Cultivation at a liquid–liquid interface prevents phenotypic heterogeneity of human induced pluripotent stem-derived retinal pigment epithelial cells

Culturing retinal pigment epithelial (RPE) cells, a valuable source for drug discovery and cell transplantation therapies, on a polystyrene solid interface often induces spontaneous phenotypic heterogeneity, including cobblestone-shaped, dome-shaped, and stratified cells within a passaged cell population. Understanding and regulating these phenotypic changes is essential for producing high-quality and safe cell sources. In this study, we developed a cultivation strategy to promote the uniform maturation of human induced pluripotent stem (hiPS)-derived RPE cells by focusing on their behavior in a culture vessel. hiPS-RPE cells cultured at the solid–liquid interface exhibited phenotypic heterogeneity, characterized by cobblestone, dome-shaped, and stratified morphologies, indicating RPE phenotype shifts associated with cellular senescence. However, replacing the Rho-associated coiled-coil kinase (ROCK) inhibitor Y27632 with forskolin, which enhances cell-cell and cell-substrate adhesion, facilitated uniform maturation of confluent hiPS-RPE cells on a laminin-332-coated liquid–liquid interface. Quantitative analysis revealed that the levels of tight junction formation, F_Z, and the homogeneity index, i.e., the degree of uniform cell distribution, H_LN, were consistent between the central and peripheral regions of the culture vessel (F_Z = 0.97, H_LN = 0.95). These findings highlight the importance of using a liquid–liquid interface to suppress spontaneous phenotypic heterogeneity by promoting uniform cell distribution. Our study presents a novel methodology for efficiently achieving uniform maturation of functional hiPS-RPE cells at the liquid–liquid interface within a culture vessel.