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In this work, the influence of parylene N film on the spheroid formation of osteoblast-like cells (MG-63) was determined and compared with that of high-hydrophilicity microenvironments, such as hydrophilic culture matrix and ultraviolet-treated parylene N film. To elucidate the change in cell properties due to the microenvironment of parylene N film, global gene expression profiles of MG-63 cells on parylene N film were analyzed. We confirmed the upregulated expression of osteoblast differentiation- and proliferation-related genes, such as Runx2, ALPL, and BGLAP and MKi67 and PCNA, respectively, using the real-time polymerase chain reaction. In addition, the differentiation and proliferation of osteoblast cells cultured on parylene N film were validated using immunostaining. Finally, the formation of spheroids and regulation of differentiation in human mesenchymal stem cells (MSCs) on parylene N film was …