Conjunctival Epithelial Cells Cultivated <i>Ex Vivo</i> from Patients with Total Limbal Stem Cell Deficiency

Abstract

PURPOSE: Reconstruction of the ocular surface is challenging. As an alternative to mucosal and limbal epithelial, we study the feasibility of cultivated human conjunctival epithelial (HCjE) cells of patients with total limbal stem cell deficiency (LSCD). METHODS: We studied superior forniceal conjunctival biopsies harvested from 9 living donors with total LSCD of several etiologies who underwent surgery for ocular surface reconstruction. The conjunctival explants were cultivated on serum and growth factor supplemented DMEM/F12 under submerged conditions on denuded human amniotic membrane and tissue culture dishes. The area of cell growth was assessed. Cell morphology was analyzed by light microscopy, impression cytology, and transmission electron microscopy. Cultures were evaluated for epithelial cytokeratins (CK3, CK19), proliferation marker (Ki-67), and putative stem cells markers (ABCG2 and p63). Confocal immunofluorescence was also performed to assess CK3, CK19, Ki-67, ABCG2, and p63. RESULTS: The HCjE cells cultivated ex vivo were successfully expanded on denuded amniotic membrane but with a slower growth than in the tissue culture dish. Transmission electron microscopy showed stratified epithelium with microvilli, desmosomes, and hemidesmosomes. Impression cytology showed PAS+ cells that resembled goblet cells. Immunocytochemical analysis showed positivity for CK3, CK19, Ki-67, ABCG2, and p63. Confocal immunofluorescence was positive for CK3, CK19, Ki-67, ABCG2, and p63. CONCLUSIONS: Our results showed that it is possible to cultivate HCjE cells ex vivo of patients with ocular surface diseases. This method is important for ocular surface reconstruction in patients with bilateral total LSCD.