The authors thank Ronald F. Renzi and Michael S. Bartsch for their contributions to the design and development of DMF devices and the DMF hub. This research was fully supported by the Laboratory Directed Research and Development program at Sandia National Laboratories. Sandia is a multi-program laboratory managed and operated by Sandia Corporation, a wholly owned subsidiary of Lockheed Martin Corporation, for the U.S. Department of Energy's National Nuclear Security Administration under contract DE-AC04-94AL85000.

This general protocol is designed to support application of the platform to a wide variety of studies; aspects specific to the representative study described in this report are separated out in brackets. Figure 2 illustrates a representative study carried out using the protocol. Note that in the protocol, all "instruct" commands are automated through use of pre-determined scripts. Note too that Step 2 can be carried out in parallel with Step 1 (e.g. during Steps 1.7 and 1.8), and that Step 2.1 i...

<ol>
	<li>Young, E. W. K., Beebe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fundamentals+of+microfluidic+cell+culture+in+controlled+microenvironments.">Fundamentals of microfluidic cell culture in controlled microenvironments.</a> Chemical Society Reviews. 39, 1036-1048 (2010).</li><li>Yeo, L. Y., Chang, H. C., Chan, P. P. Y., Friend, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+devices+for+bioapplications.">Microfluidic devices for bioapplications.</a> Small. 7, 12-48 (2011).</li><li>Zhang, B., Kim, M. C., Thorsen, T., Wang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+self-contained+microfluidic+cell+culture+system.">A self-contained microfluidic cell culture system.</a> Biomedical Microdevices. 11, 1233-1237 (2009).</li><li>Skafte-Pedersen, P., 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=A+self-contained,+programmable+microfluidic+cell+culture+system+with+real-time+microscopy+access.">A self-contained, programmable microfluidic cell culture system with real-time microscopy access.</a> Biomedical Microdevices. 14, 385-399 (2012).</li><li>Barbulovic-Nad, I., Au, S. H., Wheeler, A. 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We have developed a simple and versatile automated platform for micro-scale cell culture and stimulation experiments. The platform enables us to work with small culture volumes and cell populations (1 - 20 &mu;l and 100 - 2,000 cells, per chamber); culture sizes could be further reduced through use of microcapillaries of smaller diameter. Working at these scales reduces the cost of routine studies and makes feasible studies that require use of precious reagents and/or cells. Platform-executed experiments also sh...

The study of cells maintained in culture (in vitro analysis) has provided invaluable insight into the fundamental principles and molecular mechanisms governing complex biological systems and human health. The conventional methods for culture, stimulation, and collection of cells for analysis, which utilize Petri dishes and microtiter plates, were designed for study of large populations of cells (&ge;105) in milliliter-scale culture volumes (&ge;0.1 ml). However, there are many instances in which only limited quantities of cells are available (e.g. primary cells), or small populations of cells are desirable (e.g. to reduce cell-to-cell variability across the population), or required reagents are difficult to obtain or prohibitively expensive (e.g. purified cell-secreted factors). Such issues can be successfully addressed by scaling down culture size, which has the added benefit of reducing consumption of all reagents required for in vitro analysis1,2. Unfortunately, conventional equipment and methods do not support precise and reproducible manipulation of micro-scale cultures and the microfluidics-based automated systems currently available3-11 are too complex and specialized for routine use by most laboratories.
In this report, we describe assembly and use of a simple and versatile technology platform for automated culture, stimulation, and recovery of small populations of cells (100 - 2,000 cells) in micro-scale volumes (1 - 20 &mu;l). The platform architecture (Figure 1) is modular in design: a set of fibronectin-coated microcapillaries ("cell perfusion chambers" module) serves as the site for establishment, maintenance, and stimulation of micro-scale cultures; and a digital microfluidics (DMF)12,13 device outfitted with "transfer" microcapillaries ("central hub" module)14,15 routes cells and reagents to and from the perfusion chambers. DMF enables the user to individually address multiple droplets simultaneously and to change or re-order the manipulations (i.e. reconfigure sample processing trains) without altering the device hardware. Its tremendous flexibility is evident in its recent emergence as a key technology in a wide range of applications, including cell culture16,17, enzyme assays18,19, immunoassays20,21, DNA analysis22,23, protein processing,24,25 and clinical specimen processing.26,27 Our central hub takes advantage of the flexibility inherent to DMF devices, and further enhances it through the addition of microcapillary interfaces, which provides opportunity to carry out a subset of manipulations (e.g. cell culture) in specialized peripheral modules, rather than on the DMF device itself. Compartmentalization of processing trains in this way also simplifies design of the platform architecture (no need to build a DMF device that can carry out all processing steps) and facilitates its evolution as new functions are required (simply integrate new peripheral modules as necessary). Transport of cells and reagents within the central hub is driven by electrowetting forces generated by sequential activation of electrodes within the DMF device13,28; transport to, from, and within the perfusion chambers is powered by pressure changes generated by a high-precision syringe pump. All of these fluid movements are controlled via a simple electronic interface and automated through use of pre-determined scripts.
As a representative example, we demonstrate the use of the platform for the study of transcriptional responses elicited in immune cells upon challenge with bacteria (Figure 2). Carrying out these experiments on the platform enabled us to work with small numbers of cells (~1,000 per experimental condition), minimize experiment-to-experiment variability, conserve reagents, and re-direct hands-on labor. Given the advantages that it confers, as well as its accessibility and versatility, this platform should find use in a wide variety of laboratories and applications and prove especially useful in facilitating analysis of cells and stimuli that are available in only limited quantities.

<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

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in vitro analysis are well suited to study of large numbers of cells (&ge;105) in milliliter-scale volumes (&ge;0.1 ml). However, there are many instances in which it is necessary or desirable to scale down culture size to reduce consumption of the cells of interest and/or reagents required for their culture, stimulation, or processing. Unfortunately, conventional approaches do not support precise and reproducible manipulation of micro-scale cultures, and the microfluidics-based automated systems currently available are too complex and specialized for routine use by most laboratories. To address this problem, we have developed a simple and versatile technology platform for automated culture, stimulation, and recovery of small populations of cells (100 - 2,000 cells) in micro-scale volumes (1 - 20 &mu;l). The platform consists of a set of fibronectin-coated microcapillaries ("cell perfusion chambers"), within which micro-scale cultures are established, maintained, and stimulated; a digital microfluidics (DMF) device outfitted with "transfer" microcapillaries ("central hub"), which routes cells and reagents to and from the perfusion chambers; a high-precision syringe pump, which powers transport of materials between the perfusion chambers and the central hub; and an electronic interface that provides control over transport of materials, which is coordinated and automated via pre-determined scripts. As an example, we used the platform to facilitate study of transcriptional responses elicited in immune cells upon challenge with bacteria. Use of the platform enabled us to reduce consumption of cells and reagents, minimize experiment-to-experiment variability, and re-direct hands-on labor. Given the advantages that it confers, as well as its accessibility and versatility, our platform should find use in a wide variety of laboratories and applications, and prove especially useful in facilitating analysis of cells and stimuli that are available in only limited quantities.

As a demonstration of the automated platform, we used it to carry out a study in which small populations of immune cells were grown in micro-scale cultures, challenged with bacteria, and lysed for off-platform analysis of pro-inflammatory responses (Figure 2).
Each of six cell perfusion chambers was seeded with 104 immune cells (P388D.1 murine macrophages) resuspended in 10 &mu;l of growth medium. After an adherence period (37 &deg;C for 2 hr) and a medium exchange,...

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A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

We have developed an automated cell culture and interrogation platform for micro-scale cell stimulation experiments. The platform offers simple, versatile, and precise control in cultivating and stimulating small populations of cells, and recovering lysates for molecular analyses. The platform is well suited to studies that use precious cells and/or reagents.

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

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