Article | Published:

Quantifying cell-generated mechanical forces within living embryonic tissues

Nature Methods volume 11, pages 183189 (2014) | Download Citation

  • A Corrigendum to this article was published on 27 February 2014

This article has been updated

Abstract

Cell-generated mechanical forces play a critical role during tissue morphogenesis and organ formation in the embryo. Little is known about how these forces shape embryonic organs, mainly because it has not been possible to measure cellular forces within developing three-dimensional (3D) tissues in vivo. We present a method to quantify cell-generated mechanical stresses exerted locally within living embryonic tissues, using fluorescent, cell-sized oil microdroplets with defined mechanical properties and coated with adhesion receptor ligands. After a droplet is introduced between cells in a tissue, local stresses are determined from droplet shape deformations, measured using fluorescence microscopy and computerized image analysis. Using this method, we quantified the anisotropic stresses generated by mammary epithelial cells cultured within 3D aggregates, and we confirmed that these stresses (3.4 nN μm−2) are dependent on myosin II activity and are more than twofold larger than stresses generated by cells of embryonic tooth mesenchyme, either within cultured aggregates or in developing whole mouse mandibles.

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Change history

  • 05 February 2014

    In the version of this article initially published, the current affiliation of author Ralph Sperling was not included. His current affiliation is the Fraunhofer ICT-IMM, Mainz, Germany. The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

We thank the SysCODE consortium for postdoctoral financial support for O.C. and for interesting discussions with several of its members. We thank C. Jorcyk (Boise State University) for providing premalignant mammary epithelial M28 cells and B. Ristenpart for the Matlab code used to analyze data obtained with the pendant drop method. O.C. thanks all members of the Ingber lab for their help and support, J. Gros for help with imaging, and F. Aguet for help with SteerableJ plugins. R.A.S. gratefully acknowledges funding from the German Research Foundation (Sp 1282/1-1). This work was supported by US National Institutes of Health grant RL1 DE019023-01 (to D.E.I.), the Wyss Institute for Biologically Inspired Engineering at Harvard University, the MacArthur Foundation and the Harvard NSF-MRSEC (L.M.).

Author information

Author notes

    • Otger Campàs
    •  & Ralph A Sperling

    Present addresses: Department of Mechanical Engineering, University of California, Santa Barbara, Santa Barbara, California, USA (O.C.); Fraunhofer ICT-IMM, Mainz, Germany (R.A.S.).

Affiliations

  1. School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts, USA.

    • Otger Campàs
    • , Ralph A Sperling
    • , David A Weitz
    • , L Mahadevan
    •  & Donald E Ingber
  2. Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, Massachusetts, USA.

    • Otger Campàs
    • , Ashley G Bischof
    • , L Mahadevan
    •  & Donald E Ingber
  3. Vascular Biology Program, Children's Hospital, Boston, Massachusetts, USA.

    • Otger Campàs
    • , Tadanori Mammoto
    • , Sean Hasso
    • , Ashley G Bischof
    •  & Donald E Ingber
  4. Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, Massachusetts, USA.

    • Otger Campàs
    •  & L Mahadevan
  5. Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.

    • Daniel O'Connell
    •  & Richard Maas
  6. Department of Physics, Harvard University, Cambridge, Massachusetts, USA.

    • David A Weitz
    •  & L Mahadevan
  7. Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA.

    • Donald E Ingber

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Contributions

D.E.I., O.C. and L.M. defined the project; O.C. conceived of the droplets as force transducers; O.C. and D.E.I. designed the technique; T.M. and D.O. provided dissected mouse mandibles; O.C. and S.H. microinjected droplets into mouse mandibles; O.C., R.A.S. and D.A.W. designed and synthesized new fluorocarbon-hydrocarbon block copolymers; O.C. and A.G.B. did the initial tests of the technique using cell aggregates; O.C. performed force measurements in cell-drop aggregates and living mouse mandibles; O.C. performed confocal measurements; O.C. analyzed the data; D.O. and R.M. provided transgenic mice; and O.C., L.M. and D.E.I. wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Otger Campàs or Donald E Ingber.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1 and 2 and Supplementary Notes 1–3

Videos

  1. 1.

    Effect of myosin II inhibition on droplet deformations

    Time-lapse showing the effect of myosin II inhibition on droplet deformations. Myosin II was inhibited using blebbistatin (see Online Methods: 'Perturbation of cellular forces with drugs'). The drug was added at t = 0. Droplets rounded up as a consequence on myosin II inhibition, indicating a substantial decrease in the ability of cells to generate forces.

  2. 2.

    Effect of actin polymerization inhibition on droplet deformations

    Time-lapse showing the effect of actin polymerization inhibition on droplet deformations. Actin polymerization was inhibited using cytochalasin D (see Online Methods: 'Perturbation of cellular forces with drugs'). The drug was added at t = 0. Droplets rounded up as a consequence on actin polymerization inhibition, indicating a substantial decrease in the ability of cells to generate forces.

  3. 3.

    Effect of cell disruption on droplet deformations

    Time-lapse showing the effect of cell disruption on droplet deformations. Cells were disrupted with the detergent sodium dodecyl sulfate (see Online Methods: 'Perturbation of cellular forces with drugs'). The drug was added at t = 0. Cell aggregates disassembled completely in the presence of the drug and droplets rounded up immediately as a consequence, indicating that cell-generated forces were causing the droplet deformations.

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DOI

https://doi.org/10.1038/nmeth.2761

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