Enhancing the Outgrowth of Oral Mucosal Cell Sheets for Cultivated Rabbit Oral Mucosal Epithelial Transplantation with ROCK Inhibitor.

PURPOSE: Cultivated oral mucosal epithelial transplantation (COMET) is an effective way to treat limbal stem cell deficiency (LSCD), but its clinical usage is limited due to prolonged cultivation time and complexity. Rho-associated protein kinase (ROCK) inhibitors have been found to enhance cell proliferation, migration, and cytoskeleton maintenance. We investigated whether ROCK inhibitor (Y-27632) can be used to enhance the growth of oral mucosal epithelial cell sheet while remaining their characteristics, optimizing COMET usage for LSCD treatment.

MATERIALS AND

METHODS

Oral mucosal epithelial cells were harvested from rabbits and cultured using a collagenase-based isolation technique. Cells were seeded in a transwell plate with or without amniotic membrane (AM) and Y-27632. The speed of cell sheet outgrowth and cell density were evaluated. Stem cell marker (p63), cytokeratin (K3, K4 and K13) and junctional protein markers (ZO-1, occludin, connexin 43) were analyzed by immunostaining with confocal microscopy.

RESULTS

Y-27632 and AM coating facilitated the outgrowth of cell sheets, resulting in a larger expansion area from day 7 to day 15 (p < 0.05). Y-27632 treatment dose-dependently increased basal cell density (p < 0.05) and inhibited the squamous differentiation of the superficial layer, while maintaining cytokeratin and junctional protein markers expression. Y-27632 increased the p63-positive cells population (p < 0.05), which may play a role in maintaining stem cell properties and inhibiting differentiation.

CONCLUSIONS

Y-27632, together with AM, provides an efficient and supportive environment for producing oral mucosal epithelial cell sheets for transplantation. They might serve as an adjunctive agent to accelerate oral epithelial cell-based therapy for the treatment of limbal stem cell deficiency.