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Stiffness-controlled three-dimensional extracellular matrices for high-resolution imaging of cell behavior

Nature Protocols volume 7, pages 2056–2066 (2012)Cite this article

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Abstract

Regulation of cell functions by the physical properties of the extracellular matrix (ECM) has emerged as a crucial contributor to development and disease. Two specific physical properties of the ECM, stiffness and dimensionality, each influence cell signaling and function. As these ECM physical properties are linked to other properties that also regulate cell behavior, e.g., integrin ligand density, parsing the specific contributions of ECM stiffness and dimensionality has proven difficult. Here we detail a simple protocol, which can be completed in 1–2 d, for combining three-dimensional (3D) ECM engagement with controlled underlying ECM stiffness. In these 'sandwich gels', cells are sandwiched between a 3D fibrillar ECM and an ECM-coupled polyacrylamide gel of defined compliance, allowing the study of the specific effects of ECM compliance on cell function in physiologically relevant 3D ECMs. This type of system enables high-resolution time-lapse imaging and is suitable for a wide range of cell types and molecular perturbations.

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Figure 1: Assembly of sandwich gels.
Figure 2: Anticipated results with sandwich gel culture setup.

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Acknowledgements

R.S.F., K.A.M. and C.M.W. are supported by the NHLBI Intramural Research Program. M.L.G. is supported by the NIH Director's Pioneer Award (DP10D00354). We thank Sergey Plotnikov for insightful discussion. We also acknowledge members of the NIH Electron Microscopy Core Facility and are grateful for the particularly excellent technical skills of M. Daniels and P. Connelly.

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Authors and Affiliations

  1. Cell Biology and Physiology Center, National Heart, Lung, and Blood Institute (NHBLI), US National Institutes of Health (NIH), Bethesda, Maryland, USA

    Robert S Fischer & Clare M Waterman

  2. Department of Biological Sciences, University of the Sciences, Philadelphia, Pennsylvania, USA

    Kenneth A Myers

  3. James Franck Institute, Chicago, Illinois, USA

    Margaret L Gardel

  4. Institute for Biophysical Dynamics, Chicago, Illinois, USA

    Margaret L Gardel

  5. Department of Physics, University of Chicago, Chicago, Illinois, USA

    Margaret L Gardel

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  1. Robert S Fischer
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  2. Kenneth A Myers
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Contributions

R.S.F. and K.A.M. conducted experimental work, M.L.G. performed rheometry measurements, and R.S.F., K.A.M. and C.M.W. wrote the manuscript.

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Correspondence to Robert S Fischer or Clare M Waterman.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Figure 1

Edge ruffles of 1.4 kPa polyacrylamide gel attached to 22 mm2 glass coverslip. Note that despite ruffling along edges, tears and rips in gel are not observed, and the gel remains evenly attached to coverslip. Bar equals 2 mm. (PDF 340 kb)

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Fischer, R., Myers, K., Gardel, M. et al. Stiffness-controlled three-dimensional extracellular matrices for high-resolution imaging of cell behavior. Nat Protoc 7, 2056–2066 (2012). https://doi.org/10.1038/nprot.2012.127

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