör geri yerleştirilir. Şimdi, cihazlar nöronların tohumlama için hazırız. </li></ol>

<ol>
	<li>Park, J. W., Vahidi, B., , A. M., Rhee, S. W., Jeon, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+culture+platform+for+neuroscience+research..">Microfluidic culture platform for neuroscience research..</a> Nat Protoc. 1, 2128-2136 (2006).</li><li>Taylor, A. M., Blurton-Jones, M., Rhee, S. W., Cribbs, D. H., Cotman, C. W., Jeon, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microfluidic+culture+platform+for+CNS+axonal+injury,+regeneration+and+transport.">A microfluidic culture platform for CNS axonal injury, regeneration and transport.</a> Nat Methods. 2, 599-605 (2005).</li></ol>

Bu video, cihaz bize hücre gövdeleri, dendrit ve akson bir karışımını içeren bölmesi microgrooves ayrı bir saf aksonal kesir elde etmek için içeri kültür nöronlar için kullanabileceğiniz bir PDMS mikroakışkan cihaz yapmak ve hazırlamak için nasıl göstermiştir . Bu cihazlar, araştırmacı, çeşitli tedavilerin etkilerini incelemek yanı sıra fiziksel yaralanma aksonlar yürütmek ve bu tedaviler nöronlar üzerindeki etkileri gözlemlemek için bir platform sağlamak için izin verir. Cihaz, aynı zamanda taşıma çalışmaları kolaylaştırmak yardımcı kültür nöronlar için bir düzeyi sağlar. Buna ek olarak, hücre kültürü bölmesi akson bölmesi ayrı microgrooves, mümkün cihazın diğer tarafında direkt tedavisi ile etkili olmadan bir cihazın yan tedavisinde iki bölmeleri arasında akışkan izolasyon sağlar. Bu özellik, araştırmacı, sinyal iletimi ve ulaşım tedavi sonucunda ortaya çıkan etkileri çalışma sağlar.

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>PDMS</td>
<td>Reagent</td>
<td>Dow Corning</td>
<td>&nbsp;</td>
<td>Sylgard 184 with curing agent

Please consult Dow Corning to find a vendor near you</td>
</tr>
<tr>
<td>Plasam Cleaner</td>
<td>Tool</td>
<td>Harrick</td>
<td>&nbsp;</td>
<td>http://www.harrickplasma.com/</td>
</tr>
<tr>
<td>Neural Basal Media</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>21103-049</td>
<td>Neural Basal Media is obtained by Invitrogen under their Gibco line of cell culture reagents.</td>
</tr>
<tr>
<td>B27</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>17504-044</td>
<td>B27 is a proprietary supplement available through Invitrogen under their Gibco line of cell culture reagents.</td>
</tr>
<tr>
<td>Glutamax</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>35050-061</td>
<td>Glutamax is available through Invitrogen under their Gibco line of celll culture reagents.</td>
</tr>
<tr>
<td>Cornig No. 1 Cover Glass</td>
<td>cover glass</td>
<td>Corning</td>
<td>Corning No. X2935 244</td>
<td>Available through Fisher Scientific, Fish Catalog number 12-531D</td>
</tr>
<tr>
<td>60 mm Petri Dish</td>
<td>Tool</td>
<td>Fisher Scientific</td>
<td>08-757-13A</td>
<td>60 mm polystyrene sterile petri dishes are used to house the device bound to glass.</td>
</tr>
<tr>
<td>BD Falcon 50 ml Tube</td>
<td>Tool</td>
<td>BD Falcon</td>
<td>Falcon No. 352098</td>
<td>Available through Fisher Scientific catalog number 14-959-49A</td>
</tr>
<tr>
<td>BD Falcon 15 ml tube</td>
<td>Tool</td>
<td>BD Falcon</td>
<td>Falcon No. 352097</td>
<td>Available through Fisher Scientific catalog number 14-959-70C</td>
</tr>
<tr>
<td>Poly-L-Lysine</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P-1274</td>
<td>&nbsp;</td>
<tr>
<td>New Item</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
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fabrication of a microfluidic device for the compartmentalization of neuron soma and axons

Bu video, Polidimetil siloksan (PDMS) kültür nöronlar için mikroakışkan bir cihaz imal etmek için kullanılan yumuşak litografi tekniği göstermektedir. Daha önce, bir silikon yonga tasarımı ile SU-8 ve fotolitografi bir ana kalıp oluşturmak için, ya da biz sadece bir &quot;master&quot; olarak ne anlama kullanarak nöron mikroakışkan cihaz için desenli oldu. Sonra, silikon polimer PDMS sonra 1 saat için 80 ° C PDMS ısıtma ile tedavi ustanın üstüne dökün. PDMS cihazın olumsuz bir kalıp oluşturur. PDMS sonra dikkatlice kesilmiş ve ana uzak kaldırdı. Delik rezervuarları nerede olacak yumrukladı ve aşırı PDMS cihazdan kesilmiş. Azot cihazdan herhangi bir aşırı enkaz darbe için kullanılır. Bu noktada cihazlar artık kullanıma hazırdır ve plazma sterilizatör / temiz ya da geri dönüşümlü cam kapak cihaz sadece kapak cam üstüne koyarak bağlı olabilir ya Corning No: 1 cam kapak yapıştırılır. Geri dönüşümlü yapıştırma cam cihazın ayrı bir video kapalı ve cihaz% 70 etanol veya otoklav ile sterilize edilmelidir ilk gerektirir. Böylece başka tedaviye gerek yoktur Plazma tedavi cihazları sterilize eder. Bu yüzeyler hidrofilik iken, 10 dakika içinde plazma tedavi cihazlar sıvı eklemek için cihazlar, plazma tedavi ederken, ancak, önemlidir. Cihaz sıvı eklemek için 10 dakikadan daha uzun bir süre beklemek zor sıvı cihaz girmek için yapar. Nöron cihazlar genellikle cam ve 0.5 mg / ml pH 8.5 borat tamponu poli-L-lisin (PLL) kapsayacak şekilde plazma bağlı cihaz hemen eklenir. PLL ile inkübe en az 3 saat sonra, cihazlar her yıkama arasında en az 15 dakika dH2O su ile en az 3 kez yıkanır. Sonra, su çıkarılır ve taze medya cihazına eklenir. Bu noktada cihaz kullanıma hazır. Tüm medya cihazdan çıkarmak hiç bu noktada hatırlamak önemlidir. Her zaman ana kanal medya bırakın.

Watch this Scientific Journal Video about Fabrication of a Microfluidic Device for the Compartmentalization of Neuron Soma and Axons at JoVE.com

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

Bu video kültür nöronlar için mikroakışkan bir cihaz farbricate için kullanabileceğiniz Polidimetil siloksan (PDMS) ile yumuşak litografi tekniği göstermektedir.

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 ...

fabrication-microfluidic-device-for-compartmentalization-neuron-soma

Research

JoVE Journal

Biology

35.2K Görüntüleme.  University of California, Irvine (UCI). Bu video kültür nöronlar için mikroakışkan bir cihaz farbricate için kullanabileceğiniz Polidimetil siloksan (PDMS) ile yumuşak litografi tekniği göstermektedir.

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

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 ...

Biyoloji

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 ...

PDMS Device Fabrication and Surface Modification

PDMS Cihaz İmalat ve Yüzey Modifikasyonu

Cell Capture Using a Microfluidic Device

Hücre Yakalama mikroakışkan Cihaz Kullanımı

Non-plasma Bonding of PDMS for Inexpensive Fabrication of Microfluidic Devices

In this video, we demonstrate how to use the neuron microfluidic device without plasma bonding.  In some cases it may be desirable to reversibly bond devices to the Corning No. 1 cover glass.  This could be due, perhaps, to a plasma cleaner not being available.  In other instances, it may be desirable to remove the device from the glass after the culturing of neurons for certain types of microscopy or for immunostaining, though it is not necessary to remove the device for immunostaining since the neurons can be stained in the device.  Some researchers, however, still prefer to remove the device.  In this case, reversible bonding of the device to the cover glass makes that possible.  There are some disadvantages to non-plasma bonding of the devices in that not as tight of a seal is formed.  In some cases axons may grow under the grooves rather than through them.  Also, because the glass and PDMS are hydrophobic, liquids do not readily enter the device making it necessary at times to force media and other reagents into the device.  Liquids will enter the device via capillary action, but it takes significantly longer as compared to devices that have been plasma bonded.  The plasma cleaner creates temporary hydrophilic charges on the glass and device that facilitate the flow of liquids through the device after bonding within seconds.  For non-plasma bound devices, liquid flow through the devices takes several minutes.  It is also important to note that the devices to be used with non-plasma bonding need to be sterilized first, whereas plasma treated devices do not need to be sterilized prior to use because the plasma cleaner will sterilize them.

In this video we demonstrate how to use the neuron microfluidic device without plasma bonding.

Ucuz mikroakışkan Cihazlar İmalat PDMS Non-plazma Yapışma

In this video, we demonstrate how to use the neuron microfluidic device without plasma bonding.  In some cases it may be desirable to reversibly bond ...

Fabrication of the Thermoplastic Microfluidic Channels

In our lab, we have successfully isolated nucleic acids directly from microliter and submicroliter volumes of human blood, urine and stool using polymer/nanoparticle composite microscale lysis and solid phase extraction columns. The recovered samples are concentrated, small volume samples that are PCRable, without any additional cleanup. Here, we demonstrate how to fabricate thermoplastic microfluidic chips using hot embossing and heat sealing. Then, we demonstrate how to use in situ light directed surface grafting and polymerization through the sealed chip to form the composite solid phase columns. We demonstrate grafting and polymerization of a carbon nanotube/polymer composite column for bacterial cell lysis. We then show the lysis process followed by solid phase extraction of nucleic acids from the sample on chip using a silica/polymer composite column. The attached protocols contain detailed instructions on how to make both lysis and solid phase extraction columns.

Here we demonstrate how to fabricate thermoplastic microfluidic chips using hot embossing and heat sealing. Then we demonstrate how to use in situ light directed surface grafting and polymerization through the sealed chip to form the composite solid phase columns.

Termoplastik mikroakışkan Kanallar imalatı

In our lab, we have successfully isolated nucleic acids directly from microliter and submicroliter volumes of human blood, urine and stool using ...

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 ...

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations at risk. In the process of attempting to identify novel genetic factors associated with PD, scientists have generated many lists of candidate genes, polymorphisms, and proteins that represent important advances, but these leads remain mechanistically undefined. Our work is aimed toward significantly narrowing such lists by exploiting the advantages of a simple animal model system. While humans have billions of neurons, the microscopic roundworm Caenorhabditis elegans has precisely 302, of which only eight produce dopamine (DA) in hemaphrodites. Expression of a human gene encoding the PD-associated protein, alpha-synuclein, in C. elegans DA neurons results in dosage and age-dependent neurodegeneration.
Worms expressing human alpha-synuclein in DA neurons are isogenic and express both GFP and human alpha-synuclein under the DA transporter promoter (Pdat-1). The presence of GFP serves as a readily visualized marker for following DA neurodegeneration in these animals. We initially demonstrated that alpha-synuclein-induced DA neurodegeneration could be rescued in these animals by torsinA, a protein with molecular chaperone activity 1. Further, candidate PD-related genes identified in our lab via large-scale RNAi screening efforts using an alpha-synuclein misfolding assay were then over-expressed in C. elegans DA neurons. We determined that five of seven genes tested represented significant candidate modulators of PD as they rescued alpha-synuclein-induced DA neurodegeneration 2. Additionally, the Lindquist Lab (this issue of JoVE) has performed yeast screens whereby alpha-synuclein-dependent toxicity is used as a readout for genes that can enhance or suppress cytotoxicity. We subsequently examined the yeast candidate genes in our C. elegans alpha-synuclein-induced neurodegeneration assay and successfully validated many of these targets 3, 4.
Our methodology involves generation of a C. elegans DA neuron-specific expression vector using recombinational cloning of candidate gene cDNAs under control of the Pdat-1 promoter. These plasmids are then microinjected in wild-type (N2) worms, along with a selectable marker for successful transformation. Multiple stable transgenic lines producing the candidate protein in DA neurons are obtained and then independently crossed into the alpha-synuclein degenerative strain and assessed for neurodegeneration, at both the animal and individual neuron level, over the course of aging.

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson&#039;s Disease Genes

This video demonstrates how to use C. elegans to assess dopaminergic neuron neurodegeneration as a model for Parkinson's disease. Furthermore, genetic screens are used to identify factors that either enhance degeneration or are neuroprotective.

Potansiyel Parkinson Hastalığı Genler Doğrulama için C. elegans Dopamin Nöron Dejenerasyonu Testi Uygulama

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations ...

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 ...