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Designing Microfluidic Devices for Studying Cellular Responses Under Single or Coexisting Chemical/Electrical/Shear Stress Stimuli

Published: August 13th, 2016



1Department of Agricultural Chemistry, National Taiwan University, 2Department of Physics, Fu-Jen Catholic University, 3Research Center for Applied Sciences, Academia Sinica

Micro-fabricated devices integrated with fluidic components provide an in vitro platform for cell studies mimicking the in vivo micro-environment. We developed polymethylmethacrylate-based microfluidic chips for studying cellular responses under single or coexisting chemical/electrical/shear stress stimuli.

Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other physical or chemical stimuli. They have been commonly used for in vitro cell studies by providing in vivo like surroundings. Especially, how cells response to chemical gradients, electrical fields, and shear stresses has drawn many interests since these phenomena are important in understanding cellular properties and functions. These microfluidic chips can be made of glass substrates, silicon wafers, polydimethylsiloxane (PDMS) polymers, polymethylmethacrylate (PMMA) substrates, or polyethyleneterephthalate (PET) substrates. Out of these materials, PMMA substrates are cheap and can be easily processed using laser ablation and writing. Although a few microfluidic devices have been designed and fabricated for generating multiple, coexisting chemical and electrical stimuli, none of them was considered efficient enough in reducing experimental repeats, particular for screening purposes. In this report, we describe our design and fabrication of two PMMA-based microfluidic chips for investigating cellular responses, in the production of reactive oxygen species and the migration, under single or coexisting chemical/electrical/shear stress stimuli. The first chip generates five relative concentrations of 0, 1/8, 1/2, 7/8, and 1 in the culture regions, together with a shear stress gradient produced inside each of these areas. The second chip generates the same relative concentrations, but with five different electric field strengths created within each culture area. These devices not only provide cells with a precise, controllable micro-environment but also greatly increase the experimental throughput.

In vivo cells are surrounded by a variety of biomolecules including extracellular matrix (ECM), carbohydrates, lipids, and other cells. They functionalize by responding to micro-environmental stimuli such as interactions with ECM and responses to chemical gradients of various growth factors. Traditionally, in vitro cell studies are conducted in cell culture dishes where the consumption of cells and reagents is large and cells grow in a static (non-circulating) environment. Recently, micro-fabricated devices integrated with fluidic components have provided an alternative platform for cell studies in a more controllable way. Such devices are capable of....

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1. Chip Design and Fabrication

  1. Draw patterns to be ablated on PMMA substrates and double-side tapes using commercial software 24.
    1. To study the effects of chemical concentrations and shear stresses, draw a "Christmas tree" pattern with a varying width at its end in each of the five culture areas (Figure 1A and 1B).
    2. To study the effects of chemical concentrations and electric fields, draw a "Christmas tree" pattern with two more.......

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The Chemical-shear Stress (CSS) Chip

The CSS chip is made of three PMMA sheets, each of thickness 1 mm, attached together via two double-sided tapes, each of thickness 0.07 mm (Figure 1A and 1B). The "Christmas tree" structure generates five relative concentrations of 0, 1/8, 1/2, 7/8, and 1 in the five culture areas. By designing the culture area as a triangle, a shear stress gradient, .......

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PMMA-based chips are fabricated using laser ablation and writing which are cheaper and easier methods when compared to PDMS-based chips which require more complicated soft lithography. After designing a microfluidic chip, the fabrication and assembly can be done within just 5 min. There are some critical steps that attention should be paid to in performing the experiment. The first is the "assembling" issue. The adaptors should be glued properly to the top-most layer of the chip. Glue could leak into the fluidic .......

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This work was financially supported by the Ministry of Science and Technology of Taiwan under Contract No. MOST 104-2311-B-002-026 (K. Y. Lo), No. MOST 104-2112-M-030-002 (Y. S. Sun), and National Taiwan University Career Development Project (103R7888) (K. Y. Lo). The authors also thank the Center for Emerging Material and Advanced Devices, National Taiwan University, for the use of the cell culture room.


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Name Company Catalog Number Comments
Dulbecco's Modified Eagle Medium (DMEM) Gibco 11965-092 Cell culture medium
Trypsin Gibco 25300-054 detach cell from the dish
Fetal bovine serum (FBS) Gibco 10082147 Cell culture medium
10-cm cell culture Petri dish Nunc 150350 Cell culture
Bright-Line Hemacytometer Sigma Z359629 Cell Counting Equipment
PMMA Customized Customized Microfluidic chip
Adaptor Customized Customized Microfluidic chip
0.07/0.22 mm double-sided tape  3M 8018/9088 Microfluidic chip
Low melting point agarose Sigma A9414 Salt bridge
2'-7'-dichlorodihydrofluoresce diacetate Sigma D6883 Intracellular ROS measurement
Indium tin oxide (ITO) glass Merck 300739 Heater
Proportional-integral-derivative controller  JETEC Electronics Co. TTM-J4-R-AB Temperature controller
Thermal coupler TECPEL TPK-02A Temperature controller
CO2 laser scriber Laser Tools & Technics Corp. ILS2 Microfluidic chip fabrication
Syringe pumps New Era NE-300 Pumping medium and chemicals into the chip
Power supply Major Science  MP-300V Supplying direct currents
Inverted microscope Olympus CKX41 Monitoring cell migration
Inverted fluorescent microscope Nikon TS-100 Monitoring cell migartion and fluorescencent signals
DSLR camera Canon 60D Recording bright-field images 
CCD camera Nikon DS-Qi1 Recording fluorescent images 
super glue 3M Scotch 7004 Attaching adaptors to PMMA substrates
AutoCAD Autodesk Inc. Designing microfluidic chips
DMSO Sigma D8418 Dissolving DCFDA
ImgeJ National Institutes of Health Quantifying fluorescent intensities and cell migration

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