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Investigation of the interactions between cells and material surfaces is important not only for the understanding of cell biology but also for the development of smart biomaterials. In this study, we investigated the substrate-related effects on the interaction between cell and polymeric ultrathin film (nanosheet) by modulating the mechanical properties of the nanosheet with a metal substrate or mesh. A freestanding polymeric nanosheet with tens-of-nanometers thickness composed of poly(l-lactic acid) (PLLA nanosheet) was fabricated by combination of a spin-coating technique and a water-soluble sacrificial layer. The freestanding PLLA nanosheet was collected on a stainless steel mesh (PLLA–mesh) and subsequently used for cell adhesion studies, comparing the results to the ones on a control SiO2substrate coated with an ultrathin layer of PLLA (PLLA–substrate). The adhesion of rat cardiomyocytes (H9c2) was evaluated on both samples after 24 h of culture. The PLLA–mesh with the tens-of-nanometers thick nanosheets induced an anisotropic adhesion of H9c2, while H9c2 on the PLLA–substrate showed an isotropic adhesion independent from the nanosheet thickness. Interestingly, an increment in the nanosheet thickness in the PLLA–mesh samples reduced the cellular anisotropy and led to a similar morphology to the PLLA–substrate. Considering the huge discrepancy of Young’s modulus between PLLA nanosheet (3.5–4.2 GPa) and metal substrate (hundreds of GPa), cell adhesion was mechanically regulated by the Young’s modulus of the underlying substrate when the thickness of the PLLA nanosheet was tens of nanometers. Modulation of the stiffness of the polymeric nanosheet by utilizing a rigid underlying material will allow the constitution of a unique cell culture environment. [ABSTRACT FROM AUTHOR]