We report the establishment of a library of micromolded elastomeric micropost arrays to modulate substrate rigidity independently of effects on adhesive and other material surface properties. We demonstrate that micropost rigidity impacts cell morphology, focal adhesions, cytoskeletal contractility, and stem cell differentiation. Furthermore, early changes in cytoskeletal contractility predicted later stem cell fate decisions at the single cell level.Cell function is regulated primarily by extracellular stimuli, including soluble and adhesive factors that bind to cell surface receptors. Recent evidence suggests that mechanical properties of the extracellular matrix (ECM), particularly rigidity, can also mediate cell signaling, proliferation, differentiation, and migration 1,2 . Culturing cells on hydrogels derived from natural ECM proteins at different densities has dramatic effects on cell adhesion, morphology, and function 3 . However, changing densities of the gels impacts not only mechanical rigidity, but also the amount of ligand, leaving uncertainty as to the relevant contribution of these two matrix properties on the observed cellular response. Synthetic ECM analogs such as polyacrylamide or polyethylene glycol gels, which vary rigidity by modulating the amount of cross-linker, has revealed that substrate rigidity alone can modulate many cellular functions, including stem cell differentiation 4-6 . However, altered cross-linker amount impacts not only bulk mechanics, but also molecular-scale material properties including porosity, surface chemistry, backbone flexibility, and binding properties of immobilized adhesive ligands 7,8 . Consequently, whether cells transduce substrate rigidity at the microscopic scale (eg sensing the rigidity between adhesion sites) or the nanoscopic scale (eg sensing local alterations in receptor-ligand binding characteristics) remains an open question 7,8 . While hydrogels will continue to play a major role in characterizing and controlling cell-material interactions, alternative approaches are necessary to further elucidate the basis by which cells sense changes in substrate rigidity.

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Cells from many different tissues sense the stiffness and spatial patterning of their microenvironment to modulate their shape and cortical stiffness. It is currently unknown how substrate stiffness, cell shape, and cell stiffness modulate or interact with one another. Here, we use microcontact printing and microfabricated arrays of elastomeric posts to independently and simultaneously control cell shape and substrate stiffness. Our experiments show that cell cortical stiffness increases as a function of both substrate stiffness and spread area. For soft substrates, the influence of substrate stiffness on cell cortical stiffness is more prominent than that of cell shape, since increasing adherent area does not lead to cell stiffening. On the other hand, for cells constrained to a small area, cell shape effects are more dominant than substrate stiffness, since increasing substrate stiffness no longer affects cell stiffness. These results suggest that cell size and substrate stiffness can interact in a complex fashion to either enhance or antagonize each other's effect on cell morphology and mechanics.