AbstractPurposeTo establish a new ex vivo model and study the labial mucosal epithelium grafting as a potential approach for cornea re‐epithelization in patients with bilateral limbal stem cell deficiency.MethodsFour human donor corneal buttons were collected from the Eye tissue bank in corneal cold storage solution and left at 4°C for 32–52 days. Labial oral mucosa strips were dissected from four patients during fornix reconstruction after they signed an informed consent. The labial mucosa epithelial graft was prepared by dissecting substantia propria. The resulted graft was sutured near the limbus of the corneal button with running sutures (construct). Each construct was cultured under the standard conditions with the concave side upwards. After 21 days of culture, constructs were removed, washed and fixed. First half of the constructs were stained with hematoxylin and eosin (HE). 5 um crosscut cryo‐sections of the second half were stained with anti‐ keratin 13, 19, and p63, and paired secondary fluorescent antibodies. Nuclei were counterstained with Hoechst. Sections were accessed and photographed.ResultsAfter the culture, all constructs were integral with the attached epithelium graft and non‐loosened sutures. The graft became slightly flattened and brighter. HE staining demonstrated absence of the native cells in donor corneas. The labial mucosa grafts were located on the surface of the Bowman's membrane (BM) at the cornea periphery. Epithelial outgrowths were found to be transit from the labial mucosa graft to the BM. Cellular outgrows of the constructs had positive staining for keratin 13 and 19. Few cells were p63‐positive in nuclei.ConclusionsIn the study, a new ex vivo model composed of processed human donor cornea and live human donor labial mucosal epithelium graft was successfully established. The study confirmed the consistency of the new approach for corneal re‐epithelization by transplantation of an oral mucosal epithelial graft.