Partie 1: Préparation de l&#39;appareil neurone microfluidique <ol><li> A 3 &quot;wafer de silicium avec un motif faite de SU-8 par photolithographie est utilisée pour lancer le polydiméthylsiloxane (PDMS) moule microfluidique. Nous nous référons à l&#39;SU-8 plaquette de silicium à motifs comme moules maître, ou« maîtres ». </li><li> PDMS est mélangé avec l&#39;agent de durcissement à p / p ratio de 10:1, qui est, pour 10 grammes de PDMS, 1 gramme de durcisseur est ajouté. </li><li> Le PDMS est ensuite bien mélangé et versé sur la plaquette de silicium. La plaquette de silicium est contenu dans un 10 cm de polystyrène boîte de Petri. Il faut environ 12 g à 15 g de PDMS afin de couvrir le maître d&#39;une épaisseur de 4 mm environ. </li><li> Le maître avec le PDMS est ensuite placé dans un dessiccateur à vide avec le couvercle de la boîte de Petri, et le vide est appliqué. Ceci est fait pour aider à éliminer les bulles d&#39;air du PDMS qui ont été introduites au cours de l&#39;agitation dans de l&#39;agent de durcissement. Il faut environ 15 minutes pour les bulles d&#39;air d&#39;être enlevé. </li><li> Le maître avec PDMS est retiré de la dessiccateur. Un pistolet à air avec un filtre de 0,45 um est utilisé pour enlever les bulles restantes. </li><li> Le maître est alors placé sur une plaque de 80 ° C pendant 1 heure chaude de guérir. </li></ol> Remarque: Si un dessicateur sous vide n&#39;est pas disponible, le maître peut être laissé à température ambiante pendant plusieurs heures. Les bulles d&#39;air éventuellement dissiper par leurs propres moyens. Si une plaque chauffante n&#39;est pas disponible, le PDMS se durcir à température ambiante après 24 heures. Remarque: Il est également important de s&#39;assurer que le maître est de niveau pendant le processus de durcissement. Partie 2: Découpage du périphérique neurone microfluidique <ol><li> Une fois que le PDMS est guéri sur le maître, il est pris d&#39;une hotte propre. </li><li> En utilisant une lame chirurgicale, un cercle est coupé autour du périmètre de ces dispositifs. Quand on insère la lame dans le PDMS, il est important de prendre contact avec la plaquette de silicium, mais de ne pas mettre trop de pression sur la plaquette, sinon le maître va se fissurer. </li><li> Avec un cercle coupé tout autour des appareils (il ya 4 appareils par chacun 3 &quot;Maître de silicium), le PDMS est soigneusement décollé le maître. Cela peut être démarré en tordant avec précaution la lame chirurgicale car il est inséré dans la coupe avant . </li><li> Ensuite, les réservoirs sont perforées dans l&#39;appareil en utilisant un 8 mm biopsie punch. </li><li> Les appareils sont ensuite écartelé et l&#39;excès de PDMS parés de suite afin que le dispositif s&#39;adapte parfaitement sur Corning Couvercle en verre (n ° 1) 24 mm x 40 mm. L&#39;azote de l&#39;air est utilisé pour souffler les débris excès. Les débris tenaces peuvent aussi être retirés à l&#39;aide de 3M Scotch 471 ruban vinyle. </li></ol> Partie 3: Plasma collage des dispositifs à lamelles de verre. <ol><li> Nettoyant plasma est utilisé pour coller une Harrick les appareils neurone à l&#39;lamelles. Brièvement, les appareils sont placés dans le nettoyeur plasma et une pompe à vide est utilisée pour évacuer l&#39;air de la chambre. </li><li> Après la dépression a atteint 300 milliTorr, est sous tension à la bobine électrique et un réglage élevé. </li><li> La bobine électrique crée le plasma &quot;, un gaz partiellement ionisé composé d&#39;électrons, des ions et des atomes ou molécules neutres» - <a href="http://www.harrickplasma.com/plasma_physics.php">http://www.harrickplasma.com/plasma_physics.php</a> , sur les molécules d&#39;air restant dans la chambre de gauche. Le plasma crée des espèces réactives à la surface du verre et du dispositif qui, lorsque mis ensemble forment une liaison permanente. Le plasma se transforme aussi les surfaces hydrophiles, ce qui facilite l&#39;ajout de liquides. De plus, le nettoyeur de plasma stérilise les dispositifs. La surface de l&#39;appareil PDMS qui contient le dispositif microfluidique est placée face visible dans le nettoyeur de plasma avec les lames de verre. Les surfaces traitées plasmatique du verre et du dispositif microfluidique sont assemblés en contact les uns avec les autres sur un banc propre, puis placé dans stériles boîtes de 60 mm. Les surfaces traitées former un lien étroit irréversible. </li><li> Dans les 10 minutes de plasma liant les périphériques au verre, poly-L-Lysine (PLL) est ajouté à l&#39;appareil. Après 10 minutes, l&#39;hydrophilie de l&#39;appareil diminue ce qui rend difficile pour le PLL pour entrer dans le dispositif. Note: Il est important de vérifier les bulles d&#39;air dans l&#39;appareil après l&#39;ajout de la PLL. Les bulles d&#39;air dans le canal principal sont indésirables, comme ils le feraient oxydent les cellules. Une méthode facile pour enlever les bulles d&#39;air existante est d&#39;utiliser une pipette remplie P200 avec 150 ul de liquide (dans ce cas PLL), placez la pointe directement à l&#39;ouverture du canal principal, et appuyer sur le P200 avec force. Ce peut être nécessaire de répéter plusieurs fois, mais finalement la force de la pipette conduira les bulles. </li><li> Les appareils contenant PLL sont autorisés à incuber pendant la nuit dans une norme incubateur de culture tissulaire à 37 ° C avec 5% de CO 2. </li><li> Le lendemain, le devices sont rincées deux fois rapidement avec autoclave dH2O. Pendant ce processus, le liquide n&#39;est jamais entièrement retiré des canaux principaux, seulement à partir des réservoirs. En enlevant le liquide des canaux principaux apparition de bulles. Après deux rinçages rapides, les périphériques sont remplis avec autoclave dH2O et replacée dans l&#39;incubateur pour un minimum de 3 heures ou toute la nuit. </li><li> Le lendemain, les appareils sont à nouveau rincées 2 fois rapidement avec autoclave dH2O. Ensuite, les médias neurones basale, contenant les suppléments nécessaires B27 et glutamax pour les neurones primaires de rat en culture, est ajoutée aux périphériques. Les appareils sont replacés dans l&#39;incubateur pour une utilisation future. Les appareils sont maintenant prêts pour l&#39;ensemencement des neurones. </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>

Dans cette vidéo, nous avons démontré comment faire et préparer un dispositif microfluidique en PDMS que nous utilisons pour la culture des neurones po L&#39;appareil nous permet d&#39;obtenir une fraction pure axonale séparés par microsillons du compartiment contenant un mélange de corps cellulaires, des dendrites et des axones . Ces dispositifs permettent au chercheur d&#39;étudier les effets des différents traitements ainsi que de fournir une plate-forme pour mener à bien des blessures physiques sur les axones et observer les effets de ces traitements ont sur les neurones. L&#39;appareil offre également un niveau d&#39;ordre pour les neurones en culture qui contribue à faciliter les études de transport. En outre, les microsillons, qui séparent le compartiment de culture cellulaire à partir du compartiment axonal, permet d&#39;isoler fluidique entre les deux compartiments permettant de traiter d&#39;un côté de l&#39;appareil sans affecter l&#39;autre côté de l&#39;appareil directement avec le traitement. Cette propriété permet au chercheur d&#39;étudier les effets tels que la transduction du signal et de transport qui résultent du traitement.

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		<table border="1">
		<thead>
		<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>
		</table>
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fabrication of a microfluidic device for the compartmentalization of neuron soma and axons

Dans cette vidéo, nous démontrons la technique de lithographie douce polydiméthylsiloxane (PDMS) que nous utilisons pour fabriquer un dispositif microfluidique pour les neurones en culture. Auparavant, une tranche de silicium a été modelé à la conception pour le neurone dispositif microfluidique en utilisant SU-8 et la photolithographie à créer un moule maître, ou tout simplement ce que nous appelons un &quot;maître&quot;. Ensuite, nous versons le silicium polymère PDMS sur le dessus du maître, qui est ensuite durci par chauffage de l&#39;PDMS à 80 ° C pendant 1 heure. Le PDMS forme un moule négatif de l&#39;appareil. Le PDMS est ensuite soigneusement coupés et levé loin du maître. Des trous sont perforés où les réservoirs seront et l&#39;excès de PDMS parés loin de l&#39;appareil. L&#39;azote est utilisé pour souffler les débris excès de l&#39;appareil. A ce point les appareils sont maintenant prêts à utiliser et peut soit lié à Corning n ° 1 couvercle en verre avec un plasma stérilisateur / nettoyant ou peut être liée de façon réversible à l&#39;couvercle en verre en plaçant simplement l&#39;appareil sur le dessus du couvercle en verre. Le collage réversible de l&#39;appareil est recouvert de verre dans une vidéo séparée et exige d&#39;abord que le dispositif soit stérilisés soit à l&#39;éthanol 70% ou par autoclavage. Plasma traitement stérilise les périphériques ainsi aucun autre traitement n&#39;est nécessaire. Il est cependant important, quand plasma traitant les dispositifs, d&#39;ajouter du liquide à l&#39;appareil dans les 10 minutes de traitement par plasma alors que les surfaces sont encore hydrophile. Attendre plus longtemps que 10 minutes pour ajouter du liquide à l&#39;appareil, il est difficile pour le liquide d&#39;entrer dans le dispositif. Les dispositifs de neurones sont généralement plasma lié à couvercle en verre et 0,5 mg / ml de poly-L-lysine (PLL) dans un tampon borate pH 8,5 est immédiatement ajouté à l&#39;appareil. Après un minimum de 3 heures d&#39;incubation avec PLL, les appareils sont lavés à l&#39;eau dH2O un minimum de 3 fois avec au moins 15 minutes entre chaque lavage. Ensuite, l&#39;eau est éliminée et les médias frais est ajouté à l&#39;appareil. A ce point l&#39;appareil est prêt à l&#39;emploi. Il est important de se rappeler à ce stade de ne jamais supprimer tous les médias de l&#39;appareil. Toujours laisser les médias dans le chenal principal.

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

Dans cette vidéo, nous démontrons la technique de lithographie douce polydiméthylsiloxane (PDMS) que nous utilisons pour farbricate un dispositif microfluidique pour les neurones en culture.

Fabrication d&#39;un dispositif microfluidique pour la compartimentation du Neuron Soma et des axones

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 vues.  University of California, Irvine (UCI). Dans cette vidéo, nous démontrons la technique de lithographie douce polydiméthylsiloxane (PDMS) que nous utilisons pour farbricate un dispositif microfluidique pour les neurones en culture.

Vidéo: 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.

Un dispositif microfluidique avec des modèles Groove pour étudier le comportement cellulaire

We describe a microfluidic device with microgrooved patterns for studying cellular behavior.  This microfluidic platform consists of a top fluidic channel ...

Recherche

Biologie

A Gradient-generating Microfluidic Device for Cell Biology

 The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described.  A microfluidic platform is an enabling experimental tool, because it can precisely manipulate fluid flows, enable high-throughput experiments, and generate stable soluble concentration gradients.  Compared to conventional gradient generators, poly(dimethylsiloxane) (PDMS)-based  microfluidic devices can generate stable concentration gradients of growth factors with well-defined profiles.  Here, we developed simple gradient-generating microfluidic devices with three separate inlets.  Three microchannels combined into one microchannel to generate concentration gradients.  The stability and shape of growth factor gradients were confirmed by fluorescein isothyiocyanate (FITC)-dextran with a molecular weight similar to epidermal growth factor (EGF).  Using this microfluidic device, we demonstrated that fibroblasts exposed to concentration gradients of EGF migrated toward higher concentrations.  The directional orientation of cell migration and motility of migrating cells were quantitatively assessed by cell tracking analysis.  Thus, this gradient-generating microfluidic device might be useful for studying and analyzing the behavior of migrating cells.

We describe a protocol for the microfabrication of the gradient-generating microfluidic device that can generate spatial and temporal gradients in well-defined microenvironment. In this approach, the gradient-generating microfluidic device can be used to study directed cell migration, embryogenesis, wound healing, and cancer metastasis.

Un dispositif microfluidique Dégradé génératrices de biologie cellulaire

 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.

Préparation E18 neurones de rat corticale compartimentation dans un dispositif microfluidique

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

Fabrication de dispositifs en PDMS et modification de surface

Cell Capture Using a Microfluidic Device

Capturez cellule à l&#39;aide d&#39;un dispositif microfluidique

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.

Non du plasma Collage de PDMS pour fabrication bon marché de dispositifs microfluidiques

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.

La fabrication des canaux microfluidiques thermoplastique

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.

Évaluer la motilité Neural Stem Cell utilisant un gel d&#39;agarose à base dispositif microfluidique

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.

Application d&#39;une Assay C. elegans dégénérescence des neurones dopaminergiques pour la validation du potentiel de la maladie de Parkinson gènes

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.

Un compartiment Multi-SNC neurone-glie co-culture de la plate-forme microfluidique

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