Y-27632 and dual media culture approach promote the construction and transplantation of rabbit limbal epithelial cell sheets via cell spheroid culture and auto-bioprinting.

The shortage of corneal donors and the limitations in tissue engineering grafts, such as biocompatibility and mechanical properties, pose significant challenges in corneal transplantation. Here, for the first time, we investigate the effect of Rho kinase inhibitor Y-27632 and a dual media culture approach, including proliferative media (M1) and stabilizing media (M2), on rabbit limbal epithelial stem cells (LESCs), aiming to explore the feasibility of constructing corneal cell sheets in vitro through auto-bioprinting and assessing their corneal wound healing capacity in vivo. Y-27632 has primarily demonstrated significantly enhanced LESCs growth, proliferation, and reduced apoptosis. The dual media culture approach combined with Y-27632 improved LESCs proliferation while maintaining stemness. In spheroid culture, Y-27632 decreased cell death and promoted proliferation. Immunofluorescent staining and RNA sequencing revealed upregulation of genes related to tight junctions and cell adhesion and downregulation of genes associated with aging and cell cycle. Using a bioprinter, LESC spheroids were auto-bioprinted onto a custom-made curved collagen membrane, creating a bioactive, transplantable, tissue-engineered anterior corneal sheet. Anterior superficial corneal transplantation with these LESC sheets significantly accelerated epithelial wound healing in rabbit limbal stem cell deficiency (LSCD) models. Overall, the integration of Y-27632, dual-media culture, and spheroid cell culture led to the development of a highly bioactive and therapeutically promising bio-ink derived from LESCs. Auto-bioprinting these LESC spheroids produced a bioactive, transplantable corneal cell sheet, presenting a promising therapeutic option for LSCD. STATEMENT OF SIGNIFICANCE: The renewal and wound healing of the corneal epithelium are essential for maintaining normal vision and refractive function. Limbal stem cell deficiency (LSCD) is a major cause of blinding keratopathy, and current treatment options are limited. In this study, for the first time, we developed a highly bioactive and therapeutically potent bio-ink for ocular surface regeneration by integrating Y-27632, a dual-media culture approach, and spheroid cell culture. Additionally, using auto-bioprinting technology, the limbal epithelial stem cell (LESC) spheroid bio-ink was precisely auto-bioprinted onto the curved surface of the corneal membrane, significantly accelerating corneal epithelial healing in an LSCD rabbit model.