准备神经元细胞培养的微流体装置 1）清洁康宁第一24毫米x 40毫米玻片<ul><li> 30分钟超声水浴中sonicator玻片</li><li>用70％乙醇冲洗玻片</li><li>洗净幻灯片DH 2 O 3倍</li><li>组织培养罩在几个小时，最好是在一夜之间干幻灯片</li></ul> 注：我们有特殊的金属托盘，我们加载的幻灯片。幻灯片分开单独插槽放置在这个金属架。然后我们就可以放置在一个玻璃皿这些金属装入纸盘，我们与水，在水浴sonicator中进行填写，并超声30分钟。随后的清洗都进行了这盘。 2）准备的PDMS设备<ul><li>从硅片切割的PDMS模具，打孔PDSM投季度， </li><li>清洁的PDMS设备先吹惰性气体（氩气或氮气）。然后用3M苏格兰品牌471磁带升空任何剩余的碎片</li></ul> 注：重要的是要保持设备面对。最初，当我们从硅片切PDMS的距离，与晶圆接触的一面是干净的一面：它包含了微流体通道。我们尝试要小心，因为我们可以以不损坏设备，并保持清洁面朝直到我们准备等离子粘接。 <ul><li>在塑料容器的高压灭菌设备</li><li>经过高压灭菌，清洁1号康宁玻璃幻灯片和等离子治疗设备使用一个Harrick品牌等离子清洁， <a href="http://www.harrickplasma.com/" target="_blank">www.harrickplasma.com</a> </li><li>放置设备的清洁载玻片上的一面朝下。 </li></ul>该设备是目前保税载玻片等离子。 3）涂层设备与聚- L -赖氨酸（PLL） <ul><li> PLL被添加到每个设备端，并允许流入未来以及通过良好</li></ul> =&gt; 150μL较小井大井和30μ升PLL PLL的 。它不准确，只要井满。 注意：如果你在一个设备，你会看到4口井，每两个连接。你想，使其流入未来以及通过设备把在一个PLL。 <ul><li>允许流经设备的PLL约10分钟，然后加入更多的PLL的水井，以填补他们</li><li>放置至少4小时在孵化器的装置，含有锁相环（隔夜可取）在37 ° C </li><li>真空治疗后多余的PLL，但小心，不要吸的设备所有的液体</li></ul> 注意：您不希望引入气泡渠道或分庭。只要真空井多余的PLL。 <ul><li>添加蒸压生署2 O以及每个设备两侧，并允许流量通过毛细作用等以及</li></ul>图1是一个微流体装置图。请注意蓝（SOMA方）如何连接红（轴突方）连接。当我们说的PLL，或水或媒体，以及每个设备端，我们的意思，例如，是：将150蒸压DH 2 O的 UL在一个蓝色的那么到位150μL灭菌的dh 2 0成一个红井。 ，水（或PLL，或媒体，或细胞），将流经该设备其他相应的井。 <img src="/files/ftp_upload/old/410_2008_8_27_3/410_Figure-01.png" alt="图1" width="362" height="216" /> 图1 注意：请注意，从现在起，当我说“的地方媒体，细胞，水或在TOP井PLL的”，我指的是以上的SOMA将在左边的图和轴突将增长到权利。 <ul><li>吸过的水（再小心不要完全从设备中取出所有的液体），然后加入150μL另一个蒸压DH 2 O接好每一个，并允许它流动到相应的。 </li><li>将设备包含在一个孵化器2 O卫生署在37 ° C为1小时，然后重复清洗步骤。洗净设备与DH 2 O三次，每次一小时，这是总的。 </li></ul> 注意：由于从PLL硼酸可以吸收到的PDMS，在全度妍实验室的一些建议后，我夫妇的孵化设备蒸压生署 2过夜的可能性nitial快速冲洗。这将确保所有免费硼酸，将滤出的PDMS前加载的细胞。如果不这样做，有可能到媒体发布的硼酸，随着时间的推移，这可能导致细胞毒性。 <ul><li>添加必要的因素（glutamax，B27，PenStrep）顶部井神经基底媒体（NBM） </li></ul> 注：该设备可以孵育过夜和镀细胞第二天，或电镀前的细胞培养与NMB +因素至少3小时。 准备装载到设备的神经元我们使用18日龄胎鼠大脑皮层，我们可以称为脑位或在这里为我们准备在校园的公司购买。我们希望得到组织的新鲜。 <ol><li>获得1个胎儿大脑皮质组织（2件）存储在休眠Ë </li><li>以3个长玻璃滴管，并先后火成较小的尺寸每个使用酒精灯</li><li>删除从休眠发送一个玻璃吸管在2毫升的NMB因素在无菌15毫升管组织。 </li><li> Trituration：使用一个灯泡和玻璃吸管皮层是通过向上和向下约5至10倍。然后，下一个较小的吸管，用吸管向上和向下的组织。最后，最小的吸管，用吸管向上和向下的组织。我们要确保有不可见的大块。 注：近日，全度妍实验室开始比较存储在Hibernate发送准备从最初在50％与0.125％胰蛋白酶处理的组织细胞，组织细胞CA +自由和Mg + +免费清扫缓冲区。的共识是准备使用胰蛋白酶产量比组织更可行的最初放置在Hibernate E.如果你不打算马上使用组织的细胞，组织，那么你就需要存储在Hibernate E.组织 如果你是打算向使用组织马上和想提高生存能力，然后用胰蛋白酶处理组织机械trituration如下：1）稀释1毫升的钙+ +免费镁+ +免费1毫升0.25％胰蛋白酶（Gibco公司，Invitrogen公司）夹层缓冲液（最终胰蛋白酶= 0.125％）到15毫升管（保持冰冷）; 2）缓冲液中进行组织和孵化立即为8分钟，在37 ° C; 3）添加DMEM/10毫升10％胎牛血清停止胰蛋白酶反应，继续下面的协议。 </li><li>在1分钟1100转离心细胞</li><li>送气媒体小心关闭细胞沉淀</li><li>加入1毫升的NMB因素重新悬浮颗粒和它的吹打</li><li>经过重力流通过一个过滤器删除任何团块（屋宇署猎鹰细胞过滤40微米尼龙）重悬细胞</li><li>计数和板块细胞： <ul><li>到离心管中，加入60μLNMB +因素，再拌入20μL细胞悬液，台盼蓝和20μL </li><li>新增约10μL此解决方案的血球计数活细胞（蓝色） </li><li>调整浓度为2.5 - 4.5 × 10 6细胞/ ml 注：我们通常获得浓度约为2.5 - 4.5 × 106细胞/ ml。根据卷上的细胞悬浮（通常为1毫升的NBM + B27/Glutamax/PenStrep percortex）。如果组织准备，与胰蛋白酶的产量可能会有所增加。 </li></ul></li></ol>电镀设备<ol><li>装入到一个很好的设备细胞（初级神经元） </li><li>板5μL％的小型设备和大设备每20μL 注：您可以负载设备的顶部和10μL设备的底部（总量的一半）10μL的细胞，或直接所有的细胞somal以及在顶部的20μL（5μL以及在顶部somal小型设备）。 </li><li>孵育10分钟在37℃培养箱中培养，使细胞可以开始重视</li><li>新增约150/30μLNMB +的因素以及对设备的大小取决于每个 注：卷可能会有所不同厚度的PDMS。重要的是，井已满。 </li></ol>补充在使用无血浆粘合设备没有等离子治疗，一名技术人员，它的成功没有血浆每周，克里斯蒂娜涂说，只是马上载入细胞。 记住删除SOMA方井（轴突方都不会有问题，如果你离开媒体）媒体。它的流体的流动，使这些细胞进入主渠道。如果你有在水井的媒体，你不会得到流体的流动。

<ol>
	<li>Park, J. W., Vahidi, B., Taylor, 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 (4), 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 (8), 599-605 (2005).</li></ol>

The authors have nothing to disclose.

在这里，我们展示了如何使用，而不需要等离子体清洁的神经元微流体装置。我们参考这种非等离子的粘接。

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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>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>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>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>Poly-L-Lysine</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P-1274</td>
<td>&nbsp;</td>
<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>		</tbody>
		</table>
		</div>
			<div>
					</div>

non-plasma bonding of pdms for inexpensive fabrication of microfluidic devices

在这个视频中，我们展示了如何在不使用等离子粘接的神经元微流体装置。在某些情况下，它可能是可取的可逆债券设备，康宁公司的1号玻璃盖。这可能是因为，也许，等离子清洁，不可用的。在其他情况下，它可能需要从玻璃中删除设备后显微镜或为某些类型的免疫的神经元的培养，虽然这是没有必要因为免疫染色的神经元可在设备中删除设备。然而，一些研究人员仍然倾向于删除该设备。在这种情况下，设备可逆的粘接到玻璃盖可能。非等离子设备，而不是作为一个密封紧形成的粘接，也有一些缺点。轴突在某些情况下可能会下的沟槽，而不是通过他们的成长。此外，因为玻璃和PDMS是疏水，液体不容易进入设备使得有必要在次强行进入设备的媒体和其他试剂。通过毛细作用，液体将进入设备，但它需要显着较长相比已血浆保税设备。等离子体清洁玻璃和设备上创建的临时的亲水性收费，以便通过设备后粘接在几秒钟内液体流动。对于非等离子体约束装置，通过设备的液体流动需要几分钟时间。同样重要的是要注意设备与非血浆用于粘接需要先进行消毒，而等离子体处理设备不需要进行消毒，使用前因为等离子体清洁消毒。

Watch this Scientific Journal Video about Non-plasma Bonding of PDMS for Inexpensive Fabrication of Microfluidic Devices at JoVE.com

Non-plasma Bonding of PDMS for Inexpensive Fabrication of Microfluidic Devices

在这段视频中，我们演示了如何使用没有等离子粘接的神经元微流体装置。

PDMS微流体装置的廉价制造的非等离子键合

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 ...

non-plasma-bonding-pdms-for-inexpensive-fabrication-microfluidic

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

34.3K 次观看.  University of California, Irvine (UCI). 在这段视频中，我们演示了如何使用没有等离子粘接的神经元微流体装置。

视频: Non-plasma Bonding of PDMS for Inexpensive Fabrication of Microfluidic Devices

Fabrication of a Microfluidic Device for the Compartmentalization of Neuron Soma and Axons

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for culturing neurons. Previously, a silicon wafer was patterned with the design for the neuron microfluidic device using SU-8 and photolithography to create a master mold, or what we simply refer to as a "master". Next, we pour the silicon polymer PDMS on top of the master which is then cured by heating the PDMS to 80&deg;C for 1 hour. The PDMS forms a negative mold of the device. The PDMS is then carefully cut and lifted away from the master. Holes are punched where the reservoirs will be and the excess PDMS trimmed away from the device. Nitrogen is used to blow away any excess debris from the device. At this point the devices are now ready for use and can either bonded to corning No. 1 cover glass with a plasma sterilizer/cleaner or can be reversibly bound to the cover glass by simply placing the device on top of the cover glass. The reversible bonding of the device to glass is covered in a separate video and requires first that the device be sterilized either with 70% ethanol or by autoclaving. Plasma treating sterilizes the devices so no further treatment is necessary. It is, however, important, when plasma-treating the devices, to add liquid to the devices within 10 minutes of the plasma treatment while the surfaces are still hydrophilic. Waiting longer than 10 minutes to add liquid to the device makes it difficult for the liquid to enter the device. The neuron devices are typically plasma-bound to cover glass and 0.5 mg/ml poly-L-lysine (PLL) in pH 8.5 borate buffer is immediately added to the device. After a minimum of 3 hours incubating with PLL, the devices are washed with dH2O water a minimum of 3 times with at least 15 minutes between each wash. Next, the water is removed and fresh media is added to the device. At this point the device is ready for use. It is important to remember at this point to never remove all the media from the device. Always leave media in the main channel.

In this video we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to farbricate a microfluidic device for culturing neurons.

条块索玛的神经元和轴突的一个微流体设备制造

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for ...

科研

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.

一个渐变产生的微流体装置，细胞生物学

 The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described.  A microfluidic platform is an ...

PDMS Device Fabrication and Surface Modification

PDMS的设备制造和表面改性

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.

制备热塑性微流体通道

In our lab, we have successfully isolated nucleic acids directly from microliter and submicroliter volumes of human blood, urine and stool using ...

Microfluidic Applications for Disposable Diagnostics

In this interview, Dr. Klapperich discusses the fabrication of thermoplastic microfluidic devices and their application for development of new diagnostics.

In this interview, Dr. Klapperich discusses the fabrication of thermoplastic microfluidic devices and their application for development of new diagnostics.

微流控一次性诊断的应用

In this interview, Dr. Klapperich discusses the fabrication of thermoplastic microfluidic devices and their application for development of new ...

Fabrication of Amperometric Electrodes

Carbon fiber electrodes are crucial for the detection of catecholamine release from vesicles in single cells for amperometry measurements.  Here, we describe the techniques needed to generate low noise (&lt;0.5 pA) electrodes.  The techniques have been modified from published descriptions by previous researchers (1,2).  Electrodes are made by preparing carbon fibers and threading them individually into each capillary tube by using a vacuum with a filter to aspirate the fiber.  Next, the capillary tube with fiber is pulled by an electrode puller, creating two halves, each with a fine-pointed tip.  The electrodes are dipped in hot, liquid epoxy mixed with hardener to create an epoxy-glass seal.  Lastly, the electrodes are placed in an oven to cure the epoxy.  Careful handling of the electrodes is critical to ensure that they are made consistently and without damage.  This protocol shows how to fabricate and cut amperometric electrodes for recording from single cells.

This protocol describes how to generate carbon fiber electrodes. The electrodes are subsequently used to detect catecholamine release from vesicles with carbon fiber amperometry.

安培电极的制备

Carbon fiber electrodes are crucial for the detection of catecholamine release from vesicles in single cells for amperometry measurements.  Here, we ...

Fabrication of Myogenic Engineered Tissue Constructs

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to the restrictions imposed by a child's small size and their inevitable growth. Consequently, there is a genuine need for innovative therapies designed specifically for pediatric patients with cardiac rhythm disorders. We propose that a conductive biological alternative consisting of a collagen-based matrix containing autologously-derived cells could better adapt to growth, reduce the need for recurrent surgeries, and greatly improve the quality of life for these patients. In the present study, we describe a procedure for incorporating primary skeletal myoblast cell cultures within a hydrogel matrix to fashion a surgically-implantable tissue construct that will serve as an electrical conduit between the upper and lower chambers of the heart. Ultimately, we anticipate using this type of engineered tissue to restore atrioventricular electrical conduction in children with complete heart block. In view of that, we isolate myoblasts from the skeletal muscles of neonatal Lewis rats and plate them onto laminin-coated tissue culture dishes using a modified version of established protocols[2, 3]. After one to two days, cultured cells are collected and mixed with antibiotics, type 1 collagen, Matrigel&trade;, and NaHCO3. The result is a viscous, uniform solution that can be cast into a mold of nearly any shape and size[1, 4, 5]. For our tissue constructs, we employ type 1 collagen isolated from fetal lamb skin using standard procedures[6]. Once the tissue has solidified at 37&deg;C, culture media is carefully added to the plate until the construct is submerged. The engineered tissue is then allowed to further condense through dehydration for 2 more days, at which point it is ready for in vitro assessment or surgical-implantation.

Here, we demonstrate fabrication of collagen-based, tissue constructs containing skeletal myoblasts. These 3-D engineered constructs may be used to replace or repair tissues in vivo. For our purposes, we have designed these as an atrioventricular electrical conduit for the repair of complete heart block[1].

制作生肌工程的组织结构

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to ...

High Speed Droplet-based Delivery System for Passive Pumping in Microfluidic Devices

A novel microfluidic system has been developed that uses the phenomenon of passive pumping along with a user controlled droplet based fluid delivery system. Passive pumping is the phenomenon by which surface tension induced pressure differences drive fluid movement in closed channels. The automated fluid delivery system consists of a set of voltage controlled valves with micro-nozzles connected to a fluid reservoir and a control system. These voltage controlled valves offer a volumetrically precise way to deliver fluid droplets to the inlet of a microfluidic device in a high frequency manner. Based on the  dimensions demonstrated in the current study example, the system is capable of flowing 4 milliliters per minute (through a 2.2mm by 260um cross-sectional channel). Based on these same channel dimensions, fluid exchange of a point inside the channel can be achieved in as little as eight milliseconds. It is observed that there is interplay between momentum of the system (imparted by a combination of the droplets created by the valves and the fluid velocity in the channel), and the surface tension of the liquid. Where momentum provides velocity to the fluid flow (or vice-versa), equilibration of surface tension at the inlet provides a sudden stop to any flow. This sudden stop allows the user to control the flow characteristics of the channel and opens the door for a variety of biological applications, ranging anywhere from reagent delivery to drug-cell studies. It is also observed that when nozzles are aimed at the inlet at shallow angles, the droplet momentum can cause additional interesting fluid phenomena, such as mixing of multiple droplets in the inlet.

A novel microfluidic system has been developed using the phenomenon of passive pumping and a user controlled fluid delivery system. This microfluidic system has the potential to be used in a wide variety of biological applications given its low cost, ease of use, volumetric precision, high speed, repeatability and automation.

基于液滴在微流体装置的被动抽水高速输送系统

A novel microfluidic system has been developed that uses the phenomenon of passive pumping along with a user controlled droplet based fluid delivery ...

Automated Microfluidic Blood Lysis Protocol for Enrichment of Circulating Nucleated Cells

In this report the protocol for an automated microfluidic blood lysis device is detailed. Circulating nucleated cells (CNCs), including leukocytes and endothelial cells, provide an ideal platform for an updated status on the immune condition of an individual. The microfluidic protocol allows for enrichment of CNCs without selective cell loss and sample preparation variability due to user-mediated steps. Briefly, the protocol includes device fabrication, sample collection, device setup, and running blood through the microfluidic chamber. Within the device whole blood is rapidly mixed with deionized water for approximately 10 seconds in a 50 micron x 150 micron microfluidic channel. In this time span erythrocytes are lysed due to hypotonic conditions. Herringbone structures on the bottom of the channel ensure thorough mixing and exposure of cells to a constant environment. Remaining cells are returned to isotonic conditions at the exit of the device, fixed using 2% paraformaldehyde, centrifuged to separate erythrocyte debris from CNCs, and suspended in flow buffer for staining and analysis by flow cytometry. Results show clean flow cytometry scatter plots with CNC populations saved. Significance of this device and protocol comes in the study and understanding of disease pathogenesis by analysis of CNC populations. Hence, automation, effectiveness, and simplicity of the microfluidic protocol are demonstrated.

An automated microfluidic device was developed for circulating nucleated cell enrichment from peripheral blood via erythrocyte lysis that ensures isolation of high quality sample without cell loss.

微流控自动化血液循环核细胞富集的裂解协议

In this report the protocol for an automated microfluidic blood lysis device is detailed. Circulating nucleated cells (CNCs), including leukocytes and ...

Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures 

Oxygen is a key modulator of many cellular pathways, but current devices permitting in vitro oxygen modulation fail to meet the needs of biomedical research. The hypoxic chamber offers a simple system to control oxygenation in standard culture vessels, but lacks precise temporal and spatial control over the oxygen concentration at the cell surface, preventing its application in studying a variety of physiological phenomena. Other systems have improved upon the hypoxic chamber, but require specialized knowledge and equipment for their operation, making them intimidating for the average researcher. A microfabricated insert for multiwell plates has been developed to more effectively control the temporal and spatial oxygen concentration to better model physiological phenomena found in vivo. The platform consists of a polydimethylsiloxane insert that nests into a standard multiwell plate and serves as a passive microfluidic gas network with a gas-permeable membrane aimed to modulate oxygen delivery to adherent cells. The device is simple to use and is connected to gas cylinders that provide the pressure to introduce the desired oxygen concentration into the platform. Fabrication involves a combination of standard SU-8 photolithography, replica molding, and defined PDMS spinning on a silicon wafer. The components of the device are bonded after surface treatment using a hand-held plasma system. Validation is accomplished with a planar fluorescent oxygen sensor. Equilibration time is on the order of minutes and a wide variety of oxygen profiles can be attained based on the device design, such as the cyclic profile achieved in this study, and even oxygen gradients to mimic those found in vivo. The device can be sterilized for cell culture using common methods without loss of function. The device's applicability to studying the in vitro wound healing response will be demonstrated.