Figure 4

In vitro recellularization of the decellularized human cornea (DHC) (A) Phase contrast micrographs of LEPC (i); LM (ii); and LEPC/LM scaffolds showing intermingled melanocytes (iii, white asterisk) in the epithelial cell layer on DHC. Scale bar = 20 µm. Live/dead viability assay after 24 h of cultivation of LEPC, LMSC, and LM on DHC (iv–vi). (B) Light microscopic analysis of DHC-LEPC scaffolds showing cell monolayers after 1 week (1 w) and stratified epithelium (3 w) by hematoxylin and eosin staining (H&E) on the anterior surface. Scale bar—100 µm. (C) The injected stromal cells in DHC showing the stromal cells on the posterior side at 1-week culture and migrated to the anterior side after 3 weeks by H&E staining. Scale bar—100 µm (D) DHC-LEPC/LMSC scaffold after 3 weeks cultivation showing stratified epithelium and presence of stromal cells throughout the stroma by H&E staining (E) DHC-LEPC/LM scaffold showing stratified epithelium after 3 weeks of cultivation. Scale bar—100 µm (F) Immunofluorescence staining of DHL-LEPC scaffolds showing the expression of CK15 and p63 (green) at basal layers of epithelial cells (arrowheads); CK3 expression (green) in suprabasal cells but not in basal cells (arrows); E(epithelial)-cadherin and cytokeratin (pan-CK) expression (green) in all human limbal epithelial cells on DHC. (G) DHL-LEPC/LMSC scaffolds showing the vimentin expression on stromal cells and cytokeratin (pan-CK) expression (green) on epithelial layers. The dashed line separated the epithelium and stroma. (H) DHL-LEPC/LM scaffolds showing the melan-A (red) positive melanocytes (arrowheads) in the epithelial layers (pan-CK, green). LEPC limbal epithelial progenitor cells; LMSC limbal mesenchymal stromal cells, LM limbal melanocytes, pan-CK pan-cytokeratin, CK15 cytokeratin 15, CK3 cytokeratin 3, DAPI 4′,6‐diamidino‐2‐phenylindole.