At day 21 from the start of the floating 30 culture, the กลุ่มก้อนs were dispersed in cell dispersion solution Accutase (ICT), and debris was removed using a 40 µm cell strainer .. The resulting cells were suspended in a serum• free อาหาร (200 µl), which was a 1:1 mixture of DMEM/F-12 อาหาร and Neurobasal อาหาร added with 10% KSR, 1/2 N2 35 supplement, 1/2 B27 supplement, 20 µM Y27632, and seeded in a 65 mm Boyden chamber (Transwell, Corning Incorporated) after a coating treatment with Synthemax™ at 2x105 cells per well, a อาหาร (1 ml) having the same composition was also added to a lower well, and การเพาะเลี้ยงแบบยึดเกาะ was performed at 37°C, 5% C02• s The อาหาร was exchanged every 3 days, and the level of epithelial formation was observed under a microscope at day 40 from the start of the การเพาะเลี้ยงแบบลอย. By day 40 from the start of the การเพาะเลี้ยงแบบลอย, a uniform retinal pigment epithelial cell sheet having a 10 characteristic polygonal cell form (Fig. 48) with pigment accumulation was formed (Fig. 4A).