Bioengineered Human Limbal Stem Cell–Derived Epithelial Sheets for Ocular Surface Reconstruction

Purpose

The successful cultivation and transplantation of corneal epithelial cell sheets derived from stem cell-laden limbal explants hold great promise for the treatment of corneal injuries. This study aims to engineer human corneal limbal stem cell–derived epithelial cell sheets using a smart copolymer, poly(N-isopropylacrylamide-co-glycidyl methacrylate) (pNIPA-GMA).

Methods

The pNIPA-GMA was synthesized via free radical random copolymerization of N-isopropylacrylamide and glycidyl methacrylate. Characterization included ATR-FTIR for chemical features, HPLC/GPC for molecular weight, DSC and UV–visible spectroscopy for thermoresponsive behavior, contact angle for wettability, surface profilometer for roughness, and cytotoxicity according to ISO 10993–5. Discarded human corneal samples were ethically collected, limbal regions dissected, and explants cultured on pNIPA-GMA-coated dishes. The formation and retrieval of cell sheets were followed by gene expression analysis.

Results

The pNIPA-GMA copolymer was synthesized with a yield of about 77%. ATR-FTIR confirmed the synthesis, while HPLC/GPC revealed a molecular weight of about 114 kDa. DSC and UV–visible spectroscopy demonstrated thermal transitions around 31 °C upon heating and 28 °C upon cooling. Physical property assessments indicated hydrophilicity at ambient temperature and a smooth surface of coated substrates. Cytotoxicity evaluation indicated non-cytotoxic nature of pNIPA-GMA. Human corneal tissue explants exhibited significant epithelial cell outgrowth, forming cohesive cell sheets, which were successfully retrieved from the thermoresponsive surfaces. Gene expression analysis confirmed the corneal epithelial cell phenotype, with upregulation of key epithelial markers.

Conclusion

This study validated successful synthesis of pNIPA-GMA and established its potential in fabricating human corneal epithelial cell sheets, highlighting its promise for ocular surface reconstruction in clinical applications.

Lay Summary

This study investigates the engineering of human corneal cell sheets for potential therapeutic applications in treating eye injuries and diseases. Specifically, the research focuses on developing a smart approach to grow and retrieve these cell sheets using thermoresponsive polymer. Also, it highlights transforming clinical waste (discarded cornea that were unfit for transplantation) into potential clinical products. This innovative method offers a promising avenue for advancing eye repair therapies, highlighting its potential for clinical translation in the treatment of corneal disorders.

Graphical Abstract