パート1：マイクロ流体ニューロンデバイスの準備<ol><li> 3&quot;フォトリソグラフィーによるSU - 8で作られたパターンを持つシリコンウェハは、ポリジメチルシロキサン（PDMS）マイクロ金型をキャストするために使用されます。我々は、マスター金型、またはSU - 8のパターニングシリコンウェハを参照して&quot;マスター&quot;。 </li><li> PDMSは10:1 AW / w比で硬化剤と混合され、PDMSの10グラムのために、すなわち、硬化剤の1グラムが追加されます。 </li><li> PDMSは、よく混合し、シリコンウエハ上に注がれています。シリコンウェハは、10cmのポリスチレンペトリ皿内に含まれています。それは約4mmの厚さでマスターをカバーするためにPDMSの約12グラム〜15 gを取ります。 </li><li> PDMSとマスターは、ペトリ皿の蓋付きの真空デシケーターに配置され、真空が適用されます。これは、硬化剤の中で撹拌中に導入されたPDMSから気泡を取り除くために行われている。それは、気泡のため約15分が削除されます。 </li><li> PDMSとマスターは、デシケーターから削除されます。 0.45 UMのフィルター付き空気銃は、残りの気泡を除去するために使用されます。 </li><li>マスターは、治すために1時間80℃のホットプレート上に配置されます。 </li></ol> 注 ：真空デシケーターを使用できない場合は、マスターが数時間室温で放置することができます。気泡は、最終的に自分自身で消費されます。ホットプレートが使用できない場合は、PDMSは、24時間後に室温で硬化されます。 注 ：マスターは、硬化プロセス中レベルであることを確認することも重要です。 パート2：ニューロンマイクロ流体デバイスを切り出す<ol><li> PDMSはマスターに硬化されると、それはクリーンフードに取得されます。 </li><li>外科ブレードを使用して、円はデバイスの周囲にカットされます。するときに一インサートPDMSにブレード、それはシリコンウエハとの接触を行うことがウエハ上にあまりにも多くの圧力をかけることが重要です、他のマスターは、割れの原因となる。 </li><li>円で（各3あたり4デバイス&quot;シリコンマスターが存在する）、PDMSは慎重にマスターリフトオフされデバイスの周りのすべての方法をカット。これは、前のカットに挿入されると優しく外科刃をねじることによって起動することができます。 </li><li>次に、貯水池は、8 mmの組織生検パンチを使用してデバイスにパンチされています。 </li><li>デバイスは、四つと過剰PDMSデバイスがコーニングカバーガラス（1号）24ミリメートル× 40ミリメートルにきちんと収まるように離れてトリミングされます。窒素、空気が余分なゴミを吹き飛ばすために使用されます。頑固な残骸は、3Mスコッチブランド471ビニールテープを使用して削除できます。 </li></ol>パート3：プラズマボンディングガラスカバースリップのデバイス。 <ol><li>プラズマクリーナーは、カバースリップに結合するHarrickニューロンデバイスを使用されています。簡単に言えば、デバイスは、プラズマクリーナーに配置され、真空ポンプは、チャンバから空気を排気するために使用されます。 </li><li>真空圧力は300ミリトルに達した後、電源が電気コイルに電源が入っており、高に設定されています。 </li><li> -電気的なコイルは&quot;電子、イオンと中性原子または分子から成る部分的にイオン化されたガス&quot;、プラズマを作成し<a href="http://www.harrickplasma.com/plasma_physics.php">http://www.harrickplasma.com/plasma_physics.php</a> 、うち残っている空気分子のは、チャンバー内に残された。プラズマはガラスと永続的な結合を形成する一緒に置くとデバイスの表面に反応種を作成します。プラズマはまた、液体の付加を促進する助け親水性の表面を、になります。さらに、プラズマクリーナーは、デバイスを滅菌する。 マイクロ流体デバイスを含むPDMSデバイスの表面はガラスのスライドとともにプラズマクリーナーに表向きに置かれます。 ガラスとマイクロ流体デバイスの表面をプラズマ処理は、滅菌​​60 mmの培養ディッシュに入れ、クリーンベンチで互いに接触して組み立てられています。処理された表面は、タイトな不可逆的な結合を形成する。 </li><li>ガラスにデバイスを接合するプラズマの10分以内、ポリ- L -リジン（PLL）がデバイスに追加されます。 10分後、デバイスの親水性は、それが困難なPLLは、デバイスを入力できるようになっ減少します。 注 ：PLLの添加後、装置内の空気の泡をチェックすることが重要です。彼らは細胞を酸化と同じように、メインチャンネルでの気泡は、好ましくないです。あらゆる既存のAIRの気泡を除去する簡単な方法は、液体の150μlを（この場合は、PLL内）で満たされたP200ピペットを使用する場合、メインチャネルの開口部に直接先端を配置し、強制的にP200を押し下げる。これは繰り返すことが必要になることがありますが、最終的にはピペットからの力が気泡​​を追い出すでしょう。 </li><li> PLLを含むデバイスは、37標準組織培養インキュベーターで一晩インキュベートされている° C、5％のCO 2。 </li><li>翌日、デ悪徳は、オートクレーブdH2Oで二回迅速にすすぎ洗いされています。このプロセスの間に、液体を完全にのみ貯水池から、メインチャンネルから削除されることはありません。メインチャンネルから液体を削除すると、泡をご紹介します。 2クイック洗浄した後、デバイスは、オートクレーブdH2Oを充填し、約3時間のインキュベーターに戻し入れ、または一晩されています。 </li><li>翌日、デバイスが再度オートクレーブdH2Oで素早く2回リンスしています。その後、培養初代ラットの神経細胞のために必要なサプリメントB27とグルタミンを含む神経基本培地は、デバイスに追加されます。デバイスは、将来の使用のためにインキュベーターに戻って配置されます。デバイスは現在、神経細胞の播種のための準備が整いました。 </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>

このビデオでは、デバイスは、私たちは細胞体、樹状突起、および軸索のミックスを含むコンパートメントからmicrogroovesで区切られた純粋な軸索の画分を得ることができますインチ我々は文化の神経細胞に使用するPDMSマイクロ流体デバイスを作成し、準備する方法を示している。これらのデバイスは、研究者が様々な治療の効果を研究するだけでなく、軸索上の物理的な傷害を実施し、これらの治療は、神経細胞にどのような効果を観察するためのプラットフォームを提供することができます。デバイスはまた、輸送の研究を促進支援する培養神経細胞にするためのレベルを提供します。さらに、軸索のコンパートメントから細胞培養のコンパートメントを分離microgroovesは、、それが可能な治療法と直接デバイスの反対側に影響を及ぼすことなく、デバイスの一方の側を治療することつの区画の間に流体を分離することができます。このプロパティは、研究者が治療の結果そのシグナル伝達や輸送などの効果を研究することができます。

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

fabrication of a microfluidic device for the compartmentalization of neuron soma and axons

このビデオでは、私たちは培養神経細胞のためのマイクロ流体デバイスを作製するために使用するポリジメチルシロキサン（PDMS）とソフトリソグラフィーの手法を示しています。以前は、シリコンウェハは、SU - 8とフォトリソグラフィーマスターの金型を作成するために、または何を我々は単に&quot;マスター&quot;として参照を使用してニューロンマイクロ流体デバイスの設計をパターン化した。次に、我々は、° Cで1​​時間80 PDMSを加熱することにより硬化されているマスターの上にシリコンポリマーPDMSを注ぐ。 PDMSは、デバイスの負の金型を形成している。 PDMSは、慎重にカットし、マスターから離れて持ち上げられる。穴は貯水池があるところ打ち抜き、過剰PDMSは、デバイスからトリムされます。窒素は装置から余分なゴミを吹き飛ばすために使用されます。この時点でデバイスを使用する準備が整いましたので、どちらもプラズマ滅菌器/クリーナーとコーニング第1カバーガラスに接着したり、可逆的に単にカバーガラスの上にデバイスを配置することにより、カバーガラスにバインドすることができます。ガラスへのデバイスの可逆的結合は、別のビデオで覆われ、デバイスは70％エタノールでまたはオートクレーブのいずれかによって滅菌される最初の必要です。それ以上の処置は必要ないので、プラズマの治療には、デバイスを滅菌する。それは、表面がまだ親水性である一方、プラズマ処理の10分以内にデバイスに液体を追加するデバイスを、プラズマが、治療時に、、しかし、重要です。デバイスに液体を追加する10分以上待つことが困難液体がデバイスを入力できるようになります。ニューロンデバイスは、通常のガラスとpH8.5のホウ酸緩衝液に0.5 mg / mlのポリ- L -リジン（PLL）をカバーするためにプラズマバインドされたが、すぐにデバイスに追加されます。 PLLでインキュベート3時間の最小値の後に、デバイスが各洗浄の間に少なくとも15分にdH2Oの水で最低3回洗浄する。次に、水は除去され、新鮮なメディアがデバイスに追加されます。この時点で、デバイスの使用準備ができています。デバイスからすべてのメディアを取り外すことは決してして、この時点で覚えておくことが重要です。常にメインチャネルのメディアを残す。

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

このビデオでは、我々は、培養神経細胞のためのマイクロ流体デバイスをfarbricateするために使用するポリジメチルシロキサン（PDMS）とソフトリソグラフィーの手法を示しています。

ニューロン相馬と軸索のコンパートメントのためのマイクロ流体デバイスの作製

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 ビュー.  University of California, Irvine (UCI). このビデオでは、我々は、培養神経細胞のためのマイクロ流体デバイスをfarbricateするために使用するポリジメチルシロキサン（PDMS）とソフトリソグラフィーの手法を示しています。

ビデオ: 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.

細胞挙動を研究するための溝パターンを用いたマイクロ流体デバイス

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

生物学

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.

細胞生物学の勾配を生むマイクロ流体デバイス

 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.

マイクロ流体デバイスでコンパートメントのためのE18脳ラットのニューロンの準備

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デバイスの作製と表面改質

Cell Capture Using a Microfluidic Device

マイクロ流体デバイスを用いた細胞のキャプチャ

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.

マイクロ流体デバイスの低コスト作製のためのPDMSの非プラズマボンディング

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.

熱可塑性マイクロ流体チャネルの作製

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.

アガロースゲルベースの​​マイクロ流体デバイスを用いた神経幹細胞運動の評価

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.

潜在的なパーキンソン病の遺伝子を検証するための線虫のドーパミンニューロンの変性のアッセイのアプリケーション

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.

マルチコンパートメントのCNSニューロングリア共培養マイクロ流体プラットフォーム

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