Figure 1: Cultivation of KY sheet for 1 year.

(a) Epithelial cells from same donor were cultured without DEQ (upper panels) or organotypic cultured with DEQ (lower panels). Digital camera photographs (left panels) and invert microscopy using Objx20 (right panels). (b) Influence of air lift culture. KY sheets from same donor were submerged for 3 months or cultured at air/liquid interface for last 1 month. (c) Morphology of KY sheet cultured for 1 year as submerged. Arrows indicate desquamated cells floating on the superficial layer. (d) Basal cell density (left panel, n = 4–6) and number of desquamated cells (right panel, n = 6–22) at each time point. (e) Fluorescent microscopy of primary culture using corneal and limbal epithelium-related markers. Culture periods are indicated in each panel. PAX6, TJP1, and KRT12 are shown in red and KRT3, TP63, and KRT15 are shown in green. Nuclei were counterstained with DAPI shown in blue. White arrows indicate the linear expression of TJP1 between superficial cells. (f) Number of cell layers (n = 4–7), ratio of total cell numbers to basal cell numbers (n = 4–8), and turnover rate (n = 6–22) at each time points. (g) Colony formation of epithelial cells dissociated from 1 year culture. Colonies in ϕ100 mm indicator dishes were stained with Rhodamin B (red). Error bars indicate S.D.