Cortical cell stiffness is independent of substrate mechanics

Cell stiffness is a key cellular material property that changes locally and temporally during many cellular functions including migration, adhesion, and growth. Currently, it is widely accepted that cells adapt their mechanical properties to the stiffness of their surroundings. The link between cortical cell stiffness and substrate mechanics was hypothesized based on atomic force microscopy (AFM) indentation measurements of cells cultured on deformable substrates. Here we show that the force applied by AFM can result in a significant deformation not only of the cell surface but also of the underlying substrate if it is sufficiently soft. This 9soft substrate effect9 leads to an underestimation of a cell9s elastic modulus on substrates softer than the cells when fitting the indentation data using a standard Hertz model, as confirmed by finite element modelling (FEM) and AFM measurements of calibrated polyacrylamide beads, microglial cells, and fibroblasts. To account for this substrate deformation, we developed the 9composite cell-substrate model9 (9CoCS9 model), which does not require any knowledge about the cell-substrate geometry, and which can be implemented in any standard AFM indentation measurement. Our results provide a new formalism to analyze indentation data obtained for cells cultured on soft matrices, and they suggest that cortical cell stiffness is largely independent of substrate mechanics, which has significant implications for our interpretation of many physiological and pathological processes.