Purpose
The corneal limbus contains cytokeratin 15 (K15) positive basal epithelial cells and melanocytes that form units with basal epithelial cells. We confirmed whether long-term cultivated epithelial cell sheets can reconstruct melanocytes-epithelial cell units.
Methods
Human limbal epithelial cells were isolated from eyebank eyes, seeded on fibrin-coated cell culture inserts, and 3 dimensional co-cultured with feeder cells. Cells were fed with supplemented hormonal epithelial medium with EGF or KGF and a ROCK inhibitor Y-27632 at day 3, day 5, and every day after day 7 for up to 3 months. Cryosections and whole cell sheets were fixed with ice-cold 4% paraformaldehyde in PBS, and stained with melanocyte marker MART-1 and epithelial precursor marker K15.
Results
MART-1 positive melanocytes were observed in whole mounted epithelial cell sheets in all culture conditions. Immunohistochemisrty showed that melanocytes were scattered as MART-1 positive cells in the basal layer of cultured epithelium. Melanocytes showed dendritic morphology, some of which were proximal to K15 highly positive epithelial cell clusters. In sheets derived from dense-pigmented limbus, melanin pigments were observed in basal cells around the melanocytes.
Conclusions
Melanocytes and limbal epithelial cells can self-assemble melanin units in vitro, mimicking the limbal niche in vivo
Keywords: 482 cornea: epithelium •
447 cell-cell communication