这项工作得到了玛丽·居里国际重返社会补助金。

1。设备制造<ol><li>购买DTPA-D SU-8个控制模具和模具SPR220-7流从斯坦福大学的微流体铸造厂（ <a href="http://www.stanford.edu/group/foundry" target="_blank">www.stanford.edu /组/晶圆代工</a> ）。 </li><li>三甲基氯硅烷（TMCS）蒸汽和10分钟，以促进弹性体释放后的烘烤步骤9公开的硅氧烷模具。 </li><li>基于有机硅弹性体和固化剂（拌匀）在两个不同的比率5:1和20:1的控制和流量模具，分别准备的混合物。的不同比率的多个层进行适当的接合是必要的。 </li><li>倒入5:1 PDMS控制层（约5毫米高）。德加的控制层和烤30分钟，在80°C。旋涂（劳雷尔美国）的20:1的PDMS混合物在2600转，60秒的流动层上，然后在80℃下烘烤30分钟。旋涂机速度优化的阀门致动器的压力为15 psi。更快的纺丝将导致在一个更薄的层具有较低的活化压力和反之亦然。我们所使用的器件有一个限制在控制和25磅10 psi的流中，从基板的剥离，可能会发生上述。 </li><li>从模具中分离控制层。一定要慢慢做，并注意不要削皮SU8模式。然后切围绕其周边的​​设备和打孔访问控制渠道。 </li><li>对齐的流量和手动控制层的立体镜。开始“通过对准的左上角;按钮阀（控制层）必须是在该反应室（流动层）的中间。接下来，第一行对齐，轻轻地释放控制层的行由行。确保所有的按钮阀是在反应室的中间的地址和输入阀跨越在正确的位置上的流路。本地该过程可重复，直到所有的行都被对齐。最后，释放任何紧张在的PDMS BŸ提升小心翼翼地从边和角。不要提太多或可能发生错位。 </li><li>的温度下烤2小时，在80℃下</li><li>剪切的周边周围的设备和剥离两个层器件（芯片），从流模具。打孔访问的流路（ 图1）。 </li></ol> 2。 DNA排列和装置对准<ol><li>生产合成基因的PCR装配。合成基因组成，T7启动子，核糖体结合位点（RBS），ORF与两个表位标签（每端各一个），和T7终止子4。该基因可以具有不同的长度从100 bp的最多，至少，5,000 bp的。 </li><li>准备的合成基因组阵：制备聚的乙烯glycole（1.25％），D-海藻糖二水合物（125毫克/毫升）的混合物，和分配2微升每反应到384孔板。此解决方案将减少10对齐过程中的DNA不可逆地结合到玻璃上，以及用于可视化。 </lI&gt; <li>合成的基因转移到384孔板。一般DNA的浓度范围可以从10纳克/微升100纳克/微升的最终浓度。添加蒸馏水2 O至终体积为20微升（依赖于微阵列和针型）。 </li><li>现货环​​氧树脂涂层的玻璃基板，使用微阵列上的一系列的合成基因。我们使用微网610（生物机器人技术），SMT-S75硅胶引脚的（并行合成，USA）。联系印刷的DNA，与这些特定的标签，与直径约100μm的玻璃表面上的斑点中的结果。约100个​​点，每个引脚的负载是足够的。确保对应于所使用的特定的移动设备的列和行间距。我们所使用的装置，包含16列和40行，分别由320微米，680微米的间距。 </li><li>下的立体镜，手动对齐的基因芯片的微流体装置。 DNA斑点应该是在中间的DNA室。通过定位点b的第一行开始的对准的DNA室elow第一行。然后，其余的行线完成整个设备的微调。一旦对准阵列应小心，以减轻任何应力，通过在PDMS本地提起。如果有紧张留在PDMS债券的芯片。 </li><li>最后，债券的载玻片上培养过夜的热板上的移动设备以在80℃下</li></ol> 3。启动与激活设备<ol><li>从计算机上使用LabVIEW驱动的阀门控制层。 LabVIEW的脚本​​控制了一系列的电磁微阀通过一个电子控制盒（购斯坦福大学微流体铸造）。的电磁阀歧管被连接到压缩空气，控制成在设备上的控制阀的空气流量和压力。 </li><li>将设备连接到的电磁阀歧管，使用柔性塑料管，内部直径为0.02“（聚乙烯）和不锈钢引脚（新英国小管公司）。 </li><li>填充管与DDW和插入销插入的控制层的访问孔。确保每个管被连接到其对应的控制信道。 </li><li>在计算机上运行的LabVIEW应用程序启动控制渠道。我们通过从LabVIEW脚本启动阀，适用于空气压力，这反过来将水推到PDMS控制通道。设置的空气压力至5磅。建议填写的“三明治”的地址阀，以识别有缺陷的设备的控制通道之间的跨会谈。交叉会谈的情况下，控制线的“三明治”的致动将导致到颈部或按钮控制线的致动。这是类似于一个短的电气电路中。与串扰的装置是有缺陷的，不能使用。 </li><li>后，所有的控制通道和阀充满DDW，阀门底漆和意图DY封锁下他们的流动渠道。增加空气压力至15 psi。通过一组的通/断开关连接到个别的电磁阀，控制阀启动从一个LabVIEW程序。每个开关控制一个特定的控制线，通过其相应的电磁阀。开启的开关按钮（在脚本中），将应用于相应的管中的空气压力。的空气压力将推动DDW的控制线中，并会导致在膨胀的微流体阀，阻断的通道下面。关闭控制阀，将释放的空气压力，并随后释放的各流路的阻塞。 </li><li> ，以确保所有的阀门都打开，连接管的一个流输入和流量在4-5磅的装置的空气进入。这将释放任何粘性从载玻片上的阀门。 </li><li>最后，该设备是底漆并准备就绪。关闭所有的输入和颈部的阀门，开始表面化学。 </li></ol><p=“jove_title”的4级。表面化学<ol><li>为了便利的蛋白阵列的表面上的自组装体，并防止非特异性吸附的微流体装置内，表面化学改性： </li><li>要流过组件，通过该装置，一个新的管连接到一个设备中的流动通道与所需的解决方案。手动歧管的自由侧管连接，并打开空气压力流（5 psi）的。 </li><li>加载加入40μl生物素标记的BSA（1微克/微升）在一个新的管和流量约一半，通过该装置的20分钟，波塞将绑定到的环氧树脂表面。 </li><li>使用的Hepes（50毫摩尔）洗涤未反应的基板彼此之间的不同的表面化学步骤。 </li><li>流25的微升Strepavidin（0.5微克/微升），生物素化的牛血清白蛋白的顶部的20分钟。 </li><li>洗净用Hepes 5分钟。 </li><li>关闭“按钮”的阀和流的其余部分的生物素化的牛血清白蛋白（作为所记述床段），钝化的表面周围的按钮。 </li><li>洗净用Hepes 5分钟。 </li><li>释放的“按钮”阀和流量30μl的五-生物素（0.16微克/微升），20分钟。的抗体将结合到暴露strepavidin;具体创建一个抗His-标签阵列的“按钮”下的面积。 </li></ol> 5。蛋白表达<ol><li>在设备上使用兔网织红细胞的快速转录和翻译反应，表达的蛋白质。创建一个数组，在设备上的斑点合成的基因蛋白表达的蛋白质使用。例如，一个这样的用途的蛋白质结合的屏幕。 </li><li>打开&#39;脖子&#39;阀门和流量兔网织红细胞快速加上转录和翻译反应，通过设备到DNA室。下一步，关闭阀“三明治”，并从环境中每个基因的分离。孵育的移动设备在30℃的热板上进行2.5小时细胞表达PR的oteins从DNA室扩散到反应室自发性（脖子阀是打开的）和绑定下的“按钮”阀，通​​过其C-末端标记的蛋白质固定的抗-His抗体。 </li><li>贴上标签的蛋白质与C-myc基因Cy3标记抗体。的抗体将绑定到其对应表位位于蛋白质的N-末端。 </li><li>确定蛋白质的表达水平与微阵列扫描仪（LS重装上阵，Tecan公司），使用532 nm激光和575 nm发射过滤器。 </li></ol> 6。代表性的成果 1。设备制造根据我们的实验需要的图形设计由AutoCAD创建的设备。该设计的透明薄膜上印刷由一个高分辨率的图像设定部。作为这种透明的光掩模接触光刻。一个表面微机械加工技术，在硅片上的图案刻在T确定用于创建3-D的模板他口罩使用。 硅胶模具铸造制作的有机硅弹性体，聚二甲基硅氧烷（PDMS，SYLGARD 184，道康宁，美国）（ 图1）的微流体装置。每个装置由两个对齐的PDMS层时，流程和控制层。该模具首先接触的三甲基氯硅烷（TMCS，Aldrich公司）蒸汽和10分钟，以促进弹性体释放后的烘烤步骤。基于硅氧烷的弹性体和固化剂的混合物，在两个不同的比为5:1和20:1的控制和流量模具，分别制备。控制层进行脱气并在80℃下烘烤30分钟最初是将流层旋涂（劳雷尔美国）在2600转，60秒，并在80℃下进行30分钟烘烤。控制层分离的模具和冲压控制信道接入孔。接着，对齐的流动层和控制层下立体镜手动和烘烤2小时，在80℃。 2层的移动设备被剥离fROM的流动冲压模具流道检修孔。 2。设备描述的装置容量的范围可以从500至10,000单位的细胞（ 图2）。该装置由两个层，流层（灰色）和控制层（彩色）。控制层含有多种具有不同功能的阀。的流动层中，每一个单元电池，是由一个DNA和一个反应室，和由三种类型的微机械阀控制;&#39;脖子&#39;，“按钮”，和“三明治”（ 图2A）。有斑点的“合成基因&#39;存放的DNA内室的被阻止从反应室由&#39;脖子&#39;阀（绿色）。套印和机械清洗的表面在反应室中的结合蛋白的是，所执行的“按钮”阀（蓝色）。的“三明治”的阀使每个反应发生在其自己的单元电池（红色）。地址阀门可以分割设备8个独立的部分，differeNT状况分析。另外，控制层含有的输入阀，使选择到的流层中的流体的流动。在的PDMS控制渠道与DDW。当被致动的阀的压力的增加（15 psi）的结果在扩大的PDMS。在其中的膜是足够薄的地方（即控制和流线的交叉），这是足以完全阻断的流线。平均单元电池的高度为10微米，平均单位晶胞体积是小于1，NL。 3。蛋白的表达及检测蛋白的表达在设备上，利用兔网织红细胞迅速偶联转录和翻译反应（Promega）中。表达的蛋白质在准备有约束力的屏幕中使用的蛋白质（ 图3）的阵列。表达式组合（12.5微升）被加载到该设备，然后涌入的DNA室开放的&#39;脖子&#39;阀门。接下来，“三明治”阀门关闭离开从它的相邻小区和该装置分离的各单元电池在30℃下温育2.5小时的热板上表达的蛋白通过基因室扩散到反应室，在那里它们的C-末端His标记能够结合的抗-His抗体（Qiagen公司制）下的“按钮”阀的蛋白质固定到表面本地化。蛋白与C-myc的Cy3标记（Sigma公司），这势必其对应表位位于蛋白质的N-末端，并贴上标记。蛋白表达水平与微阵列扫描仪（LS重装上阵，Tecan公司），使用532 nm激光和575 nm过滤器。由此产生的蛋白质阵列由不同的蛋白表达水平。一般，约20％的基因库不表达可检测水平。与蛋白质大小无明显相关性，观察3。背景水平进行了测定室，没有发现与DNA。因此，从相应的蛋白质室的信号是由于是n瓦兹或的标记的抗体的非特异性吸附。 <img src="/files/ftp_upload/3849/3849fig1.jpg" alt="图1" /> 图1。芯片制造。（A）模具处理与TMCS蒸汽10分钟。 （B）PDMS浇铸控制和流量硅模具上。 （C）的两个控制和流量模具，在80℃下进行30分钟烘烤。 （D）的控制层的模具去皮，切成一定尺寸和控制进气口被冲。 （E）在控制层对准立视镜下的流层，然后烘烤在80℃下2小时。以平行的PDMS装置制造，一系列的合成基因的microarrayed到环氧树脂涂层玻璃基板（CEL）。 （F）的设备，然后对准捕获的“合成基因”的DNA微阵列内的DNA室。 <img src="/files/ftp_upload/3849/3849fig2.jpg" alt="图2" /> 图2。的照片 （A） 的PING设备。设备的注意事项，ST的两个对齐的PDMS层（控制和流量）。流量层含有的并行DNA和反应室（灰色）由微机械阀（“按钮”，“夹心”，“颈缩”），控制层中的控制。 （B）的层的印刷的微阵列，该方案各DNA室与一个单一的点对齐。这消除了任何潜在的污染。 <img src="/files/ftp_upload/3849/3849fig3.jpg" alt="图3" /> 图3。荧光的蛋白阵列创建与微流控芯片图像印刷点的基因表达的蛋白质内的设备（在DNA腔）被拉低到表面（下面的“按钮”的阀，在反应室中），通过各自的C-末端他的标签。使用C-myc蛋白N-端标签和特异性荧光抗体检测蛋白的表达。不同的信号强度显示不同的蛋白表达水平。联合国斑室作为对照的背景乐“竞争”。

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没有利益冲突的声明。

在本文中，我们提出了一个方法代高通量的蛋白质阵列使用微流体平台。上述排列生成是基于微阵列打印的DNA模板， 在体外蛋白质表达的微流体装置内的DNA。 我们的新型微流体平台有几个重要的优点，比目前使用的方法，这使得它有前途和蛋白质组学工具。的一个优点是与膜结合蛋白， 在体外蛋白质合成使用哺乳动物的网织红细胞裂解物在微粒体膜11?...

<table border="1"><tbody><tr><td> 试剂/设备 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> PDMS-SYLGARD 184 </td><td>美国道康宁</td><td>的ESSEX-DC </td></tr><tr><td>三甲基氯硅烷（TMCS </td><td> Sigma-Aldrich公司</td><td> C72854 </td></tr><tr><td>环氧涂层的玻璃基板</td><td> CEL Associates公司（美国） </td><td> VEPO-25C </td></tr><tr><td>保利：乙烯glycole（PEG） </td><td> Sigma-Aldrich公司</td><td> 81260 </td></tr><tr><td> D-海藻糖二水合物</td><td> Sigma-Aldrich公司</td><td> T9531 </td></tr><tr><td>生物素-BSA </td><td>刺穿</td><td> PIR-29130 </td></tr><tr><td> Neutravidin </td><td>刺穿</td><td> 31050 </td></tr><tr><td>五-生物素</td><td> Qiagen公司</TD&gt; <td> 34440 </td></tr><tr><td> HEPES </td><td> <a href="http://www.bioind.com/htmls/article.aspx?c0=741&amp;bsp=13384" target="_blank">生物产业</a> </td><td> 03-025-1B </td></tr><tr><td> TNT-T7 </td><td> Promega公司</td><td> L5540 </td></tr><tr><td> C-myc基因Cy3标记抗体</td><td> Sigma-Aldrich公司</td><td></td></tr><tr><td>控制箱</td><td>斯坦福大学的微流体铸造厂</td><td></td></tr><tr><td>模</td><td>斯坦福大学的微流体铸造厂</td><td></td></tr><tr><td>脚</td><td>新英国小管公司</td><td></td></tr><tr><td>聚乙烯微孔管</td><td>聚乙烯</td><td> S-54-HL </td></tr><tr><td>微阵列</td><td>生物机器人</td><td>微网610 </td></tr><tr><td>有机硅针</td><td>并行Syn的论文</td><td> SMT-S75 </td></tr></tbody></table>

high-throughput protein expression generator using a microfluidic platform

迅速增长的领域，如系统生物学，需要制定和实施新技术，使大型系统的高吞吐量和高精确度的测量。 ，微流体承诺履行这些要求的，如片上进行高通量筛选实验，当中包括生物化学，生物物理学，和基于细胞的检测1。微流体设备的初期以来，这一领域的显着发展，领导发展的微流体大规模集成2,3。该技术允许用邮资大小的足迹（ 图1）在单个设备上集成的成千上万的微机械阀。我们已经开发出一种高通量微流体平台， 在体外表达的蛋白质阵列（ 图2）PING（蛋白质相互作用网络发生器）产生。这些阵列可以作为一个模板，为许多实验如蛋白质-蛋白质4 5，蛋白质-RNA或蛋白质-DNA 6相互作用。 该装置由成千上万的反应室，单独使用微阵列编程。对齐的微流体设备方案这些印刷的微阵列，每个腔室与一个单一的点，此外，生成使用标准的微阵列斑点技术的微阵列消除潜在的污染或交叉反应性也非常模块化的，允许的蛋白质7，DNA 8，小分子的排列，甚至胶体悬浮液。微流体生物科学的潜在影响是显著的。已经许多微流控芯片为基础的检测提供了新的见解，对生物系统的结构和功能，和微流控芯片领域将继续影响生物。

Watch this Scientific Journal Video about High-throughput Protein Expression Generator Using a Microfluidic Platform at JoVE.com

High-throughput Protein Expression Generator Using a Microfluidic Platform

我们提出了一个表达的蛋白质阵列微流控方法。该装置由反应室的数以千计的由微机械阀控制。的微流体装置相配合以印刷的微阵列的基因库。然后，这些基因的转录和翻译的片上，所得的蛋白质阵列中准备实验使用。

利用微流控平台的高通量蛋白质表达发生器

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

high-throughput-protein-expression-generator-using-microfluidic

Research

JoVE Journal

Bioengineering

11.6K 次观看.  Bar-Ilan University. 我们提出了一个表达的蛋白质阵列微流控方法。该装置由反应室的数以千计的由微机械阀控制。的微流体装置相配合以印刷的微阵列的基因库。然后，这些基因的转录和翻译的片上，所得的蛋白质阵列中准备实验使用。

视频: High-throughput Protein Expression Generator Using a Microfluidic Platform

CometChip: A High-throughput 96-Well Platform for Measuring DNA Damage in Microarrayed Human Cells

DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular metabolites. Ironically, at high doses DNA damaging agents are also used to treat cancer. The ability to quantify DNA damage responses is thus critical in the public health, pharmaceutical and clinical domains. Here, we describe a novel platform that exploits microfabrication techniques to pattern cells in a fixed microarray. The &#8216;CometChip&#8217; is based upon the well-established single cell gel electrophoresis assay (a.k.a. the comet assay), which estimates the level of DNA damage by evaluating the extent of DNA migration through a matrix in an electrical field. The type of damage measured by this assay includes abasic sites, crosslinks, and strand breaks. Instead of being randomly dispersed in agarose in the traditional assay, cells are captured into an agarose microwell array by gravity. The platform also expands from the size of a standard microscope slide to a 96-well format, enabling parallel processing. Here we describe the protocols of using the chip to evaluate DNA damage caused by known genotoxic agents and the cellular repair response followed after exposure. Through the integration of biological and engineering principles, this method potentiates robust and sensitive measurements of DNA damage in human cells and provides the necessary throughput for genotoxicity testing, drug development, epidemiological studies and clinical assays.

We describe here a platform that allows comet assay detection of DNA damage with unprecedented throughput. The device patterns mammalian cells into a microarray and enables parallel processing of 96 samples. The approach facilitates analysis of base level DNA damage, exposure-induced DNA damage and DNA repair kinetics.

CometChip：一种高通量的96孔平台的微阵列人类细胞测量DNA损伤

DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular ...

科研

生物工程

High Throughput Microfluidic Rapid and Low Cost Prototyping Packaging Methods

In this work, 3 different packaging and assembly techniques are presented. They can be classified into two categories: one-time use and reusable packaging techniques.
The one-time use packaging technique employs UV-based and temperature curing epoxies to connect microtubes to access holes, wire-bonding for integrated circuit connections, and silver epoxy for electrical connections. This method is based on a robust assembly technique that can support relatively high pressure close to 1 psi and does not need any support to strengthen the microfluidic architecture.
Reusable packaging techniques consist of PDMS-based microtube interconnectors and anisotropic adhesive films for electrical connections. These devices are more sensitive and fragile. Consequently, Plexiglas support is added to the microfluidic structure to improve the electrical contact when anisotropic adhesive films are used, and also to strengthen the microfluidic architecture. In addition, a micromanipulator is needed to maintain tubes while using a thin PDMS layer to connect them to the access holes. Different PDMS layer thicknesses, ranging from 0.45-3 mm, are tested to compare the best adherence versus injection rates. Applied injection rates are varied from 50-300 &#956;l/hr for 0.45-3 mm PDMS layers, respectively. These techniques are mainly applicable for low-pressure applications. However, they can be extended for high-pressure ones through plasma-oxygen process to permanently seal the PDMS to glass substrates. The main advantage of this technique, besides the fact that it is reusable, consists of keeping the device observable when the microchannel length is very short (in the range of 3 mm or lower).

In this article we describe different techniques for microfluidic rapid prototyping platforms. The proposed techniques are based on ultraviolet (UV) sensitive and temperature curing epoxies, polydimethylsiloxane (PDMS) based tubing, wire-bonding, and anisotropic adhesive films. The assembling procedures presented are developed for both one-time use devices as well as reusable microfluidic systems.

In this work, 3 different packaging and assembly techniques are presented. They can be classified into two categories: one-time use and reusable packaging ...

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

电池挤压作为一种强大的，微流控细胞内传送平台

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected within in vivo oral biofilms. Furthermore, a system that uses natural human saliva as the nutrient source, instead of artificial media, is particularly desirable in order to support the expression of cellular and biofilm-specific properties that mimic the in vivo communities. We describe a method for the development of multi-species oral biofilms that are comparable, with respect to species composition, to supragingival dental plaque, under conditions similar to the human oral cavity. Specifically, this methods article will describe how a commercially available microfluidic system can be adapted to facilitate the development of multi-species oral biofilms derived from and grown within pooled saliva. Furthermore, a description of how the system can be used in conjunction with a confocal laser scanning microscope to generate 3-D biofilm reconstructions for architectural and viability analyses will be presented. Given the broad diversity of microorganisms that grow within biofilms in the microfluidic system (including Streptococcus, Neisseria, Veillonella, Gemella, and Porphyromonas), a protocol will also be presented describing how to harvest the biofilm cells for further subculture&#160;or DNA extraction and analysis. The limits of both the microfluidic biofilm system and the current state-of-the-art data analyses will be addressed. Ultimately, it is envisioned that this article will provide a baseline technique that will improve the study of oral biofilms and aid in the development of additional technologies that can be integrated with the microfluidic platform.

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

The goal of this methods paper is to describe the use of a microfluidic system for the development of multi-species biofilms that contain species typically identified in human supragingival dental plaque.&#160;Methods to describe biofilm architecture, biofilm viability, and an approach to harvest biofilm for culture-dependent or culture-independent analyses are highlighted.

使用高通量的<em&gt;在体外</em&gt;微流控系统开发口服多物种生物膜

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

高通量单细胞的分离和文化的微流体平台

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

A High-throughput Cell Microarray Platform for Correlative Analysis of Cell Differentiation and Traction Forces

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly controlled, high-throughput engineered system for measuring the impact of arrayed biochemical signals on cell differentiation. Recent efforts using cell microarrays have demonstrated their utility for combinatorial studies in which many microenvironmental factors are presented in parallel. However, these efforts have focused primarily on investigating the effects of biochemical cues on cell responses. Here, we present a cell microarray platform with tunable material properties for evaluating both cell differentiation by immunofluorescence and biomechanical cell&#8211;substrate interactions by traction force microscopy. To do so, we have developed two different formats utilizing polyacrylamide hydrogels of varying Young&#39;s modulus fabricated on either microscope slides or glass-bottom Petri dishes. We provide best practices and troubleshooting for the fabrication of microarrays on these hydrogel substrates, the subsequent cell culture on microarrays, and the acquisition of data. This platform is well-suited for use in investigations of biological processes for which both biochemical (e.g., extracellular matrix composition) and biophysical (e.g., substrate stiffness) cues may play significant, intersecting roles.

Cell differentiation is regulated by a host of microenvironmental factors, including both matrix composition and substrate material properties. We describe here a technique utilizing cell microarrays in conjunction with traction force microscopy to evaluate both cell differentiation and biomechanical cell&#8211;substrate interactions as a function of microenvironmental context.

细胞分化和牵引力的相关分析高通量细胞芯片平台

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly ...

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved useful as a convenient live imaging platform providing capabilities for precise control of experimental conditions in real time. In this article, we demonstrate live imaging of individual worms employing WormSpa, a previously-published custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes. To illustrate the capability, we performed proof-of-principle experiments in which worms were infected in the device with pathogenic bacteria, and the dynamics of expression of immune response genes and egg laying were monitored continuously in individual animals. The simple design and operation of this device make it suitable for users with no previous experience with microfluidic-based experiments. We propose that this approach will be useful for many researchers interested in longitudinal observations of biological processes under well-defined conditions.

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In this article, we demonstrate live imaging of individual worms employing a custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes.

秀丽线虫纵向成像的微流控平台

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved ...

A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic regulatory circuits. The analysis of synthetic genetic regulatory networks in vivo is time consuming and suffers from a lack of environmental control, with exogenous synthetic components interacting with host processes resulting in undesired behavior. To overcome these issues, cell-free characterization of novel circuitry is becoming more prevalent. In vitro transcription and translation (IVTT) mixtures allow the regulation of the experimental environment and can be optimized for each unique system. The protocols presented here detail the fabrication of a multilayer microfluidic device that can be utilized to sustain IVTT reactions for prolonged durations. In contrast to batch reactions, where resources are depleted over time and (by-) products accumulate, the use of microfluidic devices allows the replenishment of resources as well as the removal of reaction products. In this manner, the cellular environment is emulated by maintaining an out-of-equilibrium environment in which the dynamic behavior of gene circuits can be investigated over extended periods of time. To fully exploit the multilayer microfluidic device, hardware and software have been integrated to automate the IVTT reactions. By combining IVTT reactions with the microfluidic platform presented here, it becomes possible to comprehensively analyze complex network behaviors, furthering our understanding of the mechanisms that regulate cellular processes.

The fabrication process of a PDMS-based, multilayer, microfluidic device that allows in vitro transcription and translation (IVTT) reactions to be performed over prolonged periods is described. Furthermore, a comprehensive overview of the hardware and software required to automate and maintain these reactions for prolonged durations is provided.

用于长期无细胞基因表达传导的多层微流体平台

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic ...

A Microfluidic Platform for Stimulating Chondrocytes with Dynamic Compression

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth plate cartilage architecture and results in growth modulation of long bones of children. To determine the role of compressive stress in bone growth, we created a microfluidic device actuated by pneumatic pressure, to dynamically (or statically) compress growth plate chondrocytes embedded in alginate hydrogel cylinders. In this article, we describe detailed methods for fabricating and characterizing this device. The advantages of our protocol are: 1) Five different magnitudes of compressive stress can be generated on five technical replicates in a single platform, 2) It is easy to visualize cell morphology via a conventional light microscope, 3) Cells can be rapidly isolated from the device after compression to facilitate downstream assays, and 4) The platform can be applied to study mechanobiology of any cell type that can grow in hydrogels.

This article provides detailed methods for fabricating and characterizing a pneumatically actuating microfluidic device for chondrocyte compression.

微流体平台，用于用动态压缩刺激软骨细胞

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth ...

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. The tomato-P. syringae pv. tomato pathosystem is widely used to dissect the genetic basis of plant innate responses and disease resistance. While disease was successfully managed for many decades through the introduction of the Pto/Prf gene cluster from Solanum pimpinellifolium into cultivated tomato, race 1 strains of P. syringae have evolved to overcome resistance conferred by the Pto/Prf gene cluster and occur worldwide.
Wild tomato species are important reservoirs of natural diversity in pathogen recognition, because they evolved in diverse environments with different pathogen pressures. In typical screens for disease resistance in wild tomato, adult plants are used, which can limit the number of plants that can be screened due to their extended growth time and greater growth space requirements. We developed a method to screen 10-day-old tomato seedlings for resistance, which minimizes plant growth time and growth chamber space, allows a rapid turnover of plants, and allows large sample sizes to be tested. Seedling outcomes of survival or death can be treated as discrete phenotypes or on a resistance scale defined by amount of new growth in surviving seedlings after flooding. This method has been optimized to screen 10-day-old tomato seedlings for resistance to two P. syringae strains and can easily be adapted to other P. syringae strains.

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

The seedling flood assay facilitates rapid screening of wild tomato accessions for the resistance to the Pseudomonas syringae bacterium. This assay, used in conjunction with the seedling bacterial growth assay, can assist in further characterizing the underlying resistance to the bacterium, and can be used to screen mapping populations to determine the genetic basis of resistance.

高通量识别对伪单鱼的电阻syringae pv。番茄中的番茄使用苗子洪水测定

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. ...