Name: a versatile automated platform for micro-scale cell stimulation experiments
Uploaded: 2013-08-06T14:00:00
Description: Watch this Scientific Journal Video about A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments at JoVE.com

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?...

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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 ...

Research

JoVE Journal

Bioengineering

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

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 ...

科研

生物工程

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

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

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

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

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

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

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

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

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

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 ...