Parte 1: Preparando o dispositivo neurônio microfluídicos <ol><li> A 3 &quot;wafer de silício com um padrão feito de SU-8 por fotolitografia é usado para lançar o Polidimetilsiloxano molde (PDMS) microfluídicos. Referimo-nos à SU-8 padrão wafer de silício como moldes mestre, ou&quot; mestres &quot;. </li><li> PDMS é misturado com agente de cura em aw / w proporção de 10:1, ou seja, para 10 gramas de PDMS, 1 grama de agente de cura é adicionado a ele. </li><li> O PDMS é então misturado bem e derramou sobre o wafer de silício. O wafer de silício é contido dentro de um prato de poliestireno de 10 centímetros de Petri. Demora cerca de 12 g a 15 g de PDMS para cobrir o mestre com uma espessura de cerca de 4 mm. </li><li> O mestre com o PDMS é então colocada em um dessecador a vácuo com a tampa da placa de Petri, e de vácuo é aplicado. Isto é feito para ajudar a remover bolhas de ar do PDMS que foram introduzidas durante a mexer no do agente de cura. Demora cerca de 15 minutos para as bolhas de ar a ser removido. </li><li> O mestre com PDMS é removido do dessecador. Uma pistola de ar com um filtro de 0,45 hm é usado para remover as bolhas restantes. </li><li> O mestre é então colocado em uma placa de 80 ° C quente por uma hora para a cura. </li></ol> Nota: Se um dessecador a vácuo não estiver disponível, o mestre pode ser deixado em temperatura ambiente por várias horas. As bolhas de ar eventualmente se dissipar por conta própria. Se um prato quente não está disponível, o PDMS cura à temperatura ambiente após 24 horas. Nota: Também é importante certificar-se que o mestre é o nível durante o processo de cura. Parte 2: Cortar o neurônio dispositivo micro <ol><li> Uma vez que o PDMS é curado no mestre, ele é levado para uma capa limpa. </li><li> Usando uma lâmina cirúrgica, um círculo é cortado em todo o perímetro dos dispositivos. Quando alguém insere a lâmina para o PDMS, é importante fazer contato com o wafer de silício, mas para não colocar demasiada pressão sobre a bolacha, senão o mestre crack. </li><li> Com um círculo cortado a toda a volta os dispositivos (existem 4 dispositivos por cada 3 Mestre do Silício &quot;), o PDMS é cuidadosamente levantado do mestre. Isto pode ser iniciado por gentilmente torcendo a lâmina cirúrgica, uma vez que é inserido no corte antes . </li><li> Em seguida, os reservatórios são perfurados para o dispositivo usando um 8 milímetros de tecido da biópsia do perfurador. </li><li> Os dispositivos são, então, esquartejado e PDMS excesso aparado para que o dispositivo vai caber perfeitamente na tampa do Vidro de Corning (No. 1) 24 mm x 40 mm. Nitrogênio do ar é usado para afastar qualquer excesso de detritos. Os restos teimosos também pode ser removido usando 3M Scotch Marca fita de vinil 471. </li></ol> Parte 3: Plasma ligação a dispositivos para lamínulas de vidro. <ol><li> Cleaner plasma é usado para colar A Harrick os dispositivos neurônio para o lamínulas. Resumidamente, os dispositivos são colocados no limpador de plasma e uma bomba de vácuo é usada para evacuar o ar da câmara. </li><li> Após a pressão de vácuo atingiu 300 milliTorr, o poder está ligado à bobina elétrica e ajustada para alta. </li><li> A bobina elétrica cria um plasma &quot;, um gás parcialmente ionizado constituído de elétrons, íons e átomos ou moléculas neutras&quot; - <a href="http://www.harrickplasma.com/plasma_physics.php">http://www.harrickplasma.com/plasma_physics.php</a> , fora das moléculas de ar remanescente na câmara. O plasma gera espécies reativas na superfície do vidro e do dispositivo, que quando colocados juntos formam uma ligação permanente. O plasma também torna as superfícies hidrofílicas, o que ajuda a facilitar a adição de líquidos. Além disso, o aspirador de plasma esteriliza os dispositivos. A superfície do PDMS dispositivo que contém o dispositivo micro é colocado virado para cima no limpador de plasma, juntamente com as lâminas de vidro. Superfícies a plasma tratado do vidro e dispositivo micro são montados em contato uns com os outros em uma bancada limpa e, em seguida colocado em pratos estéril 60 mm. As superfícies tratadas formar uma ligação apertada irreversível. </li><li> Dentro de 10 minutos de plasma de ligação a dispositivos de vidro, Poly-L-lisina (PLL) é adicionado para o dispositivo. Após 10 minutos, a hidrofilicidade do dispositivo irá diminuir o que torna difícil para o PLL para entrar no dispositivo. Nota: É importante verificar se há bolhas de ar no dispositivo após a adição de PLL. Bolhas de ar no canal principal são indesejáveis, como eles oxidam as células. Um método fácil de remover quaisquer bolhas de ar existentes é a utilização de uma pipeta P200 cheio com 150 ul de líquido (neste caso PLL), coloque a ponta diretamente na abertura do canal principal, e pressione o P200 com força. Este pode ter de ser repetido várias vezes, mas acabou a força da pipeta irá conduzir as bolhas para fora. </li><li> Dispositivos que contêm PLL estão autorizados a incubar durante a noite em um padrão de cultura de tecidos incubadora a 37 ° C com 5% de CO 2. </li><li> No dia seguinte, o devícios são lavadas duas vezes rapidamente com dH2O autoclavado. Durante este processo, o líquido nunca é totalmente removido os canais principais, apenas a partir dos reservatórios. Remover líquido de os principais canais irá introduzir bolhas. Após 2 lavagens rápidas, os dispositivos são preenchidos com dH2O autoclavados e colocados de volta na incubadora por um período mínimo de 3 horas, ou durante a noite. </li><li> No dia seguinte, os dispositivos são novamente lavados duas vezes rapidamente com dH2O autoclavado. Então, media neural basal, contendo os suplementos necessários B27 e Glutamax para os neurônios de cultura primária de ratos, é adicionado aos dispositivos. Os dispositivos são colocados de volta na incubadora para uso futuro. Os dispositivos estão prontos para a semeadura de neurônios. </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>

Neste vídeo demonstramos como fazer e preparar um PDMS dispositivo micro que usamos para os neurônios cultura pol O dispositivo permite a obtenção de uma fração pura axonal separados por microgrooves do compartimento contendo uma mistura de corpos celulares, dendritos e axônios . Estes dispositivos permitem ao pesquisador estudar os efeitos de vários tratamentos, bem como fornecer uma plataforma para realizar dano físico no axônios e observar os efeitos que estes tratamentos têm sobre os neurônios. O dispositivo também fornece um nível de cultura a fim de neurônios que ajuda a facilitar os estudos de transportes. Além disso, o microgrooves, que separam o compartimento de cultura de células do compartimento do axônio, permite o isolamento do fluídica entre os dois compartimentos tornando possível tratar uma parte lateral do dispositivo sem afetar o outro lado do dispositivo diretamente com o tratamento. Esta propriedade permite ao pesquisador estudar os efeitos, como transdução de sinal e transporte que resultam do tratamento.

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		<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

Neste vídeo, demonstramos a técnica da litografia macia com polydimethyl siloxano (PDMS) que usamos para fabricar um dispositivo micro para os neurônios cultura. Anteriormente, uma bolacha de silício foi modelado com o projeto para o dispositivo neurônio microfluídicos usando SU-8 e fotolitografia para criar um molde mestre, ou o que nós simplesmente nos referimos como &quot;mestre&quot;. Em seguida, despeje o polímero de silicone PDMS em cima do mestre que é então curado pelo aquecimento do PDMS a 80 ° C por 1 hora. O PDMS forma um molde negativo do dispositivo. O PDMS é, então, cuidadosamente cortadas e levantou longe do mestre. Buracos são perfurados em que os reservatórios serão PDMS eo excesso aparadas longe do dispositivo. O nitrogênio é usado para afastar qualquer excesso de detritos a partir do dispositivo. Neste ponto os dispositivos estão prontos para usar e pode tanto ligado a Corning Glass cobrir No. 1 com um plasma esterilizador / limpador ou podem ser reversivelmente ligado à tampa de vidro, basta colocar o dispositivo na parte superior da tampa de vidro. A ligação reversível de que o dispositivo de vidro é coberto por um vídeo em separado e exige, primeiro, que o dispositivo seja esterilizado ou com álcool 70% ou em autoclave. Plasma tratar esteriliza os dispositivos de forma que nenhum tratamento adicional é necessário. É, no entanto, importante, quando plasma-tratamento dos dispositivos, para adicionar líquido para os dispositivos dentro de 10 minutos do tratamento de plasma, enquanto as superfícies são ainda hidrofílico. Esperando mais de 10 minutos para adicionar líquido para o dispositivo torna difícil para o líquido para entrar no dispositivo. Os dispositivos são tipicamente neurônio plasma obrigado a tampa de vidro e 0,5 mg / ml poli-L-lisina (PLL) em tampão borato pH 8,5 é imediatamente adicionado ao dispositivo. Após um mínimo de três horas de incubação com o PLL, os dispositivos são lavados com água dH2O um mínimo de 3 vezes, com pelo menos 15 minutos entre cada lavagem. Em seguida, a água é removida e novas mídias é adicionado ao dispositivo. Neste ponto, o dispositivo está pronto para uso. É importante lembrar, neste ponto, nunca remover toda a mídia do dispositivo. Sempre deixe a mídia no 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

Neste vídeo podemos demonstrar a técnica da litografia macia com polydimethyl siloxano (PDMS) que usamos para farbricate um dispositivo micro para os neurônios cultura.

Fabricação de um dispositivo micro para a compartimentação de Neuron Soma e axônios

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 visualizações.  University of California, Irvine (UCI). Neste vídeo podemos demonstrar a técnica da litografia macia com polydimethyl siloxano (PDMS) que usamos para farbricate um dispositivo micro para os neurônios cultura.

Vídeo: 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.

Um dispositivo micro com padrões Groove para estudar o comportamento celular

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

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.

Um dispositivo de geração de Gradient microfluídicos de Biologia 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.

Preparando E18 neurônios de rato para Cortical Compartimentação em um dispositivo micro

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 Fabricação de dispositivos e modificação da superfície

Cell Capture Using a Microfluidic Device

Captura de célula usando um dispositivo micro

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.

Não-plasma Colagem de PDMS para Fabricação barato e de dispositivos microfluídicos

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.

Fabricação dos canais microfluídicos Termoplásticos

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.

Avaliar motilidade celular Neural Stem Usando um dispositivo Agarose Gel baseado microfluídicos

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.

Aplicação de um elegans dopamina C. Ensaio degeneração dos neurônios de Validação do Potencial Doença 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.

Um compartimento Multi-glia do SNC Neuron-Co-cultura plataforma microfluídica

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