Cortical stiffness is an important cellular property that changes during migration, adhesion and growth. Previous atomic force microscopy (AFM) indentation measurements of cells cultured on deformable substrates have suggested that cells adapt their stiffness to that of their surroundings. Here we show that the force applied by AFM to a cell results in a significant deformation of the underlying substrate if this substrate is softer than the cell. This ‘soft substrate effect’ leads to an underestimation of a cell’s elastic modulus when analysing data using a standard Hertz model, as confirmed by finite element modelling and AFM measurements of calibrated polyacrylamide beads, microglial cells and fibroblasts. To account for this substrate deformation, we developed a ‘composite cell–substrate model’. Correcting for the substrate indentation revealed that cortical cell stiffness is largely independent of substrate mechanics, which has major implications for our interpretation of many physiological and pathological processes.
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The data underlying this study are available from the authors upon reasonable request. The AFM force–distance curves raw data can be found at https://doi.org/10.6084/m9.figshare.10732415.
Codes used for processing of AFM and confocal laser scanning microscopy raw data can be found at https://github.com/FranzeLab/AFM-data-analysis-and-processing/tree/master/Cell%20stiffness. Comsol models can be found at https://doi.org/10.6084/m9.figshare.10731869.
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We thank P. Janmey, B. Fabry and U. Schwarz for critical discussions and comments on the manuscript, T. Schäffer for personal support and A. Winkel (JPK) for technical support, as well as J. Tavares and M. Kotter for microglial cells and B. Colledge for NIH 3T3 fibroblasts. We acknowledge funding from the German Science Foundation (DFG grant numbers RH 147/1-1 to J.R., EXC 1003 CiM to T.B.), the Herchel Smith Foundation (postdoctoral fellowship to A.D.), the Royal Society (University Research Fellowship to K.J.C.), the UK EPSRC (programme grant number EP/P030017/1 to M.C.G.), the Human Frontier Science Program (HFSP grant number RGP0018/2017 to T.B.), the European Research Council (consolidator grant numbers 772798 to K.J.C., 771201 to T.B., 647186 to G.C. and 772426 to K.F.), and the UK BBSRC (equipment grant number BB/R000042/1 to G.C. and research project grant number BB/N006402/1 to K.F.).
The authors declare no competing interests.
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Supplementary Figs. 1–10, discussion and refs. 51–56.
Indentation of a microglial cell cultured on a stiff substrate by AFM.
Indentation of a microglial cell cultured on a soft substrate by AFM.
Indentation of a fibroblast cultured on a stiff substrate by AFM.
Indentation of a fibroblast cultured on a soft substrate by AFM.
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Rheinlaender, J., Dimitracopoulos, A., Wallmeyer, B. et al. Cortical cell stiffness is independent of substrate mechanics. Nat. Mater. 19, 1019–1025 (2020). https://doi.org/10.1038/s41563-020-0684-x