作者感谢罗纳德·F.壬子和Michael S. BARTSCH的为他们贡献的设计和开发的DMF设备和DMF枢纽。这项研究是完全由实验室的定向研究和发展方案在桑迪亚国家实验室的支持。桑迪亚国家实验室是一个多程序管理和经营的桑迪亚公司是洛克希德·马丁公司的全资附属公司，根据合同DE-AC04 94AL85000的美国能源部下属的国家核安全管理局的实验室。

这是一般的协议被设计为支持应用程序的平台，各种各样的研究，具体代表本报告中描述的研究方面分离出来，在括号中。 图2说明了具有代表性的研究，使用的协议进行。请注意，在协议中，所有的“指示”命令通过使用预先确定的脚本自动化。还要注意的是第2步就可以进行并行步骤1（ 例如，在步骤1.7和1.8），并往往是绕过该步骤2.1（因为的图案/涂层DMF装置板可水洗，并重新使用;见步骤5.2） 。 1。组装和填充细胞灌注室<ol><li>大衣灭菌（高压灭菌的）微毛细管（熔融石英，外径679微米，534微米的id，长15厘米）与纤连蛋白，通过指示画出一个无菌的纤连蛋白溶液（30微升的50微克/毫升的注射器泵的内表面）到每个微细管，温育（37℃，2小时），指令注射泵以退出该溶液，并让微毛细管干燥（6小时，室温（RT））。 </li><li>将每根纤维连接蛋白涂层的微毛细管（灌注室）聚碳酸酯管（外径为500μm，内径为100μm）和管的多端口的注射器泵，使用CapTite的配件（6灌注腔室，8端口注射器泵）。 </li><li>使用胶带，固定每个灌注腔体设置为所期望的温度（37℃）的热块。 </li><li>沉浸在水库中的灌注腔室的开口端（microcentifuge管或微量滴定板的孔）的含细胞（P388D.1小鼠巨噬细胞）悬浮于新鲜的生长培养基（RPMI-1640，10％胎牛血清，500单位/ ml青霉素，500微克/ ml链霉素）在所需的浓度（10 6细胞/ ml）。 </li><li>指示注射泵以绘制细胞（10微升，10 4个细胞），其次是足够的爱到每个灌注室r键将液塞（约4.5厘米）固定到散热块上的一节。 </li><li>使细胞附着到纤连蛋白涂覆的内表面的灌注室（37℃，1 - 2小时）。在此期间，从细胞储层灌注腔室的开口端传输到一个新的含有新鲜的生长培养基中的储层。 </li><li>坚持期后，指示注射器泵送液体插头（空调媒体和未婚细胞），浪费，提取新鲜培养基（10微升），并按照在坚持与足够的空气来定位新的液体插头（新鲜中等）细胞。 </li><li>继续开展中小型交流（ 即重复步骤1.7）定期（每2小时），直到细胞群体有足够的平衡和扩展（16 - 24小时的微观尺度的文化产生的种群〜1,000个细胞/室）。 </li></ol> 2。制作的的DMF设备和组装集线器架构<ol><l&gt;正如前面描述的14,29，图案46的铟锡氧化物（ITO）电极（液滴的致动的驱动程序），通过光刻和蚀刻，和5μm的聚对二甲苯-C和50的外套与DMF中的移动设备的底板上纳米的聚四氟乙烯AF。 DMF中的移动设备的顶板，通过涂布形成一个无图案的ITO玻璃基板有Teflon-AF，如上。 </li><li>使用金属压缩帧，固定到与保持板之间的间距为400μm的凹部的聚合物铸件14,30，此间距与致动电极的大小（2.5毫米2），结合在DMF中板，定义的液滴体积（2.5μl的每个电极）。需要注意的是DMF组件，可以通过简单的装置24。 </li><li> “插入”特氟龙涂层转移到DMF板之间的空间中的微毛细管（外径360微米，内径100μm; 3.5 - 4.0厘米长），定位每个公司同源的致动电极的边缘延伸。 </li><li>到对面的连接D每个传输微毛细管加贴CapTite的的拟合。 </li><li>接合电连接器与电源电压的ITO电极。电极激活序列是通过使用计算机控制的电子界面，运行预先确定的脚本自动化。 </li><li>可选：将组装的DMF轮毂上配备了电荷耦合器件（CCD）摄像机（DCR-HC96（索尼，日本）），以便跟踪液滴的显微镜（SZ-6145TR（奥林巴斯，日本））的平移阶段31 </li></ol> 3。将连接灌注钱伯斯DMF集线器和刺激细胞群体<ol><li>删除从介质油藏灌注室（见步骤1.6）的开口端，并把它们插入（见步骤2.4）的二甲基甲酰胺集线器转移微毛细管的端部贴到CapTite配件。 </li><li>指示注射器泵送液体插头（空调媒体），在灌注室浪费。 </li><li>研究所构作的DMF集线器画刺激（ 大肠杆菌生物粒子在100微克/毫升的新鲜培养基中的Pluronic F127 0.1％w / v的）从一个板上水库和提供适当大小的液滴（10微升），每个转印微细。主板上的水库是由圆筒状的隔室（1.5×1.5厘米），并通过管道连接到DMF集线器。 </li><li>指示绘制刺激转让微毛细管插入注射器泵，并按照与足够的空气灌注室的细胞群定位插头。 </li><li>培养的细胞与刺激（37℃，1 - 4小时）。可选：新鲜的刺激（ 即重复步骤3.2-3.4）定期交流。 </li></ol> 4。终止刺激并恢复细胞裂解液分析<ol><li>可选：如果刺激后需要花多长时间，交换刺激含有液体插头新鲜培养基（ 即重复步骤3.2 - 34，以新鲜培养基刺激），并继续孵化和交流需要。 </li><li>交易所的液体灌注室洗涤缓冲液（前奏曲直接的裂解模块缓冲区B）（ 即重复步骤3.2 - 3.4，以洗涤缓冲液的刺激），孵化（5分钟）插头。 </li><li>交易所液体插头（洗涤缓冲液）裂解液（前奏直接裂解模块缓冲A与0.1％的Pluronic F127 W / V）（ 即重复步骤3.2 - 3.4，裂解液代替刺激），孵化（5分钟） 。 </li><li>指导的注射器泵，送液体插头（细胞裂解液）的DMF集线器。 </li><li>拆卸的DMF枢纽，删除的DMF装置的顶板（注意不要倾斜板横盘整理，因为这可能会导致液滴混合），并收集裂解的Pipetman使用或注射器为平台（基因表达谱分析通过定量PCR）。 </li></ol> 5。清洁平台硬件重新使用<ol><li>浸入转印微毛细管和CapTite的管件在10％的漂白剂（在水中的体积/体积）和10分钟，在RT，以及与去离子水和干燥的，用氮气冲洗。 </li><li> DMF中的移动设备板浸入10％的漂白剂在RT和10分钟，用异丙醇冲洗，然后用去离子水和干燥用氮气在160℃下烘烤10分钟。 </li><li>的注射器泵的聚碳酸酯管，和DMF的移动设备的聚合物铸造和压缩帧，可以重新使用无清洗。微毛细管灌注室每次实验后丢弃。 </li></ol>

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H., Oliver, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarrays,+deep+sequencing+and+the+true+measure+of+the+transcriptome.">Microarrays, deep sequencing and the true measure of the transcriptome.</a> BMC Biology. 9, (2011).</li><li>Wu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidically-unified+cell+culture,+sample+preparation,+imaging+and+flow+cytometry+for+measurement+of+cell+signaling+pathways+with+single+cell+resolution.">Microfluidically-unified cell culture, sample preparation, imaging and flow cytometry for measurement of cell signaling pathways with single cell resolution.</a> Lab on a Chip. 12, 2823-2831 (2012).</li><li>Srivastava, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fully+integrated+microfluidic+platform+enabling+automated+phosphoprofiling+of+macrophage+response.">Fully integrated microfluidic platform enabling automated phosphoprofiling of macrophage response.</a> Analytical Chemistry. 81, 3261-3269 (2009).</li><li>Herr, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+immunoassays+as+rapid+saliva-based+clinical+diagnostics.">Microfluidic immunoassays as rapid saliva-based clinical diagnostics.</a> Proceedings of the National Academy of Sciences of the United States of America. 104, 5268-5273 (2007).</li></ol>

作者宣称，他们有没有竞争经济利益。

我们已经开发了一个简单而灵活的自动化平台，为微大规模细胞培养和刺激实验。该平台使我们的工作与小培养体积和细胞群（1 - 20μl和100 - 2,000细胞， 每室），文化的尺寸可以进一步降低通过使用较小直径的微毛细管。工作在这些尺度的常规研究，并降低了成本，使需要使用珍贵的试剂和/或细胞的可行性研究。平台上执行的实验也显示较少的可变性，推测可能是由于细胞和体液处理自动...

维持在文化研究的细胞体外分析的基本原则和复杂的生物系统和人类健康的分子机制提供了宝贵的洞察。文化，刺激和收集细胞进行分析，利用培养皿，酶标板，传统的方法是大量人口毫升规模培养体积的细胞（≥10）（≥0.1毫升）的研究设计。但是，也有许多实例中，只有有限数量的细胞（ 例如，原代细胞），或小的细胞群体是可取的（ 例如，在人口减少细胞到细胞的变异），或很难获得所需的试剂或昂贵（ 如纯化细胞分泌的因素）。这样的问题可以顺利解决文化尺寸的缩减，减少消费，这有利于增加所需试剂在体外分析1,2。不幸的是，传统的设备和方法不支持微观尺度的文化和基于微流体的自动化系统目前可用3-11过于复杂，专门用于日常使用的大多数实验室的精确和可重复的操作。 在这份报告中，我们描述了一个简单而灵活的技术平台，为自动化的文化，刺激和恢复小的细胞群体（100 - 2,000细胞）在微观尺度册（1 - 20微升）的组装和使用。采用模块化设计，该平台架构（ 图1）：一组纤维连接蛋白涂层的微毛细管（“细胞灌注腔”模块）作为该网站的建立，维护和刺激微观尺度的文化和数字微流体（DMF ）12,13设备配备“转移”微毛细管（“中央枢纽”模块）14,15航线细胞和试剂从灌注室。二甲基甲酰胺使用户单独处理多个液滴同时不改变的移动设备的硬件的情况下改变或重新排序的操纵（ 即重新配置检测体处理列车）。其巨大的灵活性是显而易见的，其最近出现的一项关键技术在广泛的应用，包括细胞培养16,17，酶化验18,19，20,21免疫测定，DNA分析，22,23，蛋白处理，24,25我们的中心枢纽和临床标本的处理。26,27利用DMF装置所固有的灵活性，进一步增强它通过添加微细的接口，它提供了契机，开展了专门的外设操作的一个子集（ 如细胞培养）模块，而不是在DMF中的移动设备本身。列车以这种方式处理的条块分割也简化了设计解放军TForm的架构（无需修建的DMF装置，可以进行所有的处理步骤），并促进其演变为新功能（简单地集成新的必要的外设模块）。是由电润湿力的DMF装置13,28内的电极所产生的顺序激活，运输到，从运输的中心轮毂内的细胞和试剂，于灌注室是由一个高精度的注射器产生的压力变化泵。所有这些流体运动控制，通过一个简单的电子接口，通过使用预先确定的脚本和自动化​​。 作为一个有代表性的例子中，我们演示了如何使用该平台的研究转录反应引起的免疫细胞细菌挑战时（ 图2）。这些实验平台上开展，使我们能够与小数量的细胞（〜1,000 元实验，TAL条件），减少实验的实验变异，节约试剂，并重新直接动手劳动。给定的优点，它赋予，以及它的可访问性和多功能性，该平台应该找到实验室和应用程序中使用各种各样的，并证明在促进细胞和刺激可只在有限数量的分析特别有用。

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Prelude Direct Lysis Module</td>
 <td>NuGEN</td>
 <td>1400-24</td>
 <td></td>
 </tr>
 <tr>
 <td>Trypan Blue (0.4% w/v)</td>
 <td>GIBCO</td>
 <td>15250-061</td>
 <td></td>
 </tr>
 <tr>
 <td>Cell Stripper</td>
 <td>Cellgro</td>
 <td>25-056-C1</td>
 <td></td>
 </tr>
 <tr>
 <td>Ovation PicoSL WTA</td>
 <td>NuGEN</td>
 <td>3310-048</td>
 <td></td>
 </tr>
 <tr>
 <td>Agencourt RNAClean XP</td>
 <td>Beckman Coulter Genomics</td>
 <td>A63987</td>
 <td></td>
 </tr>
 <tr>
 <td>pHrodo BioParticles</td>
 <td>Invitrogen</td>
 <td>P35361</td>
 <td></td>
 </tr>
 <tr>
 <td>CCL4 TaqMan qRT-PCR assay</td>
 <td>Applied Biosystems</td>
 <td>Mm00443111_m1</td>
 <td></td>
 </tr>
 <tr>
 <td>CCL5 TaqMan qRT-PCR assay</td>
 <td>Applied Biosystems</td>
 <td>Mm01302428_m1</td>
 <td></td>
 </tr>
 <tr>
 <td>PTGS2 TaqMan qRT-PCR assay</td>
 <td>Applied Biosystems</td>
 <td>Mm00478374_m1</td>
 <td></td>
 </tr>
 <tr>
 <td>TNF TaqMan qRT-PCR assay</td>
 <td>Applied Biosystems</td>
 <td>Mm00443258_m1</td>
 <td></td>
 </tr>
 <tr>
 <td>GAPDH TaqMan qRT-PCR assay</td>
 <td>Applied Biosystems</td>
 <td>Mm99999915_g1</td>
 <td></td>
 </tr>
 <tr>
 <td>Pluronic F127</td>
 <td>Sigma Chemical</td>
 <td>2594628</td>
 <td></td>
 </tr>
 <tr>
 <td>Fluorinert FC-40</td>
 <td>Sigma Chemical</td>
 <td>51142-49-5</td>
 <td></td>
 </tr>
 <tr>
 <td>Parylene C dimer</td>
 <td>Specialty Coating Systems</td>
 <td>28804-46-8</td>
 <td></td>
 </tr>
 <tr>
 <td>Teflon-AF</td>
 <td>DuPont</td>
 <td>AF1600</td>
 <td></td>
 </tr>
 <tr>
 <td>Polyimide tape</td>
 <td>ULINE</td>
 <td>S-11928</td>
 <td></td>
 </tr>
 <tr>
 <td>Indium tin oxide (ITO) coated glass substrates </td>
 <td>Delta Technologies </td>
 <td>CB-40IN-1107</td>
 <td></td>
 </tr>
 <tr>
 <td>DMF hub Teflon-coated fused-silica microcapillaries</td>
 <td>Polymicro Technologies</td>
 <td>TSU100375</td>
 <td></td>
 </tr>
 <tr>
 <td>Perfusion chamber microcapillaries</td>
 <td>Polymicro Technologies</td>
 <td>TSP530700</td>
 <td></td>
 </tr>
 <tr>
 <td>Tubing and microcapillary fittings</td>
 <td>Sandia National Laboratories</td>
 <td>&nbsp;</td>
 <td></td>
 </tr>
 <tr>
 <td>Polycarbonate tubing</td>
 <td>Paradigm Optics</td>
 <td>CTPC100-500-5</td>
 <td></td>
 </tr>
 <tr>
 <td>8-port precision syringe pump equipped with 30 mm (500 &mu;l capacity) syringes</td>
 <td>Hamilton Company</td>
 <td>54848-01</td>
 <td></td>
 </tr>
 <tr>
 <td>Parylene-C vapor deposition instrument</td>
 <td>Specialty Coating Systems</td>
 <td>PDS 2010 Labcoter 2</td>
 <td></td>
 </tr>
 <tr>
 <td>High-voltage function generator</td>
 <td>Trek</td>
 <td>615A-1 615-3</td>
 <td></td>
 </tr>
 <tr>
 <td>MVX10 microscope</td>
 <td>Olympus</td>
 <td></td>
 <td>Optional (facilitates tracking of droplets on DMF hub)</td>
 </tr>
 <tr>
 <td>QIClick digital CCD camera</td>
 <td>QImaging</td>
 <td>QIClick-F-CLR-12</td>
 <td>Optional (facilitates tracking of droplets on DMF hub)</td>
 </tr>
</tbody>
</table>

a versatile automated platform for micro-scale cell stimulation experiments

培养的细胞在体外分析中研究复杂的生物系统提供了重要的洞察。常规方法和设备，用于在体外分析中非常适合研究毫升尺度的量（≥0.1毫升）中的大量的细胞（≥10 5）。不过，也有许多情况下，它是必要或适当的缩减培养规模，降低消耗细胞的利息和/或他们的文化，刺激，或加工所需的试剂。不幸的是，传统的方法不支持精确和可重复操作的微观尺度的文化，基于微流体的自动化系统目前过于复杂，专门用于日常使用的大多数实验室。为了解决这个问题，我们已经开发出一种简单和通用的技术平台，为自动化的文化，刺激和复苏在微观尺度量小的细胞群体（100 - 2,000细胞）（1 - 20微升）。该平台由一组纤维连接蛋白涂层的微毛细管（“细胞灌注腔”），内微观尺度的文化是建立，维护和刺激;数字微流体（DMF）设备配备“转移”微毛细管（“中央枢纽“），航线细胞和试剂并从灌注室;一个高精度的注射器泵，权力灌注室和中心位置之间的材料运输和一个电子接口，它提供控制材料运输，这是协调和自动化通过预先确定的脚本。作为一个例子，我们使用的平台，以便研究转录反应引起的免疫细胞与细菌的挑战时。使用的平台使我们能够减少细胞和试剂的消耗，尽量减少实验实验变异，再直接动手劳动。的优点在于它赋予，以及它的可访问性和多功能性，邻的乌尔平台，应使用在各种各样的实验室和应用程序，并在促进细胞和刺激可只在有限数量的分析证明是特别有用的。

自动化平台作为示范，我们用它来 ​​开展一项研究中，人口较少的免疫细胞生长在微观尺度的文化，挑战与细菌，裂解为平台促炎症反应的分析（ 图2 ）。 每6个细胞的灌注腔10 4免疫细胞（P388D.1小鼠巨噬细胞）的生长培养基中再悬浮于10μl接种。 〜500个细胞粘附期（37℃，2小时）和介质的交换后，仍然连接到每一个腔室（ 图3A）的内表面?...

Watch this Scientific Journal Video about A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments at JoVE.com

A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

微观尺度细胞刺激实验，我们已经开发了一个自动细胞培养和审讯平台。该平台提供简单，功能多样，培育和刺激小的细胞群体，并恢复裂解分子分析和精确的控制。该平台非常适合使用珍贵的细胞和/或试剂的研究。

一种多功能自动化平台微尺度细胞刺激实验

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in ...

a-versatile-automated-platform-for-micro-scale-cell-stimulation

Research

JoVE Journal

Bioengineering

10.5K 次观看.  Sandia National Laboratories. 微观尺度细胞刺激实验，我们已经开发了一个自动细胞培养和审讯平台。该平台提供简单，功能多样，培育和刺激小的细胞群体，并恢复裂解分子分析和精确的控制。该平台非常适合使用珍贵的细胞和/或试剂的研究。

视频: A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and metastasis of tumor cells. Migration has been studied on glass coverslips in order to make cellular dynamics amenable to investigation by light microscopy. However, it has become clear that many aspects of cell migration depend on features of the local environment including its elasticity, protein composition, and pore size, which are not faithfully represented by rigid two dimensional substrates such as glass and plastic 1. Furthermore, interaction with other cell types, including stromal fibroblasts 2 and immune cells 3, has been shown to play a critical role in promoting the invasion of cancer cells. Investigation at the molecular level has increasingly shown that molecular dynamics, including response to drug treatment, of identical cells are significantly different when compared in vitro and in vivo 4. 
Ideally, it would be best to study cell migration in its naturally occurring context in living organisms, however this is not always possible. Intermediate tissue culture systems, such as cell derived matrix, matrigel, organotypic culture (described here) tissue explants, organoids, and xenografts, are therefore important experimental intermediates. These systems approximate certain aspects of an in vivo environment but are more amenable to experimental manipulation such as use of stably transfected cell lines, drug treatment regimes, long term and high-resolution imaging. Such intermediate systems are especially useful as proving grounds to validate probes and establish parameters required to image the dynamic response of cells and fluorescent reporters prior to undertaking imaging in vivo 5. As such, they can serve an important role in reducing the need for experiments on living animals.

A method is described for the preparation of a 3-dimensional matrix consisting of collagen type I and primary human fibroblasts. This organotypic gel serves as a useful substrate to assess invasive cell migration because it mimics basic features of tissue stroma and is amenable to many forms of microscopy.

器官Ⅰ型胶原的含量可延展的平台，以评估细胞行为的一个3维的情况下

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

科研

生物工程

GENPLAT: an Automated Platform for Biomass Enzyme Discovery and Cocktail Optimization

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid transportation fuels such as ethanol. We have developed an integrated high throughput platform, called GENPLAT, for the discovery and development of novel enzymes and enzyme cocktails for the release of sugars from diverse pretreatment/biomass combinations. GENPLAT comprises four elements: individual pure enzymes, statistical design of experiments, robotic pipeting of biomass slurries and enzymes, and automated colorimeteric determination of released Glc and Xyl. Individual enzymes are produced by expression in Pichia pastoris or Trichoderma reesei, or by chromatographic purification from commercial cocktails or from extracts of novel microorganisms. Simplex lattice (fractional factorial) mixture models are designed using commercial Design of Experiment statistical software. Enzyme mixtures of high complexity are constructed using robotic pipeting into a 96-well format. The measurement of released Glc and Xyl is automated using enzyme-linked colorimetric assays. Optimized enzyme mixtures containing as many as 16 components have been tested on a variety of feedstock and pretreatment combinations.
GENPLAT is adaptable to mixtures of pure enzymes, mixtures of commercial products (e.g., Accellerase 1000 and Novozyme 188), extracts of novel microbes, or combinations thereof. To make and test mixtures of &tilde;10 pure enzymes requires less than 100 &mu;g of each protein and fewer than 100 total reactions, when operated at a final total loading of 15 mg protein/g glucan. We use enzymes from several sources. Enzymes can be purified from natural sources such as fungal cultures (e.g., Aspergillus niger, Cochliobolus carbonum, and Galerina marginata), or they can be made by expression of the encoding genes (obtained from the increasing number of microbial genome sequences) in hosts such as E. coli, Pichia pastoris, or a filamentous fungus such as T. reesei. Proteins can also be purified from commercial enzyme cocktails (e.g., Multifect Xylanase, Novozyme 188). An increasing number of pure enzymes, including glycosyl hydrolases, cell wall-active esterases, proteases, and lyases, are available from commercial sources, e.g., Megazyme, Inc. (www.megazyme.com), NZYTech (www.nzytech.com), and PROZOMIX (www.prozomix.com).
Design-Expert software (Stat-Ease, Inc.) is used to create simplex-lattice designs and to analyze responses (in this case, Glc and Xyl release). Mixtures contain 4-20 components, which can vary in proportion between 0 and 100%. Assay points typically include the extreme vertices with a sufficient number of intervening points to generate a valid model. In the terminology of experimental design, most of our studies are "mixture" experiments, meaning that the sum of all components adds to a total fixed protein loading (expressed as mg/g glucan). The number of mixtures in the simplex-lattice depends on both the number of components in the mixture and the degree of polynomial (quadratic or cubic). For example, a 6-component experiment will entail 63 separate reactions with an augmented special cubic model, which can detect three-way interactions, whereas only 23 individual reactions are necessary with an augmented quadratic model. For mixtures containing more than eight components, a quadratic experimental design is more practical, and in our experience such models are usually statistically valid.
All enzyme loadings are expressed as a percentage of the final total loading (which for our experiments is typically 15 mg protein/g glucan). For "core" enzymes, the lower percentage limit is set to 5%. This limit was derived from our experience in which yields of Glc and/or Xyl were very low if any core enzyme was present at 0%. Poor models result from too many samples showing very low Glc or Xyl yields. Setting a lower limit in turn determines an upper limit. That is, for a six-component experiment, if the lower limit for each single component is set to 5%, then the upper limit of each single component will be 75%. The lower limits of all other enzymes considered as "accessory" are set to 0%. "Core" and "accessory" are somewhat arbitrary designations and will differ depending on the substrate, but in our studies the core enzymes for release of Glc from corn stover comprise the following enzymes from T. reesei: CBH1 (also known as Cel7A), CBH2 (Cel6A), EG1(Cel7B), BG (&beta;-glucosidase), EX3 (endo-&beta;1,4-xylanase, GH10), and BX (&beta;-xylosidase).
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GENPLAT (GLBRC Enzyme Platform) is an automated platform for discovery and optimization of enzyme cocktails for biomass degradation. It can be adapted to multiple feedstocks and mixtures of enzymes containing multiple components.

GENPLAT：生物质酶的发现和鸡尾酒优化的自动化平台

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid ...

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 ...

A Novel Stretching Platform for Applications in Cell and Tissue Mechanobiology

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have contributed dramatically to the understanding of basic mechanobiology. These techniques have been extensively used to demonstrate how the onset and progression of various diseases are heavily influenced by mechanical cues. This article presents a multi-functional biaxial stretching (BAXS) platform that can either mechanically stimulate single cells or quantify the mechanical stiffness of tissues. The BAXS platform consists of four voice coil motors that can be controlled independently. Single cells can be cultured on a flexible substrate that can be attached to the motors allowing one to expose the cells to complex, dynamic, and spatially varying strain fields. Conversely, by incorporating a force load cell, one can also quantify the mechanical properties of primary tissues as they are exposed to deformation cycles. In both cases, a proper set of clamps must be designed and mounted to the BAXS platform motors in order to firmly hold the flexible substrate or the tissue of interest. The BAXS platform can be mounted on an inverted microscope to perform simultaneous transmitted light and/or fluorescence imaging to examine the structural or biochemical response of the sample during stretching experiments. This article provides experimental details of the design and usage of the BAXS platform and presents results for single cell and whole tissue studies. The BAXS platform was used to measure the deformation of nuclei in single mouse myoblast cells in response to substrate strain and to measure the stiffness of isolated mouse aortas. The BAXS platform is a versatile tool that can be combined with various optical microscopies in order to provide novel mechanobiological insights at the sub-cellular, cellular and whole tissue levels.

We present in this article a novel stretching platform that can be used to investigate single cell responses to complex anisotropic biaxial mechanical deformation and quantify the mechanical properties of biological tissues.

在细胞和组织力学生物学应用一种新型的拉伸平台

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have ...

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 ...

Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential mechanosensing characteristics. Engineering and analysis of these tissue types remain a challenge. Layered hydrogel constructs provide an opportunity for investigating the interactions among multiple cell populations within single constructs. Alginate hydrogels are both biocompatible and allow for easy isolation of cells after experimentation. Here, we describe a method for the development of small sized dual layered alginate hydrogel discs. This process maintains high cell viability of human mesenchymal stem cells during the formation process and these layered discs can withstand unconfined cyclic compression, commonly used for stimulation of hMSCs undergoing chondrogenesis. These layered constructs can potentially be scaled up to include additional levels, and also be used to segregate cell populations initially after layering. This dual layer alginate hydrogel culture platform can be used for many different applications including engineering and analysis of cells of load bearing tissues and co-cultures of other cell types.

Engineering and analysis of load bearing tissues with heterogeneous cell populations are still a challenge. Here, we describe a method for creating bi-layered alginate hydrogel discs as a platform for co-culture of diverse cell populations within one construct.

分层海藻酸钠构造：一个异质细胞群的共培养平台

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential ...

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 Versatile Method of Patterning Proteins and Cells

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning techniques work well only with simple shapes, small areas and selected bio-materials. This article describes a method to distribute cell suspensions as well as substrate solutions into complex, long, closed (dead-end) polydimethylsiloxane (PDMS) microchannels using negative pressure. This method enables researchers to pattern multiple substrates including fibronectin, collagen, antibodies (Sal-1), poly-D-lysine (PDL), and laminin. Patterning of substrates allows one to indirectly pattern a variety of cells. We have tested C2C12 myoblasts, the PC12 neuronal cell line, embryonic rat cortical neurons, and amphibian retinal neurons. In addition, we demonstrate that this technique can directly pattern fibroblasts in microfluidic channels via brief application of a low vacuum on cell suspensions. The low vacuum does not significantly decrease cell viability as shown by cell viability assays. Modifications are discussed for application of the method to different cell and substrate types. This technique allows researchers to pattern cells and proteins in specific patterns without the need for exotic materials or equipment and can be done in any laboratory with a vacuum.

This report describes a simple, easy to perform technique, using low pressure vacuum, to fill microfluidic channels with cells and substrates for biological research.

图案蛋白质和细胞的通用的方法

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning ...

DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of flexible polymers nor of rigid rods. Underlying filaments remain outstretched due to their non-vanishing backbone stiffness, which is quantified via the persistence length (lp), but they are also subject to strong thermal fluctuations. Their finite bending stiffness leads to unique, non-trivial collective mechanics of bulk networks, enabling the formation of stable scaffolds at low volume fractions while providing large mesh sizes. This underlying principle is prevalent in nature (e.g., in cells or tissues), minimizing the high molecular content and thereby facilitating diffusive or active transport. Due to their biological implications and potential technological applications in biocompatible hydrogels, semiflexible polymers have been subject to considerable study. However, comprehensible investigations remained challenging since they relied on natural polymers, such as actin filaments, which are not freely tunable. Despite these limitations and due to the lack of synthetic, mechanically tunable, and semiflexible polymers, actin filaments were established as the common model system. A major limitation is that the central quantity lp cannot be freely tuned to study its impact on macroscopic bulk structures. This limitation was resolved by employing structurally programmable DNA nanotubes, enabling controlled alteration of the filament stiffness. They are formed through tile-based designs, where a discrete set of partially complementary strands hybridize in a ring structure with a discrete circumference. These rings feature sticky ends, enabling the effective polymerization into filaments several microns in length, and display similar polymerization kinetics as natural biopolymers. Due to their programmable mechanics, these tubes are versatile, novel tools to study the impact of lp on the single-molecule as well as the bulk scale. In contrast to actin filaments, they remain stable over weeks, without notable degeneration, and their handling is comparably straightforward.

Semiflexible polymers display unique mechanical properties that are extensively applied by living systems. However, systematic studies on biopolymers are limited since properties such as polymer rigidity are inaccessible. This manuscript describes how this limitation is circumvented by programmable DNA nanotubes, enabling experimental studies on the impact of filament rigidity.

DNA 纳米管作为研究半聚合物的万能工具

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of ...

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 ...