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, FZ, and the homogeneity index, i.e., the degree of uniform cell distribution, HLN, were consistent between the central and peripheral regions of the culture vessel (FZ = 0.97, HLN = 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.
扫码关注我们
求助内容:
应助结果提醒方式:
