Fig 2: 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 ?100 mm indicator dishes were stained with Rhodamin B (red). Error bars indicate S.D.

Fig 3: Corneal stromal SLATEs as substrates for human corneal epithelial cell growth. Epithelial cells isolated from human corneas were grown on A- and R-SLATEs for 2 weeks with Cnt-7 medium, followed by an additional 2 weeks period with SHEM in air-lifting conditions, and then imaged by immunofluorescence confocal microscopy. Expression of A) CK3 and collagen-IV, B) CK15 and ABCG2, C) ?Np63 and ß1-integrins, D) YAP and ZO-1, or E) laminin and collagen-VII was analyzed by tridimensional reconstruction of a 300 × 300 µm area (large panels) and corresponding cross-section views of projected composites (small panels). Cell nuclei were identified by DAPI staining. F) Signal specificity was evaluated using secondary antibody-only controls.

Fig 4: Postoperative evaluation at days 60 and 180 in a rabbit model of lamellar keratoplasty. A) Images of cornea implants taken by slit lamp after operation at days 60 and 180 in a rabbit keratectomy model (FL means fluorescein staining). B) Typical hematoxylin and eosin (H&E) staining in the control wound group, convex group, and normal group on days 60 and 180 postsurgery. The black marked parts are defects and the blue parts are magnified. The scale bars of the upper images are 200 µm, and the scale bars of the magnified images are 50 µm. C,D) Thickness of the total and the epithelium layer of cornea at 60/180 days postsurgery obtained from the H&E image. E) Immunofluorescent staining in all groups at days 60 and 180 postsurgery including CK3 (green), Col-V (green), ZO-1 (red), vinculin (red), and a-SMA (green) antibodies. The white triangle indicates typical differences. In all images, nuclei were costained with DAPI (blue). F) Immunofluorescent staining of corneal neurons (green) in all groups at day 180. The white arrows indicate typical neurons, Tuj 1 indicates beta-III tubulin. The scale bars in (E,F) are 100 µm.