1 부 : microfluidic 신경 장치를 준비 <ol><li> 3 &quot;석판술하여 SU - 8으로 만들어진 패턴으로 실리콘 웨이퍼는 Polydimethylsiloxane (PDMS) microfluidic 금형을 주조하는 데 사용됩니다. 우리는 마스터 금형, 또는 SU - 8 패턴 실리콘 웨이퍼를 참조&quot;마스터 &quot;. </li><li> PDMS가 10시 1분의 오 / W 비율로 치료 요원과 함께 혼합되어 그 PDMS의 10g를 위해이다, 치료 요원의 1g 그것에 추가됩니다. </li><li> PDMS 다음 잘 혼합하여 실리콘 웨이퍼 위에 부어 있습니다. 실리콘 웨이퍼는 10cm의 폴리스티렌 페트리 접시 안에 들어 있습니다. 그것은 약 4 mm의 두께로 마스터를 충당하기 위해 PDMS의 약 12​​g에 15g 걸립니다. </li><li> PDMS와 함께 마스터는 다음 페트리 접시에서 뚜껑이있는 진공 dessicator에 배치하고, 진공이 적용됩니다. 이것은 치료 에이전트에 감동하는 동안 도입된 PDMS에서 공기 방울을 제거하는 데 도움 이루어집니다. 이것은 기포가 생겼는 지 약 15 분 제거하는 정도 소요됩니다. </li><li> PDMS와 함께 마스터는 dessicator에서 제거됩니다. 0.45 음 필터와 공기총은 나머지 거품을 제거하는 데 사용됩니다. </li><li> 마스터가 다음 치료 1 시간 80 ° C 뜨거운 접시에 배치됩니다. </li></ol> 참고 : 진공 dessicator를 사용할 수없는 경우, 마스터가 몇 시간 동안 실온에서 남아있을 수 있습니다. 공기 방울은 결국 자신에 분산됩니다. 뜨거운 접시를 사용할 수없는 경우, PDMS 24 시간 후 실온에서 치료됩니다. 참고 : 마스터가 치료 과정 수준에 있는지 확인하는 것도 중요하다. 2 부 : 뉴런 microfluidic 장치를 절단 <ol><li> PDMS가 마스터에서 치료되면, 그것은 깨끗한 후드로 이동합니다. </li><li> 외과 블레이드를 사용하여 원형이 장치의 주위에 상처입니다. 때 하나 삽입 PDMS에 블레이드, 이것은 실리콘 웨이퍼와 접촉하도록하지만 웨이퍼에 너무 많은 부담을주지하는 것이 중요하다, 다른 마스터 균열 것입니다. </li><li> 원형의 장치 (각 3 &quot;실리콘 마스터 당 사 장치가있다) 주변의 모든 방법을 잘라와 PDMS는 신중하게 마스터를 해제합니다. 그것이 이전 절단에 삽입되는 등 이것은 부드럽게 외과 블레이드를 복잡하게 시작할 수 있습니다 . </li><li> 다음 저수지는 8mm 조직 생검 펀치를 사용하여 장치에 펀치입니다. </li><li> 장치는 다음 등분하고 초과 PDMS는 장치 코닝 유리 커버 (번호 1) 24mm X 40mm에 맞게 깔끔하게 수 있도록 거리에 손질하고 있습니다. 질소 공기 초과 파편을 날려하는 데 사용됩니다. 고집 파편 또한 3M 스카치 브랜드 471 비닐 테이프를 사용하여 제거할 수 있습니다. </li></ol> 파트 3 : 플라즈마는 유리 커버 풀렸는데에 장치를 결합. <ol><li> 플라즈마 클리너는 커버 전표에 Harrick은 신경 세포 장치 결합하는 데 사용됩니다. 간단히, 장치는 플라즈마 클리너에 배치하고 진공 펌프는 챔버에서 공기를 대피하는 데 사용됩니다. </li><li> 진공 압력은 300 milliTorr에 도달하면, 전력은 전기 코일에 켜져 고로 설정되어 있습니다. </li><li> - 전기 코일은 &quot;전자, 이온 및 중성 원자 또는 분자로 구성된 부분적으로 이온화 가스&quot;플라즈마를 생성 <a href="http://www.harrickplasma.com/plasma_physics.php">http://www.harrickplasma.com/plasma_physics.php</a> 밖으로 남아있는 공기 분자의 챔버에 남아. 플라즈마는 유리와 영구 채권을 형성합니다 함께 배치하면 장치의 표면에 반응 종을 만듭니다. 플라즈마는 액체의 추가를 용이하게하는 데 도움이 친수성 ​​표면을 켭니다. 또한, 플라즈마 청소기 장치를 sterilizes. microfluidic 장치가 들어있는 PDMS 장치의 표면은 유리 슬라이드와 함께 플라즈마 클리너로 얼굴을 배치됩니다. 유리와 microfluidic 장치의 플라즈마 표면 처리 후 무균 60mm 요리에 배치, 깨끗한 벤치에서 서로 연락을 조립하고 있습니다. 처리 표면 꽉 돌이킬 유대를 형성하고 있습니다. </li><li> 플라즈마는 유리에 장치를 결합 10 분 이내, 폴리 - L - 라이신 (PLL)가 장치에 추가됩니다. 십분 후, 장치의 친수성은 어렵게 PLL이 장치를 입력할 수 있도록하고 감소합니다. 참고 : PLL의 추가 후 장치에 기포를 확인하는 것이 중요합니다. 그들은 세포를 산화처럼 기본 채널에 공기 방울이 원하지 않는 수 있습니다. 기존의 기포를 제거하는 쉬운 방법은 액체의 150 UL (이 경우 PLL)를 가득 P200 pipet을 사용하는 것입니다, 메인 채널의 개통에 직접 팁을 장소, 강제 P200를 놓으면 되죠. 이것은 여러 번 반복해야 할 수도 있지만 pipet에서 결국 힘이 거품을 몰아 것입니다. </li><li> PLL을 포함하는 장치는 37 표준 조직 문화 인큐베이터에서 하룻밤 부화 허용 ° C와 5 % CO 2. </li><li> 다음날, 드나쁜가 autoclaved dH2O로 두 번 빠르게 씻어서 있습니다. 이 과정에서, 액체는 완전히에서만 저수지에서 메인 채널에서 제거되지 않습니다. 기본 채널에서 액체를 제거하면 거품을 소개합니다. 이 빠른 rinses 후, 장치는 autoclaved dH2O 가득한 3 시간 최소 인큐베이터에 다시 배치하거나,​​ 하룻밤 있습니다. </li><li> 그 다음날, 장치가 다시 한 번 autoclaved dH2O 2 배 빨리 씻어서 있습니다. 그런 다음, 필요한 보조제 culturing 기본 쥐의 뉴런에 B27 및 glutamax를 포함하는 신경 기저 미디어, 장치에 추가됩니다. 장치는 나중에 사용하기 위해 인큐베이터에 다시 배치됩니다. 디바이스는 이제 뉴런의 시딩에 대한 준비가되어 있습니다. </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>

이 비디오에서 우리는 장치가 우리 세포 기관, dendrites, 그리고 axons의 혼합을 포함하는 구획에서 microgrooves로 구분하여 순수한 axonal 분수를 얻을 수 있도록 인치 우리 문화 뉴런하는 데 사용되는 PDMS microfluidic 장치를 만들고 준비하는 방법을 입증해야 . 이러한 장치는 연구자가 다양한 치료의 효과를 연구뿐만 아니라 axons에 신체 부상을 수행하고 이러한 치료가 뉴런에 미칠 영향을 어떻게 관찰할 수있는 플랫폼을 제공하실 수 있습니다. 이 디바이스는 또한 수송 연구를 촉진하는 데 도움이 culturing의 뉴런을 위해 수준을 제공합니다. 또한, 축삭 구획에서 세포 배양 구획을 별도 microgrooves은, 그것이 가능한 치료와 직접 장치의 반대편을 effecting없이 장치의 한쪽을 치료하는 두 구획 사이의 유체 절연을 허용합니다. 이 속성은 연구자가 이러한 신호 전달 및 전송 치료에서 해당 결과로 효과를 공부하실 수 있습니다.

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<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

이 비디오에서는, 우리는 culturing의 뉴런에 microfluidic 장치를 조작하는 데 사용 polydimethyl 실록산 (PDMS)과 소프트 리소그래피의 기술을 보여줍니다. 이전에는 실리콘 웨이퍼는 SU - 8과 마스터 금형을 만들 석판술, 또는 우리는 단순히 &quot;마스터&quot;로 무엇을 참조를 사용하여 신경 세포 microfluidic 장치의 디자인과 패턴했다. 다음, 우리는 다음 ° C 1 시간 80 PDMS를 가열하여 치료하는 마스터 위에 실리콘 고분자 PDMS 잔. PDMS는 장치의 부정적인 곰팡이를 형성합니다. PDMS 그런 다음 조심스럽게 자르고 마스터 떨어져 해제됩니다. 구멍은 저수지가 어디 부수고 초과 PDMS 거리 장치에서 손질하고 있습니다. 질소는 장치에서 초과 파편을 날려하는 데 사용됩니다. 이 시점에서 장치는 이제 사용 준비하거나, 플라즈마 살균기 / 세척기 또는 reversibly 단순히 커버 유리 위에 장치를 배치하여 덮개 유리에 바인딩 될 수있는 작업은 다음과 코닝 제 1 커버 유리 접착 수 있습니다. 유리 장치의 가역 본딩은 별도의 비디오로 덮여 있고 장치가 70 % 에탄올이나 autoclaving을 통해 소독하는 것이 첫번째 필요합니다. 더 이상 치료가 필요하지 않도록 플라즈마 치료는 장치를 sterilizes. 이것은 표면이 아직 친수성하는 동안 플라즈마 치료 10 분 이내에 장치에 액체 추가 장치를, 플라즈마는 - 대우 때, 그러나 중요합니다. 장치에 액체 추가하는 이상보다 10 분 정도 기다리는 것은 어려운 액체가 장치를 입력할 수 있도록합니다. 신경 장치는 일반적으로 유리와 산도 8.5 borate 버퍼의 0.5 MG / ML 폴리 - L - 라이신 (PLL)를 충당하기 위해 플라즈마 - 바운드 아르 즉시 장치에 추가됩니다. PLL과 잠복기 3 시간 최소 후, 장치는 각 씻어 사이에 적어도 15 분 dH2O의 물로 3 회 최소 씻어 있습니다. 다음, 물을 제거하고 신선한 미디어 장치에 추가됩니다. 이 시점에서 장치를 사용할 준비가되었습니다. 장치에서 모든 미디어를 제거하지 않았을이 시점에서 기억하는 것이 중요합니다. 항상 메인 채널에서 미디어를 두십시오.

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

이 비디오에서 우리는 culturing의 뉴런에 microfluidic 장치를 farbricate을 사용 polydimethyl 실록산 (PDMS)과 소프트 리소그래피의 기술을 보여줍니다.

신경 소마와 Axons의 Compartmentalization위한 Microfluidic 장치의 제작

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 조회수.  University of California, Irvine (UCI). 이 비디오에서 우리는 culturing의 뉴런에 microfluidic 장치를 farbricate을 사용 polydimethyl 실록산 (PDMS)과 소프트 리소그래피의 기술을 보여줍니다.

비디오: 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.

세포 행동 유학을위한 그루브 패턴 Microfluidic 장치

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

연구

생물학

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.

세포 생물학을위한 그라디언트 생성 Microfluidic 장치

 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.

Microfluidic 장치에서 Compartmentalization에 대한 E18 두피 랫 뉴런 준비

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 장치 제작 및 표면 변경

Cell Capture Using a Microfluidic Device

Microfluidic 장치를 사용하여 셀 캡처

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.

Microfluidic 장치의 저렴한 제작에 대한 PDMS의 비 플라즈마 본딩

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.

열가 소성 Microfluidic 채널 제작

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.

아가로 오스 겔 기반 Microfluidic 장치를 사용 신경 줄기 세포의 운동성 평가

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.

잠재적인 파킨슨병의 유전자 검증을위한 C. 엘레간스 도파민 신경 세포의 변성 분석의 응용

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.

다중 구획 CNS 뉴런 - glia 공동 문화 Microfluidic 플랫폼

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