Understanding of the fundamental mechanisms that govern tight junction formation of retinal pigment epithelial (RPE) cells provides surface design strategies for promoting their maturation in culture. RPE cells were cultured to investigate their migratory behavior and the expression of tight junction protein ZO-1 in the central and peripheral regions of a culture vessel. Regardless of locational differences in the culture vessel, the cells at day 1 were elongated in shape, did not form tight junctions, and migrated actively. As the culture progressed, the cells in the central region slowly moved with morphological change of a cobblestone-like shape via interaction between contact cells and exhibiting the shift from random migration to collective movement toward the center, accompanied by tight junction formation. On the other hand, the cells in the peripheral region maintained the random migration at day 5, meaning spatial heterogeneity in maturation in the vessel. At day 5, RPE cells were incubated in medium with Rac1 inhibitor and the exposure to the Rac1 inhibitor triggered the rapid conversion of migratory behavior from random migration to collective movement toward the center of the vessel, resulting in uniform maturation. These findings indicate that the change in migratory patterns is an important cues and the collective movement toward the center causes the facilitation of uniform maturation in the vessel. (C) 2015, The Society for Biotechnology, Japan. All rights reserved.