Parte 1: Preparazione del dispositivo a microfluidi neurone <ol><li> A 3 &quot;wafer di silicio con un modello fatto di SU-8 di fotolitografia è utilizzata per lanciare l&#39;(PDMS) Polidimetilsilossano stampo microfluidica. Ci riferiamo al SU-8 wafer di silicio modellata come stampi master o&quot; maestri &quot;. </li><li> PDMS è mescolato con catalizzatore nel p / w rapporto di 10:1, cioè per 10 grammi di PDMS, 1 grammo di catalizzatore è aggiunto ad esso. </li><li> Il PDMS è poi mescolato bene e versato sul wafer di silicio. Il wafer di silicio è contenuto in un piatto di polistirolo 10 centimetri di Petri. Ci vogliono circa 12 g per 15 g di PDMS per coprire il master con uno spessore di circa 4 mm. </li><li> Il master con il PDMS viene poi posto in un essiccatore a vuoto con il coperchio della scatola di Petri, e pompa a vuoto. Questo viene fatto per aiutare a rimuovere le bolle d&#39;aria dal PDMS che sono state introdotte durante l&#39;agitazione in del catalizzatore. Ci vogliono circa 15 minuti per le bolle d&#39;aria da rimuovere. </li><li> Il master con PDMS viene rimosso dal essiccatore. Un fucile ad aria con un filtro da 0,45 um viene utilizzato per rimuovere eventuali bolle rimanenti. </li><li> Il master è quindi posto su una piastra di 80 ° C a caldo per 1 ora da curare. </li></ol> Nota: se un essiccatore a vuoto non è disponibile, il master può essere lasciato a temperatura ambiente per diverse ore. Le bolle d&#39;aria alla fine dissipare per conto proprio. Se un piatto caldo non è disponibile, il PDMS curerà a temperatura ambiente dopo 24 ore. Nota: E &#39;anche importante assicurarsi che il master sia in piano durante il processo di polimerizzazione. Parte 2: Taglio del dispositivo neurone microfluidi <ol><li> Una volta che il PDMS è curata sul master, che viene portato in una cappa pulita. </li><li> Utilizzando una lama chirurgica, un cerchio è tagliato intorno al perimetro dei dispositivi. Quando si inserisce la lama nel PDMS, è importante prendere contatto con il wafer di silicio, ma non mettere troppa pressione sul wafer, altrimenti il ​​padrone si spezzerà. </li><li> Con un cerchio tagliare tutto il modo per aggirare i dispositivi (ci sono 4 dispositivi per ogni &quot;3 Master Silicio), il PDMS è alzato attentamente fuori il padrone. Questo può essere avviato da torcere delicatamente la lama chirurgica in quanto è inserito nel sezionate prima . </li><li> Successivamente, i serbatoi sono forati nel dispositivo utilizzando un 8 mm biopsia punch. </li><li> I dispositivi sono poi squartato e tagliato via l&#39;eccesso PDMS in modo che il dispositivo si adatta perfettamente a Corning coperchio di vetro (n. 1) 24 mm x 40 mm. L&#39;azoto dell&#39;aria è usato per soffiare via tutti i detriti in eccesso. Detriti testardo può anche essere rimosso utilizzando Marca 3M Scotch 471 nastro in vinile. </li></ol> Parte 3: Plasma incollaggio i dispositivi scivola copertura in vetro. <ol><li> Cleaner plasma viene utilizzato per legare un Harrick i dispositivi neurone al coprioggetto. In breve, i dispositivi vengono inseriti nella pulizia al plasma e una pompa da vuoto è utilizzato per evacuare l&#39;aria dalla camera. </li><li> Dopo la pressione di vuoto ha raggiunto 300 milliTorr, si accende la bobina elettrica e impostato su alto. </li><li> La bobina elettrica crea plasma &quot;, un gas parzialmente ionizzato costituito da elettroni, gli ioni e atomi neutri o molecole&quot; - <a href="http://www.harrickplasma.com/plasma_physics.php">http://www.harrickplasma.com/plasma_physics.php</a> , fuori le molecole di aria residua nella camera di sinistra. Il plasma crea specie reattive sulle superfici del vetro e del dispositivo, che una volta messi insieme formeranno un legame permanente. Il plasma si trasforma anche le superfici idrofile, che aiuta a facilitare l&#39;aggiunta di liquidi. Inoltre, il dispositivo di pulizia al plasma sterilizza i dispositivi. La superficie del dispositivo PDMS che contiene il dispositivo a microfluidi è posizionato rivolto verso il filtro al plasma con le diapositive di vetro. Le superfici trattate al plasma del vetro e dispositivo a microfluidi sono assemblati in contatto con l&#39;altro in un banco pulito, poi messo in sterili 60 piatti mm. Le superfici trattate formare un legame stretto irreversibile. </li><li> Entro 10 minuti di plasma incollaggio dei dispositivi al vetro, Poli-L-lisina (PLL) viene aggiunto al dispositivo. Dopo 10 minuti, l&#39;idrofilia del dispositivo diminuisce rendendo difficile per il PLL per entrare nel dispositivo. Nota: è importante verificare la presenza di bolle d&#39;aria nel dispositivo dopo l&#39;aggiunta di PLL. Le bolle d&#39;aria nel canale principale sono indesiderabili, come farebbero ossidare le cellule. Un metodo semplice per rimuovere eventuali bolle d&#39;aria esistente è quello di utilizzare una pipetta P200 riempito con 150 ul di liquido (in questo caso PLL), posizionare la punta direttamente all&#39;apertura del canale principale, e premere il P200 con forza. Questo può essere necessario ripetere più volte ma alla fine la forza dalla pipetta guiderà le bolle fuori. </li><li> I dispositivi contenenti PLL sono autorizzati a incubare una notte in una cultura incubatore standard di tessuto a 37 ° C con 5% di CO 2. </li><li> Il giorno successivo, devizi sono sciacquate due volte rapidamente con autoclave dH2O. Durante questo processo, il liquido non è mai completamente rimossa dai canali principali, solo dai serbatoi. La rimozione di liquido dai canali principali introdurrà bolle. Dopo 2 risciacqui veloci, i dispositivi sono pieni di autoclave dH2O e reinserito nella incubatore per un minimo di 3 ore, o durante la notte. </li><li> Il giorno dopo, i dispositivi sono ancora una volta risciacquati 2 volte rapidamente con autoclave dH2O. Poi, neurali dei media basale, contenente le integrazioni necessarie B27 e glutamax per i neuroni di ratto coltura principale, si aggiunge ai dispositivi. I dispositivi sono rimesse in incubatrice per un uso futuro. I dispositivi sono ora pronti per la semina dei neuroni. </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>

In questo video abbiamo dimostrato come fare e preparare un dispositivo PDMS microfluidi che usiamo per i neuroni cultura in Il dispositivo permette di ottenere una frazione pura assonale separati da microscolpiture dal vano contenente un mix di corpi cellulari, dendriti e degli assoni . Questi dispositivi consentono al ricercatore di studiare gli effetti dei vari trattamenti, oltre a fornire una piattaforma per effettuare danni fisici allo assoni e osservare quello che questi trattamenti hanno effetti sui neuroni. Il dispositivo fornisce anche un livello di coltura per i neuroni che aiuta a facilitare gli studi di trasporto. Inoltre, il microscolpiture, che separano il vano di coltura cellulare dal vano assone, permette di isolamento fluido tra i due comparti che rende possibile il trattamento di un lato del dispositivo senza influenzare l&#39;altro lato del dispositivo direttamente con il trattamento. Questa proprietà consente al ricercatore di studiare gli effetti come la trasduzione del segnale e dei trasporti che risultano dal trattamento.

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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>
		</table>
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fabrication of a microfluidic device for the compartmentalization of neuron soma and axons

In questo video, dimostriamo la tecnica della litografia soft con polidimetil silossano (PDMS) che usiamo per fabbricare un dispositivo a microfluidi per i neuroni di coltura. In precedenza, un wafer di silicio è stato modellato con la progettazione del dispositivo neurone microfluidica utilizzando SU-8 e fotolitografia per creare uno stampo maestro, o che cosa ci riferiamo semplicemente come un &quot;master&quot;. Successivamente, versate il silicio polimero PDMS in cima al maestro che viene poi curata dal riscaldamento del PDMS a 80 ° C per 1 ora. Il PDMS forma uno stampo negativo del dispositivo. Il PDMS è poi accuratamente tagliata e sollevata dal maestro. I buchi sono forati in cui i serbatoi saranno e l&#39;eccesso PDMS tagliato fuori dal dispositivo. L&#39;azoto è usato per soffiare via i detriti in eccesso dal dispositivo. A questo punto i dispositivi sono ora pronti per l&#39;uso e può legato a corning n ° 1 bicchiere coprire con un plasma sterilizzatore / pulitore o può essere reversibile legata al vetro di copertura, semplicemente posizionando il dispositivo sulla parte superiore del vetro di copertura. Il legame reversibile del dispositivo al vetro è coperto in un altro video e richiede prima che il dispositivo da sterilizzare sia con il 70% di etanolo o in autoclave. Plasma trattamento sterilizza i dispositivi in ​​modo che nessun ulteriore trattamento è necessario. E &#39;tuttavia importante, quando plasma trattamento dei dispositivi, per aggiungere liquido al dispositivo entro 10 minuti di trattamento al plasma, mentre le superfici sono ancora idrofile. In attesa più lunghi di 10 minuti per aggiungere liquido al dispositivo rende difficile per il liquido di entrare nel dispositivo. I dispositivi sono in genere neurone plasma legato alla copertura in vetro e 0,5 mg / ml di poli-L-lisina (PLL) a pH 8,5 tampone borato è immediatamente aggiunto al dispositivo. Dopo un minimo di 3 ore di incubazione con PLL, i dispositivi vengono lavati con acqua dH2O un minimo di 3 volte con almeno 15 minuti tra ogni lavaggio. Successivamente, l&#39;acqua viene rimossa e mezzi freschi si aggiunge al dispositivo. A questo punto il dispositivo è pronto per l&#39;uso. E &#39;importante ricordare, a questo punto per rimuovere mai tutti i media dal dispositivo. Lasciare sempre i media nel canale principale.

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

In questo video ci mostrano la tecnica della litografia soft con polidimetil silossano (PDMS) che usiamo per farbricate un dispositivo a microfluidi per i neuroni di coltura.

Fabbricazione di un dispositivo a microfluidi per la compartimentazione dei Neuron Soma e assoni

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 Views.  University of California, Irvine (UCI). In questo video ci mostrano la tecnica della litografia soft con polidimetil silossano (PDMS) che usiamo per farbricate un dispositivo a microfluidi per i neuroni di coltura.

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.

Un dispositivo a microfluidi con pattern Groove per studiare il comportamento cellulare

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

Ricerca

Biologia

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 gradiente di generazione del dispositivo Microfluidic di Biologia Cellulare

 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.

Preparazione E18 neuroni corticali di ratto per compartimentazione in un dispositivo a microfluidi

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 fabbricazione del dispositivo e la modifica della superficie

Cell Capture Using a Microfluidic Device

Cattura cellula Utilizzando un dispositivo a microfluidi

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 plasmatici Incollaggio di PDMS Economico per la Fabbricazione di dispositivi microfluidici

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.

Fabbricazione dei canali termoplastico Microfluidic

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.

Valutare motilità delle cellule staminali neurali L&#39;utilizzo di un gel a base di Agarose dispositivo a microfluidi

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.

L&#39;applicazione di una degenerazione C. elegans Dopamina Saggio Neuron per la convalida dei potenziali morbo di Parkinson geni

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 multi-vano SNC Neuron-glia Co-cultura Microfluidic piattaforma

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