The research on stem cell cultures has attracted much attention due to the recent development of regenerative medicine. Therefore, higher functionalities for devices used for culturing cells are strongly demanded. In this study, we fabricated cell culture sheets using transparent polyimide (PI), parylene (PA), and polyetheretherketone (PEEK) to make polymer materials that had microstructures. We then cultured stromal marrow cells (OP9) on them and investigated the cell alignment within the microstructures. Hot embossing was used to fabricate the microstructures with a width and depth of 5 μm on the polymer substrates. Cultivation of the cells was confirmed on the transparent PI and PA sheets, however, it was not observed on the PEEK sheet. Slight alignment of the cells was also observed along with the microstructures.