As cells with aberrant force-generating phenotypes can directly lead to disease, cellular force-generation mechanisms are high-value targets for new therapies. Here, we show that single-cell force sensors embedded in elastomers enable single-cell force measurements with ~100-fold improvement in throughput than was previously possible. The microtechnology is scalable and seamlessly integrates with the multi-well plate format, enabling highly parallelized time-course studies. In this regard, we show that airway smooth muscle cells isolated from fatally asthmatic patients have innately greater and faster force-generation capacity in response to stimulation than healthy control cells. By simultaneously tracing agonist-induced calcium flux and contractility in the same cell, we show that the calcium level is ultimately a poor quantitative predictor of cellular force generation. Finally, by quantifying phagocytic forces in thousands of individual human macrophages, we show that force initiation is a digital response (rather than a proportional one) to the proper immunogen. By combining mechanobiology at the single-cell level with high-throughput capabilities, this microtechnology can support drug-discovery efforts for clinical conditions associated with aberrant cellular force generation.

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A correction to this article is available online at https://doi.org/10.1038/s41551-018-0207-0.

Change history

  • 12 March 2018

    In the version of this Article originally published, in Fig. 1a, all cells in the top schematic were missing, and in the bottom-left schematic showing multiple pattern shapes, two cells were missing in the bottom-right corner. This figure has now been updated in all versions of the Article.


  1. 1.

    Discher, D. E., Janmey, P. & Wang, Y.-L. Tissue cells feel and respond to the stiffness of their substrate. Science 310, 1139–1143 (2005).

  2. 2.

    Fournier, M. F., Sauser, R., Ambrosi, D., Meister, J.-J. & Verkhovsky, A. B. Force transmission in migrating cells. J. Cell Biol. 188, 287–297 (2010).

  3. 3.

    Burton, K. & Taylor, D. L. Traction forces of cytokinesis measured with optically modified elastic substrata. Nature 385, 450–454 (1997).

  4. 4.

    Evans, E., Leung, A. & Zhelev, D. Synchrony of cell spreading and contraction force as phagocytes engulf large pathogens. J. Cell Biol. 122, 1295–1300 (1993).

  5. 5.

    Hall, C. N. et al. Capillary pericytes regulate cerebral blood flow in health and disease. Nature 508, 55–60 (2014).

  6. 6.

    Pelaia, G. et al. Molecular mechanisms underlying airway smooth muscle contraction and proliferation: implications for asthma. Respir. Med. 102, 1173–1181 (2008).

  7. 7.

    Yemisci, M. et al. Pericyte contraction induced by oxidative-nitrative stress impairs capillary reflow despite successful opening of an occluded cerebral artery. Nat. Med. 15, 1031–1037 (2009).

  8. 8.

    Huang, X. et al. Relaxin regulates myofibroblast contractility and protects against lung fibrosis. Am. J. Pathol. 179, 2751–2765 (2011).

  9. 9.

    Valencia, A. M. J. et al. Collective cancer cell invasion induced by coordinated contractile stresses. Oncotarget 6, 43438–43451 (2015).

  10. 10.

    Gupta, S., Nahas, S. J. & Peterlin, B. L. Chemical mediators of migraine: preclinical and clinical observations. Headache 51, 1029–1045 (2011).

  11. 11.

    Munevar, S., Wang, Y. & Dembo, M. Traction force microscopy of migrating normal and H-ras transformed 3T3 fibroblasts. Biophys. J. 80, 1744–1757 (2001).

  12. 12.

    Park, C. Y. et al. High-throughput screening for modulators of cellular contractile force. Integr. Biol. (Camb.) 7, 1318–1324 (2015).

  13. 13.

    Tan, J. L. et al. Cells lying on a bed of microneedles: an approach to isolate mechanical force. Proc. Natl Acad. Sci. USA 100, 1484–1489 (2003).

  14. 14.

    Oakes, P. W., Banerjee, S., Marchetti, M. C. & Gardel, M. L. Geometry regulates traction stresses in adherent cells. Biophys. J. 107, 825–833 (2014).

  15. 15.

    Ricart, B. G., Yang, M. T., Hunter, C. A., Chen, C. S. & Hammer, D. A. Measuring traction forces of motile dendritic cells on micropost arrays. Biophys. J. 101, 2620–2628 (2011).

  16. 16.

    Legant, W. R. et al. Multidimensional traction force microscopy reveals out-of-plane rotational moments about focal adhesions. Proc. Natl Acad. Sci. USA 110, 881–886 (2013).

  17. 17.

    Polacheck, W. J. & Chen, C. S. Measuring cell-generated forces: a guide to the available tools. Nat. Methods 13, 415–423 (2016).

  18. 18.

    Myers, D. R. et al. Single-platelet nanomechanics measured by high-throughput cytometry. Nat. Mater. 16, 230–235 (2017).

  19. 19.

    Tseng, Q. et al. A new micropatterning method of soft substrates reveals that different tumorigenic signals can promote or reduce cell contraction levels. Lab Chip 11, 2231–2240 (2011).

  20. 20.

    Rape, A., Guo, W. & Wang, Y. The regulation of traction force in relation to cell shape and focal adhesions. Biomaterials 32, 2043–2051 (2011).

  21. 21.

    Wang, N., Ostuni, E., Whitesides, G. M. & Ingber, D. E. Micropatterning tractional forces in living cells. Cell Motil. Cytoskeleton 52, 97–106 (2002).

  22. 22.

    Fu, J. et al. Mechanical regulation of cell function with geometrically modulated elastomeric substrates. Nat. Methods 7, 733–736 (2010).

  23. 23.

    Kim, H. R., Appel, S., Vetterkind, S., Gangopadhyay, S. S. & Morgan, K. G. Smooth muscle signalling pathways in health and disease. J. Cell. Mol. Med. 12, 2165–2180 (2008).

  24. 24.

    Limouze, J., Straight, A. F., Mitchison, T. & Sellers, J. R.Specificity of blebbistatin, an inhibitor of myosin II. J. Muscle Res. Cell Motil. 25, 337–341 (2004).

  25. 25.

    Straight, A. F. et al. Dissecting temporal and spatial control of cytokinesis with a myosin II inhibitor. Science 299, 1743–1747 (2003).

  26. 26.

    An, S. S. et al. An inflammation-independent contraction mechanophenotype of airway smooth muscle in asthma. J. Allergy Clin. Immunol. 138, 294–297.e4 (2016).

  27. 27.

    Herington, J. L. et al. High-throughput screening of myometrial calcium-mobilization to identify modulators of uterine contractility. PLoS ONE 10, e0143243 (2015).

  28. 28.

    Wertek, F. & Xu, C. Digital response in T cells: to be or not to be. Cell Res. 24, 265–266 (2014).

  29. 29.

    McNally, A. K., Jones, J. A., Macewan, S. R., Colton, E. & Anderson, J. M. Vitronectin is a critical protein adhesion substrate for IL-4-induced foreign body giant cell formation. J. Biomed. Mater. Res. A 86, 535–543 (2008).

  30. 30.

    Labernadie, A., Thibault, C., Vieu, C., Maridonneau-Parini, I. & Charrière, G. M. Dynamics of podosome stiffness revealed by atomic force microscopy. Proc. Natl Acad. Sci. USA 107, 21016–21021 (2010).

  31. 31.

    Gordon, S. & Taylor, P. R. Monocyte and macrophage heterogeneity. Nat. Rev. Immunol. 5, 953–964 (2005).

  32. 32.

    Soon, C. F., Tee, K. S., Youseffi, M. & Denyer, M. C. T. Tracking traction force changes of single cells on the liquid crystal surface. Biosensors (Basel) 5, 13–24 (2015).

  33. 33.

    Tsang, P. H., Li, G., Brun, Y. V., Freund, L. B. & Tang, J. X. Adhesion of single bacterial cells in the micronewton range. Proc. Natl Acad. Sci. USA 103, 5764–5768 (2006).

  34. 34.

    Hind, L. E., Dembo, M. & Hammer, D. A. Macrophage motility is driven by frontal-towing with a force magnitude dependent on substrate stiffness. Integr. Biol. (Camb.) 7, 447–453 (2015).

  35. 35.

    Goh, Y. S. et al. Human IgG isotypes and activating Fcγ receptors in the interaction of Salmonella enterica serovar Typhimurium with phagocytic cells. Immunology 133, 74–83 (2011).

  36. 36.

    Kaplan, G. Differences in the mode of phagocytosis with Fc and C3 receptors in macrophages. Scand. J. Immunol. 6, 797–807 (1977).

  37. 37.

    Hackam, D. J., Rotstein, O. D. & Grinstein, S.Phagosomal acidification mechanisms and functional significance. Adv. Cell. Mol. Biol. Membr. Organelles 5, 299–319 (1999).

  38. 38.

    Schlam, D. et al. Phosphoinositide 3-kinase enables phagocytosis of large particles by terminating actin assembly through Rac/Cdc42 GTPase-activating proteins. Nat. Commun. 6, 8623 (2015).

  39. 39.

    Beemiller, P. et al. A Cdc42 activation cycle coordinated by PI 3-kinase during Fc receptor-mediated phagocytosis. Mol. Biol. Cell 21, 470–480 (2010).

  40. 40.

    Papakonstanti, E. A. et al. Distinct roles of class IA PI3K isoforms in primary and immortalised macrophages. J. Cell Sci. 121, 4124–4133 (2008).

  41. 41.

    Castellano, F., Montcourrier, P. & Chavrier, P. Membrane recruitment of Rac1 triggers phagocytosis. J. Cell Sci. 113, 2955–2961 (2000).

  42. 42.

    Massol, P., Montcourrier, P., Guillemot, J.-C. & Chavrier, P. Fc receptor†mediated phagocytosis requires CDC42 and Rac1. EMBO J. 17, 6219–6229 (1998).

  43. 43.

    Ganesan, L. P. et al. The serine/threonine kinase Akt promotes Fcγ receptor-mediated phagocytosis in murine macrophages through the activation of p70S6 kinase. J. Biol. Chem. 279, 54416–54425 (2004).

  44. 44.

    Papakonstanti, E. A., Ridley, A. J. & Vanhaesebroeck, B. The p110δ isoform of PI 3-kinase negatively controls RhoA and PTEN. EMBO J. 26, 3050–3061 (2007).

  45. 45.

    Swinney, D. C. Phenotypic vs. target-based drug discovery for first-in-class medicines. Clin. Pharmacol. Ther. 93, 299–301 (2013).

  46. 46.

    Tseng, P., Pushkarsky, I. & Carlo, D. D. Metallization and biopatterning on ultra-flexible substrates via dextran sacrificial layers. PLoS ONE 9, e106091 (2014).

  47. 47.

    Panettieri, R. A., Murray, R. K., DePalo, L. R., Yadvish, P. A. & Kotlikoff, M. I. A human airway smooth muscle cell line that retains physiological responsiveness. Am. J. Physiol. 256, C329–C335 (1989).

  48. 48.

    Koziol-White, C. J. et al. Inhibition of PI3K promotes dilation of human small airways in a rho kinase-dependent manner. Br. J. Pharmacol. 173, 2726–2738 (2016).

  49. 49.

    Yoo, E. J. et al. Gα12 facilitates carbachol-induced shortening in human airway smooth muscle by modulating phosphoinositide 3-kinase-mediated activation in a RhoA-dependent manner. Br. J. Pharmacol. 174, 4383–4395 (2017).

  50. 50.

    Morrison, S. L., Johnson, M. J., Herzenberg, L. A. & Oi, V. T. Chimeric human antibody molecules: mouse antigen-binding domains with human constant region domains. Proc. Natl Acad. Sci. USA 81, 6851–6855 (1984).

  51. 51.

    Zhuang, P. et al. Characterization of the denaturation and renaturation of human plasma vitronectin II. Investigation into the mechanism of formation of multimers. J. Biol. Chem. 271, 14333–14343 (1996).

  52. 52.

    Vanlandingham, M. R., Chang, N.-K., Drzal, P. L., White, C. C. & Chang, S.-H. Viscoelastic characterization of polymers using instrumented indentation. I. Quasi-static testing. J. Polym. Sci. B Polym. Phys. 43, 1794–1811 (2005).

  53. 53.

    Tandon, N. et al. Electrical stimulation systems for cardiac tissue engineering. Nat. Protoc. 4, 155–173 (2009).

  54. 54.

    Beussman, K. M. et al. Micropost arrays for measuring stem cell-derived cardiomyocyte contractility. Methods 94, 43–50 (2016).

  55. 55.

    Cheng, Q., Sun, Z., Meininger, G. & Almasri, M. PDMS elastic micropost arrays for studying vascular smooth muscle cells. Sens. Actuators B Chem. 188, 1055–1063 (2013).

  56. 56.

    Munevar, S., Wang, Y. & Dembo, M. Traction force microscopy of migrating normal and H-ras transformed 3T3 fibroblasts. Biophys. J. 80, 1744–1757 (2001).

  57. 57.

    Wu, H. et al. Epigenetic regulation of phosphodiesterases 2A and 3A underlies compromised β-adrenergic signaling in an iPSC model of dilated cardiomyopathy. Cell Stem Cell 17, 89–100 (2015).

  58. 58.

    Del Álamo, J. C. et al. Three-dimensional quantification of cellular traction forces and mechanosensing of thin substrata by Fourier traction force microscopy. PLoS ONE 8, e69850 (2013).

  59. 59.

    Plotnikov, S. V., Pasapera, A. M., Sabass, B. & Waterman, C. M. Force fluctuations within focal adhesions mediate ECM-rigidity sensing to guide directed cell migration. Cell 151, 1513–1527 (2012).

  60. 60.

    Goedecke, N., Bollhalder, M., Bernet, R., Silvan, U. & Snedeker, J.Easy and accurate mechano-profiling on micropost arrays. J. Vis. Exp. 2015, e53350 (2015).

  61. 61.

    Tolić-Nørrelykke, I. M. & Wang, N. Traction in smooth muscle cells varies with cell spreading. J. Biomech. 38, 1405–1412 (2005).

  62. 62.

    Liu, K. et al. Improved-throughput traction microscopy based on fluorescence micropattern for manual microscopy. PLoS ONE 8, e70122 (2013).

  63. 63.

    Colin-York, H. et al. Super-resolved traction force microscopy (STFM). Nano Lett. 16, 2633–2638 (2016).

  64. 64.

    Murrell, M., Oakes, P. W., Lenz, M. & Gardel, M. L. Forcing cells into shape: the mechanics of actomyosin contractility. Nat. Rev. Mol. Cell Biol. 16, 486–498 (2015).

  65. 65.

    Basu, R. et al. Cytotoxic T cells use mechanical force to potentiate target cell killing. Cell 165, 100–110 (2016).

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The work was supported by the National Institutes of Health Director’s New Innovator Award 1DP2OD007113, the David and Lucile Packard Fellowship and the National Institutes of Health/National Institute of Biomedical Imaging and Bioengineering R21 Award 1R21EB024081-01. The authors thank C. Walthers for providing the primary mouse intestinal SMCs used in the Supplementary Videos, Y. Wang and J. Z. Lee for providing the neonatal rat ventricular myocytes, and O. Adeyiga for performing bloods draws over the course of six months to facilitate primary macrophage culture. All microfabrication steps were completed using equipment provided by the Integrated Systems Nanofabrication Cleanroom at the California NanoSystems Institute at the University of California, Los Angeles.

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Author notes

  1. These authors contributed equally: Ivan Pushkarsky and Peter Tseng.


  1. Department of Bioengineering, University of California, Los Angeles, Los Angeles, CA, USA

    • Ivan Pushkarsky
    • , Peter Tseng
    • , Dylan Black
    • , Lyndon Warfe
    • , Jonathan Lin
    •  & Dino Di Carlo
  2. Department of Electrical Engineering and Computer Science, University of California, Los Angeles, Irvine, CA, USA

    • Peter Tseng
  3. California NanoSystems Institute, University of California, Los Angeles, Los Angeles, CA, USA

    • Bryan France
    • , Robert Damoiseaux
    •  & Dino Di Carlo
  4. Rutgers Institute for Translational Medicine and Science, Child Health Institute, Rutgers University, New Brunswick, NJ, USA

    • Cynthia J. Koziol-White
    • , William F. Jester Jr
    •  & Reynold A. Panettieri Jr
  5. Department of Microbiology, Immunology and Molecular Genetics and The Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA, USA

    • Ryan K. Trinh
    •  & Sherie L. Morrison
  6. Division of Dermatology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA

    • Philip O. Scumpia
  7. Department of Molecular and Medicinal Pharmacology, University of California, Los Angeles, Los Angeles, CA, USA

    • Robert Damoiseaux
  8. Department of Mechanical Engineering, University of California, Los Angeles, Los Angeles, CA, USA

    • Dino Di Carlo


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P.T., D.D.C. and I.P. conceived the methods. I.P., R.D., P.O.S., S.L.M., R.A.P., C.J.K.-W. and D.D.C. designed the experiments. I.P. performed all the experiments, developed the multi-well embodiment, optimized the protocols and wrote the image analysis software. D.B. assisted with the substrate preparation and macrophage differentiation procedures. L.W. assisted with the substrate preparation. R.K.T. maintained the chimeric antibody stocks. S.L.M. supplied all the chimeric antibodies. J.L. constructed the finite element method model. R.D. supplied the high-throughput screening (HTS) equipment for dose-response experiments and provided extensive guidance and technical advice on HTS procedures and developing the multi-well plate embodiment. B.F. assisted with the HTS equipment and drug administration. W.F.J. maintained the donor HASM cells. P.O.S. performed the macrophage and dendritic cells differentiation and advised the experimental procedures. I.P., R.D., P.O.S., S.L.M., R.A.P., C.J.K.-W. and D.D.C. interpreted the results. I.P. and D.D.C. wrote the manuscript. R.D., P.O.S., S.L.M., R.A.P. and C.J.K.-W. helped revise the manuscript.

Competing interests

I.P., P.T. and D.D.C. are named inventors on a patent application by the University of California, Los Angeles that covers the technology described in this study. I.P., R.D. and D.D.C. have a financial interest in Forcyte Biotechnologies, which aims to commercialize FLECS technology.

Corresponding author

Correspondence to Dino Di Carlo.

Supplementary information

  1. Supplementary Information

    Supplementary figures, tables and video captions.

  2. Life Sciences Reporting Summary

  3. Supplementary Text

    MATLAB computer code used to evaluate micropattern displacements and calcium dye intensity.


  1. Supplementary Video 1

    Fibronectin–fibrinogen patterns created using our sacrificial dextran method.

  2. Supplementary Video 2

    A second example of fibronectin–fibrinogen patterns created using the sacrificial dextran method.

  3. Supplementary Video 3

    Patterns contracted by adhered HeLa cells relax after the addition of the myosin inhibitor blebbistatin. Time-lapsed video.

  4. Supplementary Video 4

    HASM cells treated with 1 μM bradykinin contract significantly beyond tonic levels within 30 minutes.

  5. Supplementary Video 5

    HASM cells treated with 100 nM endothelin-1 contract significantly beyond tonic levels within 30 minutes.

  6. Supplementary Video 6

    Visualization of calcium flux within HASM cells seeded on arrays of FLECS micropatterns after the addition of Hist.

  7. Supplementary Video 7

    Time-lapsed video of human monocyte-derived macrophages engaged in frustrated phagocytosis of an IgG-opsonized patterned surface.

  8. Supplementary Video 8

    Representative video (real time) of a phasically contracting neonatal rat ventricular myocyte.

  9. Supplementary Video 9

    Representative video (real time) of a pattern phasically contracted by an adhered and spontaneously beating neonatal rat ventricular myocyte.

  10. Supplementary Video 10

    Representative video (real time) of a pattern phasically contracted by an adhered and beating neonatal rat ventricular myocyte paced with pulsed electric fields at frequencies of 1 Hz and 2 Hz.

  11. Supplementary Video 11

    Fibronectin–fibrinogen patterns created using adsorption rather than our sacrificial dextran method. Time-lapse video.

  12. Supplementary Video 12

    Second example of fibronectin–fibrinogen patterns created using adsorption rather than our sacrificial dextran method. Time-lapse video.

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