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Long-term microfluidic cultures of myotube microarrays for high-throughput focal stimulation

Nature Protocols volume 1, pages 1092–1104 (2006)Cite this article

Abstract

We have developed a microfluidic cell culture method that allows for the formation of linear isolated myotubes organized in a parallel microarray. Attachment and spreading of cells are confined within microtracks of cell-adherent proteins separated by a protein-repellent coating. Signaling molecules or other molecules of interest can be focally delivered to the myotubes using heterogeneous microfluidic streams. We have used the method to focally deliver agrin (a molecule implicated as a postsynaptic organizer), which leads to localized acetylcholine receptor clustering. These techniques can be modified to accommodate other cell types and can be adapted to virtually any bioactive molecule such as signaling factors or drugs. This protocol features two major techniques that can be utilized simultaneously or independently to (i) micropattern cells using surface chemical modification and (ii) use a microfluidic platform for culturing and focal stimulation of cells with molecules of interest. Device design, fabrication and assembly can be completed in 3 days.

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Figure 1: C2C12 cells in random and micropatterned microfluidic cultures.
Figure 2: Schematic illustration of cellular micropatterning in the microfluidic device.
Figure 3
Figure 4: Photomasks of the devices.
Figure 5: Procedure for protein micropatterning based on the use of PDMS microchannels.
Figure 6: Schematic illustration of the device assembly and cellular micropatterning.
Figure 7: Process flow in photolithographic fabrication of the master mold.
Figure 8: Wafer spin-coater.
Figure 9: UV aligner.
Figure 10: The microfluidic perfusion device.
Figure 11: Oxygen plasma device.
Figure 12
Figure 13: Fluidic setup used for flow experiments.

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Acknowledgements

We thank K. Healy and T. Barber for the original IPN-grafting protocol and G. Cooksey for the photographs of microfabrication equipment.

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Authors and Affiliations

  1. Department of Bioengineering, University of Washington, Box 355061, Seattle, 98195-2255, Washington, USA

    Anna Tourovskaia, Xavier Figueroa-Masot & Albert Folch

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  1. Anna Tourovskaia
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  2. Xavier Figueroa-Masot
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  3. Albert Folch
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Contributions

A.T.: study concept, design, development and preparation of manuscript. X.F.M.: preparation of manuscript. A.F.: study concept and preparation of manuscript.

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Correspondence to Albert Folch.

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The authors declare no competing financial interests.

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Tourovskaia, A., Figueroa-Masot, X. & Folch, A. Long-term microfluidic cultures of myotube microarrays for high-throughput focal stimulation. Nat Protoc 1, 1092–1104 (2006). https://doi.org/10.1038/nprot.2006.123

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