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Living cells sense and respond to their extracellular environment. Their contact guidance is affected by the underlying substrate morphology. Previous studies of the effect of substrate pattern on the mechanical behavior of living cells were only limited to the quantification of the cellular elasticity. However, how the length and time scales of the cellular mechanical properties are affected by the patterned substrates have yet to be studied. In this study, the effect of the substrate morphology on the biomechanical behavior of living cells was thoroughly investigated using indentation-based atomic force microscopy. The results showed that the cellular biomechanical behavior was affected by the substrate morphology significantly. The elasticity and viscosity of the cells on the patterned PDMS substrates were much lower compared to those cultured on flat PDMS. The poroelastic diffusion coefficient of the cells was higher on the patterned PDMS substrates, specifically on the substrate with 2D pitches. In addition, fluorescence images showed that the substrate topography directly affects the cell cytoskeleton morphology. Together, the results suggested that cell mechanical behavior and morphology can be controlled using substrates with properly designed topography. 

Cell–substrate interaction plays an important role in intracellular behavior and function. Adherent cell mechanics is directly regulated by the substrate mechanics. However, previous studies on the effect of substrate mechanics only focused on the stiffness relation between the substrate and the cells, and how the substrate stiffness affects the time-scale and length-scale of the cell mechanics has not yet been studied. The absence of this information directly limits the in-depth understanding of the cellular mechanotransduction process. In this study, the effect of substrate mechanics on the nonlinear biomechanical behavior of living cells was investigated using indentation-based atomic force microscopy. The mechanical properties and their nonlinearities of the cells cultured on four substrates with distinct mechanical properties were thoroughly investigated. Furthermore, the actin filament (F-actin) cytoskeleton of the cells was fluorescently stained to investigate the adaptation of F-actin cytoskeleton structure to the substrate mechanics. It was found that living cells sense and adapt to substrate mechanics: the cellular Young’s modulus, shear modulus, apparent viscosity, and their nonlinearities (mechanical property vs. measurement depth relation) were adapted to the substrates’ nonlinear mechanics. Moreover, the positive correlation between the cellular poroelasticity and the indentation remained the same regardless of the substrate stiffness nonlinearity, but was indeed more pronounced for the cells seeded on the softer substrates. Comparison of the F-actin cytoskeleton morphology confirmed that the substrate affects the cell mechanics by regulating the intracellular structure.