摘要： PURPOSE. Retinal pigment epithelium (RPE) and neural retina could be generated concurrently through retinal organoid induction approaches using human induced pluripotent stem cells (hiPSCs), providing valuable sources for cell therapy of retinal degenerations. This study aims to enrich and expand hiPSC-RPE acquired with this platform and explore characteristics of serially passaged RPE cells. METHODS. RPE has been differentiated from hiPSCs with a published retinal organoid induction method. After detachment of neural retina on the 4th week, the remaining mixture was scraped from the dish and subjected to suspension culture for the formation of RPE spheroids. RPE sheets were isolated and digested for expansion. The cellular, molecular, and functional features of expanded RPE cells were evaluated by different assays. RESULTS. Under suspension culture, hiPSC-RPE spheroids with pigmentation self-formed were readily enriched by removing the non-retinal tissues. RPE sheets were further dissected and purified from the spheroids. The individualized RPE cells could be passaged every week for at least 5 times in serum medium, yielding large numbers of cells with high quality in a short period. In addition, when switched to a serum-free medium, the passaged RPE cells could mature in cellular, molecular, and physiological levels, including repigmentation, markers expression, and phagocytosis. CONCLUSIONS. We developed a simple and novel RPE spheroids formation approach to enrich and expand hiPSC-RPE cells generated along with retinal neurons on a universal retinal organoid induction platform. This achievement will reduce the cost and time in producing retinal cells for basic and translational researches, in particular for retinal cell therapy.