Parte 1: Preparar el dispositivo de microfluidos neurona <ol><li> A 3 &quot;oblea de silicio con un patrón de hechos de SU-8 mediante fotolitografía se utiliza para convertir el polidimetilsiloxano (PDMS) molde de microfluidos. Nos referimos a la SU-8 de obleas de silicio estampado como moldes del amo, o&quot; maestros &quot;. </li><li> PDMS se mezcla con el agente de curado en w / w proporción de 10:1, es decir, por 10 gramos de PDMS, 1 gramo de catalizador se añade a la misma. </li><li> El PDMS se mezcla bien y se vierte sobre la oblea de silicio. La oblea de silicio se encuentra a unos 10 cm de poliestireno plato de Petri. Se tarda aproximadamente 12 a 15 g de PDMS para cubrir el principal con un espesor de aproximadamente 4 mm. </li><li> El maestro con el PDMS se coloca en un desecador de vacío con la tapa de la placa de Petri, y el vacío se aplica. Esto se hace para ayudar a eliminar las burbujas de la PDMS que se introdujeron durante la agitación en el del agente de curado. Se tarda aproximadamente 15 minutos para que las burbujas de aire que ser eliminado. </li><li> El maestro con PDMS se quita de en el desecador. Una pistola de aire con un filtro de 0,45 um se utiliza para eliminar las burbujas restantes. </li><li> El maestro se coloca sobre una placa de 80 ° C caliente durante 1 hora de curar. </li></ol> Nota: Si un desecador de vacío no está disponible, el maestro puede dejarse a temperatura ambiente durante varias horas. Las burbujas de aire con el tiempo se disipará por sí mismos. Si un plato caliente no está disponible, el PDMS se cura a temperatura ambiente, después de 24 horas. Nota: También es importante asegurarse de que el maestro es el nivel durante el proceso de curado. Parte 2: Cortar el dispositivo de microfluidos neurona <ol><li> Una vez que el PDMS se cura en el maestro, que es llevado a una campana de limpieza. </li><li> Usando una hoja de bisturí, un círculo se corta en todo el perímetro de los dispositivos. Cuando uno inserta el disco en el PDMS, es importante ponerse en contacto con la oblea de silicio, pero no poner demasiada presión sobre la lámina, de lo contrario el maestro se agrieta. </li><li> Con un círculo de cortar todo el camino alrededor de los dispositivos (hay 4 equipos por cada 3 &quot;Maestro de silicio), el PDMS es cuidadosamente levantado del maestro. Este se puede iniciar girando suavemente la hoja de bisturí, ya que se inserta en la corte antes de . </li><li> A continuación, los embalses están perforados en el dispositivo mediante un 8 mm de tejido biopsia en sacabocados. </li><li> Los dispositivos están en cuartos y luego el exceso de PDMS recorta de modo que el dispositivo se encaja perfectamente en la cubierta del cristal de Corning (N º 1) 24 mm x 40 mm. El nitrógeno se utiliza aire para soplar los residuos restantes. Restos obstinada también se pueden eliminar con la marca 3M Scotch 471 cinta de vinilo. </li></ol> Parte 3: Plasma unión de los dispositivos de cubreobjetos de vidrio. <ol><li> Limpiador de plasma se utiliza para unir una Harrick los dispositivos de neurona a la cubierta se desliza. En pocas palabras, los productos se colocan en el filtro de plasma y una bomba de vacío se utiliza para evacuar el aire de la cámara. </li><li> Después de la presión de vacío ha alcanzado los 300 miliTorr, se activa la alimentación de la bobina eléctrica y establece en alto. </li><li> La bobina eléctrica crea plasma, &quot;un gas parcialmente ionizado compuesto de electrones, iones y átomos neutros o moléculas&quot; - <a href="http://www.harrickplasma.com/plasma_physics.php">http://www.harrickplasma.com/plasma_physics.php</a> , de las moléculas de aire que queda a la izquierda en la cámara. El plasma genera especies reactivas en la superficie del vidrio y el dispositivo, que cuando se colocan juntos formarán una unión permanente. El plasma también convierte las superficies hidrófilas, que ayuda a facilitar la incorporación de líquidos. Además, el filtro de plasma esteriliza los dispositivos. La superficie del dispositivo de PDMS que contiene el dispositivo de microfluidos se coloca boca arriba en el filtro de plasma junto con las diapositivas de cristal. Las superficies de los tratados con plasma de la copa y el dispositivo de microfluidos son ensambladas en contacto unos con otros en una mesa de trabajo limpia y luego se colocan en placas de 60 mm estériles. Las superficies tratadas formar un vínculo estrecho irreversible. </li><li> A 10 minutos de plasma de unión de los dispositivos de cristal, poli-L-lisina (PLL) se añade al dispositivo. Después de 10 minutos, la hidrofilia del dispositivo se reducirá por lo que es difícil para el PLL para entrar en el dispositivo. Nota: Es importante para comprobar si las burbujas de aire en el dispositivo después de la adición de PLL. Las burbujas de aire en el canal principal no son deseables, ya que se oxidan las células. Un método fácil para eliminar las burbujas de aire existente es utilizar una pipeta P200 lleno de 150 ul de líquido (en este caso PLL), coloque la punta directamente en la apertura del canal principal, y presione el P200 con fuerza. Esto puede ser necesario repetir varias veces, pero finalmente la fuerza de la pipeta impulsará las burbujas. </li><li> Los dispositivos que contienen PLL se dejan incubar durante la noche en un estándar de cultivo de tejidos incubadora a 37 ° C con 5% de CO 2. </li><li> Al día siguiente, el devicios se lavan dos veces rápidamente con autoclave dH2O. Durante este proceso, el líquido nunca es totalmente eliminado de los canales principales, sólo a partir de los embalses. La eliminación de líquidos de los principales canales se introducen burbujas. Después de dos lavados rápidos, los dispositivos están llenos de autoclave dH2O y volver a colocar en la incubadora durante un mínimo de 3 horas, o toda la noche. </li><li> Al día siguiente, los dispositivos son una vez más, lavados 2 veces rápidamente con autoclave dH2O. Entonces, los medios de comunicación neural basal, con los suplementos necesarios B27 y glutamax para el cultivo de neuronas de rata primaria, se añade a los dispositivos. Los dispositivos se colocan de nuevo en la incubadora para su uso futuro. Los dispositivos ya están listos para la siembra de las neuronas. </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>

En este video nos han demostrado cómo hacer y preparar un dispositivo de microfluidos PDMS que utilizamos para las neuronas cultura pulg El dispositivo nos permite obtener una fracción pura axonal separados por microsurcos del compartimiento que contiene una mezcla de cuerpos celulares, dendritas y los axones . Estos dispositivos permiten al investigador para estudiar los efectos de distintos tratamientos, así como ofrecer una plataforma para llevar a cabo una lesión física en los axones y observar los efectos que estos tratamientos tienen sobre las neuronas. El dispositivo también ofrece un nivel de orden a las neuronas de cultivo que ayuda a facilitar los estudios de transporte. Además, los microsurcos, que separan el compartimiento de cultivo de células desde el compartimiento de los axones, permite el aislamiento de fluidos entre los dos compartimentos por lo que es posible tratar a un lado del dispositivo sin afectar al otro lado del dispositivo directamente con el tratamiento. Esta característica permite que el investigador para estudiar los efectos tales como la transducción de señales y el transporte que el resultado del tratamiento.

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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

En este vídeo, se demuestra la técnica de la litografía blanda, con polidimetilsiloxano (PDMS), que se utiliza para la fabricación de un dispositivo de microfluidos para el cultivo de neuronas. Anteriormente, una oblea de silicio fue modelado con el diseño del dispositivo de microfluidos con las neuronas SU-8 y la fotolitografía para crear un molde maestro, o lo que nos referimos simplemente como un &quot;maestro&quot;. A continuación, se vierte el polímero de silicio PDMS en la parte superior de la maestra, que se cura por calentamiento de la PDMS a 80 ° C durante 1 hora. El PDMS forma un molde negativo del dispositivo. El PDMS es cuidadosamente cortado y levantado del maestro. Los agujeros son perforados en los yacimientos y será el exceso de PDMS recortada desde el dispositivo. El nitrógeno es utilizado para vencer a los residuos restantes del dispositivo. En este punto, los dispositivos están listos para su uso y puede unido a Corning cubierta de vidrio N º 1 con un esterilizador de plasma / limpiador o puede ser unida reversiblemente a la cubierta de cristal con sólo colocar el dispositivo en la parte superior de la cubierta de vidrio. La unión reversible del dispositivo de vidrio está cubierto en un video por separado y requiere en primer lugar que el dispositivo se esterilizan con el 70% de etanol o en autoclave. Plasma esteriliza el tratamiento de los dispositivos por lo que no se requiere tratamiento adicional. Es, sin embargo, importante, cuando se plasma el tratamiento de los dispositivos, para añadir líquidos a los dispositivos a los 10 minutos del tratamiento de plasma, mientras que las superficies están todavía hidrofílico. Esperando más de 10 minutos para añadir líquidos en el dispositivo hace que sea difícil que el líquido entre en el dispositivo. Los dispositivos de las neuronas suelen plasma con destino a la cubierta de vidrio y 0,5 mg / ml de poli-L-lisina (PLL) en tampón borato pH 8,5 se añade inmediatamente el dispositivo. Después de un mínimo de 3 horas de incubación con PLL, los dispositivos se lavan con agua dH2O un mínimo de 3 veces con al menos 15 minutos entre cada lavado. A continuación, se elimina el agua y los medios de comunicación fresca se agrega al dispositivo. En este punto, el dispositivo está listo para su uso. Es importante recordar en este punto para no eliminar todos los medios de comunicación del dispositivo. Siempre dejan los medios de comunicación en el canal 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

En este video se demuestra la técnica de la litografía blanda, con polidimetilsiloxano (PDMS) que utilizamos para farbricate un dispositivo de microfluidos para el cultivo de neuronas.

Fabricación de un dispositivo de microfluidos para la compartimentación de las neuronas y los axones Soma

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 vistas.  University of California, Irvine (UCI). En este video se demuestra la técnica de la litografía blanda, con polidimetilsiloxano (PDMS) que utilizamos para farbricate un dispositivo de microfluidos para el cultivo de neuronas.

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 de microfluidos con los patrones de Surco para el estudio del comportamiento celular

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

Investigación

Biología

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 dispositivo de microfluidos gradiente de generación de Biología Celular

 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.

Preparación de neuronas de rata E18 cortical de compartimentación en un dispositivo de microfluidos

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 fabricación de dispositivos y modificación de la superficie

Cell Capture Using a Microfluidic Device

Captura de células utilizando un dispositivo de microfluidos

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.

No plasmáticos de unión de PDMS para la Fabricación de bajo costo de los dispositivos de microfluidos

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.

Fabricación de los canales de microfluidos termoplástico

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.

La evaluación de la motilidad de células madre neurales uso de un gel de agarosa al dispositivo basado en microfluídica

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.

Aplicación de un ensayo de C. elegans la degeneración neuronal de dopamina para la validación del potencial de la enfermedad de Parkinson genes

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 compartimiento de Multi-CNS neuronas-glia Co-cultivo de microfluidos Plataforma

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