Elastomeric sensor surfaces for high-throughput single-cell force cytometry

Abstract

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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Fig. 1: Operational principles of the general-use FLECS force cytometer.
Fig. 2: Whole-cell contractility resolves contractile changes with differentiation and drug treatment.
Fig. 3: Parallel study of fatal asthma and non-asthma patient-derived airway SMCs.
Fig. 4: Collective comparisons of all asthma versus non-asthma HASM cells.
Fig. 5: Simultaneous measurements of calcium release and contractility in patient-derived HASM single cells.
Fig. 6: Measuring phagocytic forces generated by individual human macrophages.
Fig. 7: Effects of chloroquine, cytochalasin D and CAL-101 on hMDM contractile force.

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.

References

  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).

    CAS  Article  PubMed  Google Scholar 

  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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  3. 3.

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

    CAS  Article  PubMed  Google Scholar 

  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).

    CAS  Article  PubMed  Google Scholar 

  5. 5.

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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  6. 6.

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

    Article  PubMed  Google Scholar 

  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).

    CAS  Article  PubMed  Google Scholar 

  8. 8.

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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  9. 9.

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

    Article  PubMed Central  Google Scholar 

  10. 10.

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

    Article  PubMed  PubMed Central  Google Scholar 

  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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  12. 12.

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

    Article  Google Scholar 

  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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  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).

    CAS  Article  PubMed  Google Scholar 

  17. 17.

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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. 18.

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

    CAS  Article  PubMed  Google Scholar 

  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).

    CAS  Article  PubMed  Google Scholar 

  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).

    CAS  Article  PubMed  Google Scholar 

  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).

    Article  PubMed  Google Scholar 

  22. 22.

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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  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).

    CAS  Article  PubMed  Google Scholar 

  25. 25.

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

    CAS  Article  PubMed  Google Scholar 

  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).

    Article  PubMed  PubMed Central  Google Scholar 

  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).

    Article  PubMed  PubMed Central  Google Scholar 

  28. 28.

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

    Article  PubMed  PubMed Central  Google Scholar 

  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).

    Article  PubMed  PubMed Central  Google Scholar 

  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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  31. 31.

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

    CAS  Article  PubMed  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  36. 36.

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

    CAS  Article  PubMed  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  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).

    CAS  Article  PubMed  Google Scholar 

  41. 41.

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

    CAS  PubMed  Google Scholar 

  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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  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).

    CAS  Article  PubMed  Google Scholar 

  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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  45. 45.

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

    CAS  Article  PubMed  Google Scholar 

  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).

    Article  PubMed  PubMed Central  Google Scholar 

  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).

    CAS  Article  PubMed  Google Scholar 

  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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  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).

    CAS  Article  PubMed  Google Scholar 

  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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  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).

    CAS  Article  PubMed  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  53. 53.

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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  54. 54.

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

    CAS  Article  PubMed  Google Scholar 

  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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  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).

    Article  PubMed  PubMed Central  Google Scholar 

  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).

    CAS  Article  PubMed  Google Scholar 

  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).

    Google Scholar 

  61. 61.

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

    Article  Google Scholar 

  62. 62.

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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  63. 63.

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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  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).

    CAS  Article  PubMed  Google Scholar 

  65. 65.

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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

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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Contributions

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.

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Correspondence to Dino Di Carlo.

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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.

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

Supplementary information

Supplementary Information

Supplementary figures, tables and video captions.

Life Sciences Reporting Summary

Supplementary Text

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

Videos

Supplementary Video 1

Fibronectin–fibrinogen patterns created using our sacrificial dextran method.

Supplementary Video 2

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

Supplementary Video 3

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

Supplementary Video 4

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

Supplementary Video 5

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

Supplementary Video 6

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

Supplementary Video 7

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

Supplementary Video 8

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

Supplementary Video 9

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

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.

Supplementary Video 11

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

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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Pushkarsky, I., Tseng, P., Black, D. et al. Elastomeric sensor surfaces for high-throughput single-cell force cytometry. Nat Biomed Eng 2, 124–137 (2018). https://doi.org/10.1038/s41551-018-0193-2

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