他在设备安装的援助提供DEP的驱动和米切尔Collens trifurcating渠道，我们对此表示赞赏常宇晋。这项工作一直支持由美国国家科学基金会通过赠款OISE - 0951647，由台湾国科会通过批准99-2911-1-002-007。

一个设备安装总图如图1a所示。 PCB样品的大会，是进一步详细在一个横断面示意图，在图1B。 1。印刷电路板电极的准备: <ol><li>设计PCB电极所需的几何形状，产生非均匀的电场。定制印刷电路板电极的芯片，可以责令通过商业制造设备（图1C）。 </li><li>准备预装配的印刷电路板电极焊接16号线的每一个印刷金属电极（图1D）结束。 <ol><li>广场上的电极年底丝。举办​​的印刷电路板的金属面积与热的烙铁加热丝导线到位。 </li><li>加热丝送入少量的焊锡，焊锡线，以填补。 </li><li>导线与焊料填充后，取出烙铁，导线，而焊料冷却到位。 </li></ol></li><li>每个PCB上（图1D）的电气连接，重复的焊接工艺。 </li></ol> 2。微流体通道做准备: <ol><li>准备使用184 Sylgard从道康宁硅橡胶弹性体，聚二甲基硅氧烷（PDMS）为基础的微流体通道。一个母模定义的渠道通常是通过标准的微细加工用硅片和SU - 8胶过程。 </li><li>在10:1的比例混合基复合固化剂为5分钟。 </li><li>泼到液体硅橡胶预制SU - 8母模和消除气泡暴露液态硅橡胶真空几分钟。重复真空过程中，如果需要彻底清除所有气泡。 </li><li>治愈的PDMS 70 ° 2小时。 </li><li>删除与微流体通道，从晶圆用刀片，不小心打破晶圆硅橡胶板。 </li><li>引入微流体装置的体液和细胞打孔。 （注:注射器抽，重力或表面张力的流量都可以使用DEP的保护。） </li><li>检查微流体装置，以确保它是免费的灰尘和碎屑。清洁的PDMS，就可以轻松实现使用3M苏格兰魔术贴。 </li><li>等离子体债券的PDMS微流体通道，以一个干净的0号厚度（80-130微米）盖玻片。在100 ° C热盖玻片，微流体大会至少15分钟。 </li></ol> 3。准备低电导率介质: <ol><li>低电导率介质准备混合8.5％的蔗糖+葡萄糖0.3％（W / V），在去离子（DI）水。 注:之前我们已经证实，可以在传统的细胞培养介质天培养细胞暴露后短时间（约30分钟），低电导率 11 </li></ol> 4。组装到印刷电路板电极的微流体通道: <ol><li>放置到PCB矿物油少量（约10微升），以确保印刷电路板和盖玻片之间的紧密接触。 注:为减少电极的可视化的可选步骤是大衣PCB表面用黑色记号笔或油漆层非常薄。 </li><li>将盖玻片，微流体通道上的油污PCB盖玻片形成与油的接触（盖玻片下）大会，。轻压盖玻片，微流体大会，以确保良好的接触，并尽量减少气泡，可以从细胞和珠可视减损。完成设备的一个例子是显示在图1D。 </li></ol> 5。排序和集中珠细胞使用的DEP: <ol><li>与去离子水或低电导率介质填充的微流体通道;血浆粘接工艺有利于暂时通常疏水的PDMS表面亲水性的水溶液易于加载到微流体通道。 </li><li>引入渠道水库（图1C）的细胞和/或聚苯乙烯珠。在这里，我们使用人类结肠癌细胞（HT - 29）。 </li><li>函数发生器的输出连接到交流电源放大器的输入，然后连接放大器的输出电极导线。涵盖所有的电线和表面安装用电工胶带，以保护用户，从潜在的暴露在冲击。一个设备安装示意图如图1a所示。 </li><li>设置函数发生器产生1.0-1.5 MHz的正弦波输出。射频功率放大器的输出幅度应调整，以产生一个80 - 100V输出到PCB。在这项工作中的射频功率放大器需要大约220-330毫伏的输入电压。分开的细胞和珠，层流率与DEP的力量，必须符合移动到目标通道（宽W = 100微米的主渠道（宽度w = 100微米，高度H = 27微米）内的细胞和珠高度H = 27微米）。 注意:PCB制造商进行磋商，以确定Maximum工作电压，以避免PCB过热或熔化等问题。检查函数发生器和功率放大器制造商的设备制造商的规格，以确定安全工作设置。 </li><li>启动DEP的排序细胞和珠。细胞的经验，积极的DEP的，而聚苯乙烯珠经验，在一个非均匀的电场，在指定的频率范围内负的DEP。这使DEP的力量，激活bioactuation和分离细胞和其他粒子在作为电极使用负担得起的和可重复使用的多氯联苯的微流体装置。 </li></ol> 6。代表性的成果: 当DEP是有效的驱动细胞或颗粒，在非均匀电场中的一个强大的对齐观察静态浴缸或流速缓慢。高流速的条件下，细胞或粒子的行为取决于轴流电场和流速相对方向。此外，细胞和粒子的行为也时的电场强度和电气领域内的非均匀性程度依赖。细胞或粒子的典型行为包括"珍珠链，"骑自行车，拖延，或转折点。 条件作出妥协或取消的DEP包括盐或其他流体，电场强度较弱，流速过大，盖玻片太厚，或盖玻片和印刷电路板电极之间的导电性的解决方案离子分子的存在（如可诱发裂纹盖玻片水和矿物油的混合）。 在这里，当使用0号盖玻片厚度（80-130微米）和一个电极间距（231微米），电场强度的平方梯度的HT - 29细胞和珠估计为2至-8到6日-8 V 2 /微米3 1 DEP的力量可以测量DEP的操纵静电液（图2A - B的）的粒子，速度估计。由于惯量小的粒子和微流体通道，高度粘稠的环境，流体拖曳力将会相等，但在与DEP力的方向相反。珠在低电导率介质速度平均为34.5微米/小号与DEP 93 Vpp的电压和1.5兆赫。 1水力阻力的力量，可以用下面的公式计算。 F 拖动 =6πηRv 低电导率介质的平均粘度η在20 ° C是1.27 MPA•s和珠半径R为7.5微米。聚苯乙烯微珠的流体拖曳力估计为6.19 PN。为了证明驱动微流体通道内的珠子（图2C - F）的能力，我们开始采用表面张力介导的流量低电导率介质的流量。珠在单输入通道的速度平均为1540微米/秒，而各个通道内的平均流速降低到565微米/秒（图2C）。发起的DEP，珠被保留在中央通道，而不是被释放到侧通道。因此，在这些情况下，DEP的力量足以克服流体拖曳力成两个侧通道。 同样的原理也被用来疏导的渠道和珠流的方向通过简单地改变DEP的电极（图2E - F）的珠子从中央烛到一个单端通道。通过钓鱼DEP时开始的单进风通道，两侧的金属电极上，珠引导通道一侧珠走近trifurcation点。目前，DEP的力量是足够拉入珠层流继续推动他们的通道（图2F）侧面渠道之一。 由于细胞和聚苯乙烯珠在他们有能力在非均匀的电场极化和驱动有明显的差异，我们使用DEP，证明执行能力的细胞和珠片上分离，同时。我们使用相同的微流体通道的结构和配置图2E - F的印刷电路板电极，但引入的单进风通道（图3）的HT - 29细胞和珠暂停低电导率介质。 DEP时介绍，在这些条件下，HT - 29细胞在左侧的两输出通道的保留，而珠保留在右侧的个人输出通道。在这里，细胞显示出一个特点的"珍珠链"的行为，因为它们是在左输出通道收集。偶尔珠和细胞在其中，他们收集在一起，在电池的输出渠道往往成为连接在一起。从这个实验人的数据，我们确定排序率713颗粒（细胞和珠），每分钟或相当于14,260细胞和珠在20分钟内。检索细胞和珠是相关的，有用的分子生物学，影像学，生物化学和单芯片上的实验室应用。 <img src="/files/ftp_upload/2545/2545fig1.jpg" alt="图1" /> 图1。细胞和微流体通道中的颗粒。基于PCB的DEP（一）DEP基于驱动设备的设置与函数发生器开始定义的频率和幅度的电信号，然后功率放大器来增强信号强度PCB上产生的电场。 （二）样品驱动的微流体装置大会组成的PDMS微流体通道，不可逆转地绑定到一个没有。 0盖玻片厚度（80-130微米）通过氧等离子体，非导电介质洗澡的细胞和/或珠子。 （三）印刷电路板电极使用，这里包括两个电极叉产生强烈的非均匀电场地区。 （四）完成设备:PCB与一个trifurcated单一盖玻片上的微流体装置，插图显示整个装置与电极导线。 （CD）的规模，PCB测量8.4厘米（L），2.1​​厘米（宽），5毫米宽的电极。 <img src="/files/ftp_upload/2545/2545fig2.jpg" alt="图2" /> 图2。代表图像之前和期间的DEP驱动渠道的细胞和珠（一）15微米的荧光聚苯乙烯珠在低电导率介质在PDMS微流体通道，无层流，（时间过去了，1.23秒）的解决方案。 （b）在DEP的启动，珠迁移对PCB的电极图案（黑色条纹），而不是电极之间（时间过去了，8.18秒）。 （三）珠层流下相同的渠道分为三个独立的通道（时间过去了，5.3秒）;启动DEP时（D）珠驱动只有在中央通道流（时间已过，4.3秒）。 （五）通过改变渠道的方向在PCB电极，珠可以被不同的侧面渠道（d）所示，使用DEP的，而不是中央通道（F）的指示进入。 （EF）失效时间是8.23秒和5.16秒，分别。所有比例尺= 100微米。 <img src="/files/ftp_upload/2545/2545fig3.jpg" alt="图3" /> 图3。之前和期间的DEP驱动通道 （AB） 的细胞和珠代表的图像开始前的DEP，混合溶液（HT - 29）的人结肠腺癌细胞和珠流量从一个通道3个独立的输出通道。开放的箭头标识层流和渠道的方向为方向和澄清虚线的概述。 （光盘版）诱导的DEP，珠子和细胞选择性驱动成单独的渠道确定。 HT - 29细胞退出中央和左声道，而珠退出通道。特色的珍珠链珠和细胞观察期间的DEP驱动。 （A，C）微分干涉相衬成像微细胞和珠呈现的珠子很容易看到。的发光规模强度图像相同的DIC的图像（A，C）（B，D），提高细胞通道中的可视化。反光金属电极（（A，C）光条纹（B，D）黄色和绿色）为使有效的DEP基于驱动的微电极提供了一个突出的地标。比例尺= 100微米。

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细胞在微流体装置的操作是可取的排序或单个细胞的选择性安置或2层流人口研究。阀门和泵结合使用，操纵细胞在微流体装置。但是，这些方法是具有挑战性的，需要进行详细的制造工艺和技能3离心可以简化为单元布局，但同时成像是一个挑战的要求。此外，离心通道架构必须审慎考虑在设计时所需的操作，并考虑影响4个激光镊子可用于细胞的位置，但方法是昂贵，不适合用于高通量细胞分选5然而，向心力。DEP已被证明有效的制度，作为"电镊"，为有效的细胞安置，表征和操纵。6,7 具体来说，环境保护署署长已用于片上处理生活和死亡的细胞，7-10和细胞质量的测量谐振传感器收集细胞的细胞有选择性地捕捉和排序11。之前我们已经证实增加的DEP细菌或以上的流体拖曳力珠的力量，片上的集中和聚苯乙烯粒单核细胞增生李斯特菌和V7诱捕可以 完成 12大肠杆菌混合种群大肠杆菌和L。指示菌的细菌也可通过DEP的脉冲释放。此外，较大的颗粒，可差异捕获并主要集中在基于大颗粒大小的电极，而更小的粒子都无法捕捉，但与流体流动删除13。DEP时势力不克服流体拖曳力的粒子，珠或细胞无法捕捉的，而是内流体流。如在图2C所示，珠子可以集中到足够的液体流保留在中央通道的珠中部地区。这可能是由于粒子粒子影响的综合效应，内电场流速大于拖动DEP的力量，这些组合，或其他一些不确定的效果。 接触的DEP新的进步使得 1,9接触的DEP提供的承诺，整理，收集和定位细胞微流体社会所需的最低场细胞的捕获和操纵，从而保护细胞类型，在最大程度上，在DEP的操作，最大限度地提高。在微流体装置。我们预计，随着需求的增加，并在微流体装置，进一步发现和创新的操作实施，环境保护署署长将扩大DEP的力量的理解和影响。 可以通过高容量的过程制造多氯联苯在一个合理的价格与快速制造的周转时间，使他们良好的商业化平台。此外，多氯联苯，易于使用和访问片上的细胞操作和排序的学科范围的科学家。 设计的印刷电路板电极的布局时，应考虑到所需的颗粒/细胞的运动轨迹。考虑的主要因素包括颗粒大小和类型（珠和/或细胞），细胞类型，微大小，流速，电极间距（这就决定了电场强度），盖玻片厚度，流体的电导率。这些因素的影响，所需的力和可供操作的粒子或细胞，并最终分离效率。我们这里介绍的协议表明，为有效启动微球和细胞分离的DEP设置。对于潜在的应用，用户应配合所需的细胞和珠子的流动路径，以及电极图案的微流体通道设计的结构优化为每个应用程序的效率。根据此前公布的指引，设计微流体通道布局时，电极间距和盖玻片厚度可用于1 细胞/粒子与周围介质的电导率和介电常数必须是不同的，足以促进积极或消极的DEP，同时保持细胞完好无损。 DEP的球形粒子的极性可确定真正从克劳修斯 - Mosotti DEP的电压频率ω以下因素的一个复杂的价值的一部分。 <img src="/files/ftp_upload/2545/2545eq1.jpg" alt="公式1" /> 公式1 在这个方程ε是介电常数，σ是电导率，ε*是复介电常数。标P和M分别表示粒子和媒体。当一个细胞具有较高的介电常数比媒体，或克劳Mosotti因素的真正变为正值，颗粒变得比周围介质的极化。由于非均匀的电场，粒子的极化成为非均匀，这产生了积极的DEP（DEP）的力量，向着更高的电场强度区域的绘制细胞。如果一个单元有一个比周围介质的介电常数较低，或克劳修斯 - Mosotti因素变为负的实际部分，它将接受负的DEP（DEP）的细胞是被迫走向最小的场区。如果细胞和媒体有大致相同的复介电常数，没有任何力量可以产生操纵细胞。出于这个原因，纯净水是DEP粒子操纵的首选媒体。然而，以避免对纯净水的细胞渗透胁迫，低电导率介质是制定保持导电性不变，但增加的渗透压降低渗透胁迫的细胞。传统的细胞培养介质或生理的缓冲区，如DMEM或PBS，具有很高的导电性，这是不适合的DEP操纵。 我们也有先前表明，细胞可捕获与DEP使用低电导率介质传感器。细胞附着的一个短暂的时期之后，低电导率介质可以被替换为所需的细胞培养介质，支持天的细胞生长。11 从我们的经验，荧光珠与活细胞荧光非常明亮，因此它可以是一个挑战珠强度相匹配的生活和荧光细胞强度。为了提高可视化细胞和珠，我们使用了一个直立显微镜成像都DIC的显微镜。要显示细胞和珠中，我们提出的数据，在强度焕发规模的形象，它保留在很宽的彩色光谱数据为便于观看。因此，设计研究的兴趣时，成像参数和资源必须加以考虑。 总之，我们证明能够有选择性地驱动单独使用DEP的渠道珠和细胞。随着细胞生物学，生物化学，生物工程应用的微流体通道的增加实用工具，DEP是细胞采集，安置和整理的一个理想的选择。 PCB电极制造廉价和方便，易于使用的电极，快速的制作时间是理想的实施DEP的。

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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>Sylgard 184 kit (PDMS)</td>
<td>&nbsp;</td>
<td>Elsworth Adhesives</td>
<td>184 SIL ELAST KIT 0.5KG</td>
<td>Dow Corning Sylgard</td>
</tr>
<tr>
<td>16 awg hook-up wire</td>
<td>&nbsp;</td>
<td>Belden</td>
<td>8521</td>
<td>(Type MW) Mil-W-76C-PVC</td>
</tr>
<tr>
<td>Gold seal Cover Glass </td>
<td>&nbsp;</td>
<td>Ted Pella</td>
<td>260320</td>
<td>No. 0 thick, 24 x 60 mm</td>
</tr>
<tr>
<td>Miltex Biopsy Punch</td>
<td>&nbsp;</td>
<td>VWR Scientific</td>
<td>95039-104</td>
<td>Miltex, assorted sizes</td>
</tr>
<tr>
<td>Custom PCB electrodes</td>
<td>&nbsp;</td>
<td>Bay Area Circuits </td>
<td>Custom parts</td>
<td>&nbsp;</td>
<tr>
<td>Mineral oil</td>
<td>&nbsp;</td>
<td>Fisher Scientific </td>
<td>O121-1 </td>
<td>&nbsp;</td>
<tr>
<td>Deionized water</td>
<td>&nbsp;</td>
<td>House DI water supply</td>
<td>None</td>
<td>Use filtered DI water</td>
</tr>
<tr>
<td>D-Glucose Anhydrous Granular AR</td>
<td>&nbsp;</td>
<td>Mallinckrodt Chemicals </td>
<td>4912-06</td>
<td>&nbsp;</td>
<tr>
<td>Sucrose, Crystal</td>
<td>&nbsp;</td>
<td> Mallinckrodt Chemicals</td>
<td>8360-06 </td>
<td>&nbsp;</td>
<tr>
<td>Fluorescent polymer microspheres </td>
<td>&nbsp;</td>
<td> Bangs Laboratories</td>
<td> FS07F/9277</td>
<td>15&mu;m Dragon green microspheres</td>
</tr>
<tr>
<td>HT-29 cell line</td>
<td>&nbsp;</td>
<td>ATCC</td>
<td>HTB-38D</td>
<td>Human colon adenocarcinoma</td>
</tr>
<tr>
<td>Typsin 0.05% EDTA</td>
<td>&nbsp;</td>
<td>Invitrogen </td>
<td>25300054</td>
<td>&nbsp;</td>
<tr>
<td>Eclipse E600FN upright microscope </td>
<td>&nbsp;</td>
<td> Nikon</td>
<td>Eclipse E600FN </td>
<td>&nbsp;</td>
<tr>
<td>Phantom V310 High-speed imaging camera </td>
<td>&nbsp;</td>
<td>Phantom</td>
<td> V310</td>
<td>&nbsp;</td>
<tr>
<td>BX51 upright research microscope </td>
<td>&nbsp;</td>
<td>Olympus </td>
<td>BX51</td>
<td>&nbsp;</td>
<tr>
<td>SpotFlex high resolution color camera </td>
<td>&nbsp;</td>
<td>Diagnostic Instruments </td>
<td>FX1520</td>
<td>&nbsp;</td>
<tr>
<td>Function generator</td>
<td>&nbsp;</td>
<td>Agilent </td>
<td>3325A</td>
<td>&nbsp;</td>
<tr>
<td>RF Power amplifier</td>
<td>&nbsp;</td>
<td>EIN</td>
<td>2100L</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

separating beads and cells in multi-channel microfluidic devices using dielectrophoresis and laminar flow

拥有先进的微流体装置上规模的细胞研究，处理，分类和计数细胞提供一个充满活力的流体环境的细胞研究。然而，操纵细胞内的流体域仍然是一个挑战，需要复杂的制造协议，形成阀和电极，或要求光镊等专业设备。在这里，我们表明，传统的印刷电路板（PCB），可用于细胞的非接触式操纵用人bioactuation珠和细胞层流场操纵介（DEP），并在多通道微分离细胞和珠设备。首先，我们目前的协议，用于组装的DEP电极和微流体装置，并准备DEP的细胞。然后，我们描述了与聚苯乙烯珠DEP的操作。最后，我们代表结果表明珠和细胞分离在一个多通道的微流体装置。总之，DEP是一个有效的方法，用于操纵微流体装置内的粒子（珠或细胞）。

Watch this Scientific Journal Video about Separating Beads and Cells in Multi-channel Microfluidic Devices Using Dielectrophoresis and Laminar Flow at JoVE.com

Separating Beads and Cells in Multi-channel Microfluidic Devices Using Dielectrophoresis and Laminar Flow

介电泳（DEP）是一种有效的方法操纵细胞。无接触的细胞在微流体装置操作，印刷电路板（PCB），可以提供价格低廉，可重复使用的和有效的电极。 PCB上的盖玻片相结合为基础的PDMS微流体通道，我们展示了多通道微流控装置内珠和细胞的操纵和分离。

使用介电泳分离珠和多通道微流控芯片细胞和层流

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting ...

separating-beads-cells-multi-channel-microfluidic-devices-using

Research

JoVE Journal

Bioengineering

27.4K 次观看.  University of Illinois at Urbana-Champaign. 介电泳（DEP）是一种有效的方法操纵细胞。无接触的细胞在微流体装置操作，印刷电路板（PCB），可以提供价格低廉，可重复使用的和有效的电极。 PCB上的盖玻片相结合为基础的PDMS微流体通道，我们展示了多通道微流控装置内珠和细胞的操纵和分离。

视频: Separating Beads and Cells in Multi-channel Microfluidic Devices Using Dielectrophoresis and Laminar Flow

Parallel-plate Flow Chamber and Continuous Flow Circuit to Evaluate Endothelial Progenitor Cells under Laminar Flow Shear Stress

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well quantifiable fluid shear stresses1.
Our flow chamber design and flow circuit (Fig. 1) contains a transparent viewing region that enables testing of cell adhesion and imaging of cell morphology immediately before flow (Fig. 11A, B), at various time points during flow (Fig. 11C), and after flow (Fig. 11D). These experiments are illustrated with human umbilical cord blood-derived endothelial progenitor cells (EPCs) and porcine EPCs2,3.
This method is also applicable to other adherent cell types, e.g. smooth muscle cells (SMCs) or fibroblasts.
The chamber and all parts of the circuit are easily sterilized with steam autoclaving. In contrast to other chambers, e.g. microfluidic chambers, large numbers of cells (&gt; 1 million depending on cell size) can be recovered after the flow experiment under sterile conditions for cell culture or other experiments, e.g. DNA or RNA extraction, or immunohistochemistry (Fig. 11E), or scanning electron microscopy5. The shear stress can be adjusted by varying the flow rate of the perfusate, the fluid viscosity, or the channel height and width. The latter can reduce fluid volume or cell needs while ensuring that one-dimensional flow is maintained. It is not necessary to measure chamber height between experiments, since the chamber height does not depend on the use of gaskets, which greatly increases the ease of multiple experiments. Furthermore, the circuit design easily enables the collection of perfusate samples for analysis and/or quantification of metabolites secreted by cells under fluid shear stress exposure, e.g. nitric oxide (Fig. 12)6.

We are describing a method to subject adherent cells to laminar flow shear stress in a sterile continuous flow circuit. The cells' adhesion, morphology can be studied through the transparent chamber, samples obtained from the circuit for metabolite analysis and cells harvested after shear exposure for future experiments or culture.

平行板流动腔和评估内皮祖细胞层流剪应力下连续流电路

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well ...

科研

生物工程

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are available for microfluidic devices using soft lithography techniques 1-3. Microfluidic devices have been used for sub-cellular imaging 4,5, in vivo laser microsurgery 2,6 and cellular imaging 4,7. In vivo imaging requires immobilization of organisms. This has been achieved using suction 5,8, tapered channels 6,7,9, deformable membranes 2-4,10, suction with additional cooling 5, anesthetic gas 11, temperature sensitive gels 12, cyanoacrylate glue 13 and anesthetics such as levamisole 14,15. Commonly used anesthetics influence synaptic transmission 16,17 and are known to have detrimental effects on sub-cellular neuronal transport 4. In this study we demonstrate a membrane based poly-dimethyl-siloxane (PDMS) device that allows anesthetic free immobilization of intact genetic model organisms such as Caenorhabditis elegans (C. elegans), Drosophila larvae and zebrafish larvae. These model organisms are suitable for in vivo studies in microfluidic devices because of their small diameters and optically transparent or translucent bodies. Body diameters range from ~10 &mu;m to ~800 &mu;m for early larval stages of C. elegans and zebrafish larvae and require microfluidic devices of different sizes to achieve complete immobilization for high resolution time-lapse imaging. These organisms are immobilized using pressure applied by compressed nitrogen gas through a liquid column and imaged using an inverted microscope. Animals released from the trap return to normal locomotion within 10 min.
We demonstrate four applications of time-lapse imaging in C. elegans namely, imaging mitochondrial transport in neurons, pre-synaptic vesicle transport in a transport-defective mutant, glutamate receptor transport and Q neuroblast cell division. Data obtained from such movies show that microfluidic immobilization is a useful and accurate means of acquiring in vivo data of cellular and sub-cellular events when compared to anesthetized animals (Figure 1J and 3C-F 4).
Device dimensions were altered to allow time-lapse imaging of different stages of C. elegans, first instar Drosophila larvae and zebrafish larvae. Transport of vesicles marked with synaptotagmin tagged with GFP (syt.eGFP) in sensory neurons shows directed motion of synaptic vesicle markers expressed in cholinergic sensory neurons in intact first instar Drosophila larvae. A similar device has been used to carry out time-lapse imaging of heartbeat in ~30 hr post fertilization (hpf) zebrafish larvae. These data show that the simple devices we have developed can be applied to a variety of model systems to study several cell biological and developmental phenomena in vivo.

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

A simple microfluidic device has been developed to perform anesthetic free in vivo imaging of C. elegans, intact Drosophila larvae and zebrafish larvae. The device utilizes a deformable PDMS membrane to immobilize these model organisms in order to perform time lapse imaging of numerous processes such as heart beat, cell division and sub-cellular neuronal transport. We demonstrate the use of this device and show examples of different types of data collected from different model systems.

简单的微流控芯片的<em在体内</em&gt;成像<em&gt; C.线虫</em&gt;<em&gt;果蝇</em&gt;和斑马鱼

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are ...

Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D culture of cells in collagen type I gels helps to stabilize cell morphology and function, which is necessary for creating microfluidic tissue models in microdevices. Translating traditional 3D culture techniques for tissue culture plates to microfluidic devices is often difficult because of the limited channel dimensions. In this method, we describe a technique for modifying native type I collagen to generate polycationic and polyanionic collagen solutions that can be used with layer-by-layer deposition to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These thin collagen layers stabilize cell morphology and function, as shown using primary hepatocytes as an example cell, allowing for the long term culture of microtissues in microfluidic devices.

The creation of functional microtissues within microfluidic devices requires the stabilization of cell phenotypes by adapting traditional cell culture techniques to the limited spatial dimensions in microdevices. Modification of collagen allows the layer-by-layer deposition of ultrathin collagen assemblies that can stabilize primary cells, such as hepatocytes, as microfluidic tissue models.

层与层胶原沉积在微流体装置的Microtissue稳定

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D ...

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of physiological dcEF in guiding cell movement during embryo development, cell differentiation, and wound healing has been demonstrated in many studies. By applying microfluidic chips to an electrotaxis assay, the investigation process is shortened and experimental errors are minimized. In recent years, microfluidic devices made of polymeric substances (e.g., polymethylmethacrylate, PMMA, or acrylic) or polydimethylsiloxane (PDMS) have been widely used in studying the responses of cells to electrical stimulation. However, unlike the numerous steps required to fabricate a PDMS device, the simple and rapid construction of the acrylic micro&#64258;uidic chip makes it suitable for both device prototyping and production. Yet none of the reported devices facilitate the efficient study of the simultaneous chemical and dcEF effects on cells. In this report, we describe our design and fabrication of an acrylic-based multichannel dual-electric-field (MDF) chip to investigate the concurrent effect of chemical and electrical stimulation on lung cancer cells. The MDF chip provides eight combinations of electrical/chemical stimulations in a single test. The chip not only greatly shortens the required experimental time but also increases accuracy in electrotaxis studies.

Many microfluidic devices have been developed for use in the study of electrotaxis. Yet, none of these chips allows the efficient study of the simultaneous chemical and electric-field (EF) effects on cells. We developed a polymethylmethacrylate-based device that offers better-controlled coexisting EF and chemical stimulation for use in electrotaxis research.

使用多通道双电场微流控芯片肺癌细胞Electrotaxis研究

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of ...

Using Adhesive Patterning to Construct 3D Paper Microfluidic Devices

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol adhesive through a metal stencil, the overall amount of adhesive used in assembling paper microfluidic devices can be significantly reduced. We show on a simple 4-layer planar paper microfluidic device that the optimal adhesive application technique and device construction style depends heavily on desired performance characteristics. By moderately increasing the overall area of a device, it is possible to dramatically decrease the wicking time and increase device success rates while also reducing the amount of adhesive required to keep the device together. Such adhesive application also causes the adhesive to form semi-permanent bonds instead of permanent bonds between paper layers, enabling single-use devices to be non-destructively disassembled after use. Nonplanar 3D origami devices also benefit from the semi-permanent bonds during folding, as it reduces the likelihood that unrelated faces may accidently stick together. Like planar devices, nonplanar structures see reduced wicking times with patterned adhesive application vs uniformly applied adhesive.

We demonstrate the use of patterned aerosol adhesives to construct 3D paper microfluidic devices. This method of adhesive application forms semi-permanent bonds between layers, enabling single-use devices to be non-destructively disassembled after use and to ease folding complex nonplanar structures.

使用粘性图案构建3D纸微流体装置

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol ...

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of (indirect) in vitro experiments, but none of these is as comprehensive as microfluidic flow chambers. In this protocol, the reconstitution of thrombocytopenic fresh blood with stored blood bank platelets is used to simulate platelet transfusion. Next, the reconstituted sample is perfused in microfluidic flow chambers which mimic hemostasis on exposed subendothelial matrix proteins. Effects of blood donation, transport, component separation, storage and pathogen inactivation can be measured in paired experimental designs. This allows reliable comparison of the impact every manipulation in blood component preparation has on hemostasis. Our results demonstrate the impact of temperature cycling, shear rates, platelet concentration and storage duration on platelet function. In conclusion, this protocol analyzes the function of blood bank platelets and this ultimately aids in optimization of the processing chain including phlebotomy, transport, component preparation, storage and transfusion.

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Platelet transfusion and hemostasis was modeled using blood reconstitution and microfluidic flow chambers to investigate the function of blood banking platelets. The data demonstrate the consequences of platelet storage lesion on hemostasis, in vitro.

微流体流钱伯斯再造使用血止血建模和血小板输注<em&gt;体外</em

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of ...

Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to entry for non-specialists who would benefit greatly from the ability to develop their own microfluidic devices to address research questions. Particularly lacking has been the open dissemination of protocols related to photolithography, a key step in the development of a replica mold for the manufacture of polydimethylsiloxane (PDMS) devices. While the fabrication of single height silicon masters has been explored extensively in literature, fabrication steps for more complicated photolithography features necessary for many interesting device functionalities (such as feature rounding to make valve structures, multi-height single-mold patterning, or high aspect ratio definition) are often not explicitly outlined. 
Here, we provide a complete protocol for making multilayer microfluidic devices with valves and complex multi-height geometries, tunable for any application. These fabrication procedures are presented in the context of a microfluidic hydrogel bead synthesizer and demonstrate the production of droplets containing polyethylene glycol (PEG diacrylate) and a photoinitiator that can be polymerized into solid beads. This protocol and accompanying discussion provide a foundation of design principles and fabrication methods that enables development of a wide variety of microfluidic devices. The details included here should allow non-specialists to design and fabricate novel devices, thereby bringing a host of recently developed technologies to their most exciting applications in biological laboratories.

Multilayer microfluidic devices often involve the fabrication of master molds with complex geometries for functionality. This article presents a complete protocol for multi-step photolithography with valves and variable height features tunable to any application. As a demonstration, we fabricate a microfluidic droplet generator capable of producing hydrogel beads.

多级可变高度的光刻多层带瓣微流体装置

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to ...

Mammalian Cell Encapsulation in Alginate Beads Using a Simple Stirred Vessel

Cell encapsulation in alginate beads has been used for immobilized cell culture in vitro as well as for immunoisolation in vivo. Pancreatic islet encapsulation has been studied extensively as a means to increase islet survival in allogeneic or xenogeneic transplants. Alginate encapsulation is commonly achieved by nozzle extrusion and external gelation. Using this method, cell-containing alginate droplets formed at the tip of nozzles fall into a solution containing divalent cations that cause ionotropic alginate gelation as they diffuse into the droplets. The requirement for droplet formation at the nozzle tip limits the volumetric throughput and alginate concentration that can be achieved. This video describes a scalable emulsification method to encapsulate mammalian cells in 0.5% to 10% alginate with 70% to 90% cell survival. By this alternative method, alginate droplets containing cells and calcium carbonate are emulsified in mineral oil, followed by a decrease in pH leading to internal calcium release and ionotropic alginate gelation. The current method allows the production of alginate beads within 20 min of emulsification. The equipment required for the encapsulation step consists in simple stirred vessels available to most laboratories.

This video and manuscript describe an emulsion-based method to encapsulate mammalian cells in 0.5% to 10% alginate beads which can be produced in large batches using a simple stirred vessel. The encapsulated cells can be cultured in vitro or transplanted for cellular therapy applications.

使用简单搅拌容器在藻酸盐珠中的哺乳动物细胞包封

Cell encapsulation in alginate beads has been used for immobilized cell culture in vitro as well as for immunoisolation in vivo. Pancreatic islet ...

Fabrication of Three-dimensional Paper-based Microfluidic Devices for Immunoassays

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and directed within a layer of paper. Moreover, stacking multiple layers of patterned paper creates sophisticated three-dimensional microfluidic networks that can support the development of analytical and bioanalytical assays. Paper-based microfluidic devices are inexpensive, portable, easy to use, and require no external equipment to operate. As a result, they hold great promise as a platform for point-of-care diagnostics. In order to properly evaluate the utility and analytical performance of paper-based devices, suitable methods must be developed to ensure their manufacture is reproducible and at a scale that is appropriate for laboratory settings. In this manuscript, a method to fabricate a general device architecture that can be used for paper-based immunoassays is described. We use a form of additive manufacturing (multi-layer lamination) to prepare devices that comprise multiple layers of patterned paper and patterned adhesive. In addition to demonstrating the proper use of these three-dimensional paper-based microfluidic devices with an immunoassay for human chorionic gonadotropin (hCG), errors in the manufacturing process that may result in device failures are discussed. We expect this approach to manufacturing paper-based devices will find broad utility in the development of analytical applications designed specifically for limited-resource settings.

We detail a method to fabricate three-dimensional paper-based microfluidic devices for use in the development of immunoassays. Our approach to device assembly is a type of multilayer, additive manufacturing. We demonstrate a sandwich immunoassay to provide representative results for these types of paper-based devices.

三维纸基微流体装置的制造用于免疫分析

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and ...

Polydimethylsiloxane-polycarbonate Microfluidic Devices for Cell Migration Studies Under Perpendicular Chemical and Oxygen Gradients

This paper reports a microfluidic device made of polydimethylsiloxane (PDMS) with an embedded polycarbonate (PC) thin film to study cell migration under combinations of chemical and oxygen gradients. Both chemical and oxygen gradients can greatly affect cell migration in vivo; however, due to technical limitations, very little research has been performed to investigate their effects in vitro. The device developed in this research takes advantage of a series of serpentine-shaped channels to generate the desired chemical gradients and exploits a spatially confined chemical reaction method for oxygen gradient generation. The directions of the chemical and oxygen gradients are perpendicular to each other to enable straightforward migration result interpretation. In order to efficiently generate the oxygen gradients with minimal chemical consumption, the embedded PC thin film is utilized as a gas diffusion barrier. The developed microfluidic device can be actuated by syringe pumps and placed into a conventional cell incubator during cell migration experiments to allow for setup simplification and optimized cell culture conditions. In cell experiments, we used the device to study migrations of adenocarcinomic human alveolar basal epithelial cells, A549, under combinations of chemokine (stromal cell-derived factor, SDF-1&#945;) and oxygen gradients. The experimental results show that the device can stably generate perpendicular chemokine and oxygen gradients and is compatible with cells. The migration study results indicate that oxygen gradients may play an essential role in guiding cell migration, and cellular behavior under combinations of gradients cannot be predicted from those under single gradients. The device provides a powerful and practical tool for researchers to study interactions between chemical and oxygen gradients in cell culture, which can promote better cell migration studies in more in vivo-like microenvironments.

The control of chemical and oxygen gradients is essential for cell cultures. This paper reports a polydimethylsiloxane-polycarbonate (PDMS-PC) microfluidic device capable of reliably generating combinations of chemical and oxygen gradients for cell migration studies, which can be practically utilized in biological labs without sophisticated instrumentation.

细胞迁移研究在垂直化学和氧梯度聚二甲基硅氧烷的聚碳酸酯微流体装置

This paper reports a microfluidic device made of polydimethylsiloxane (PDMS) with an embedded polycarbonate (PC) thin film to study cell migration under ...

使用介电泳分离珠和多通道微流控芯片细胞和层流

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