Evaluation of Protective Effects of Trehalose on Desiccation of Epithelial Cells in Three Dimensional Reconstructed Human Corneal Epithelium

Abstract

Purpose: Trehalose has been shown to protect epithelial cells from desiccation damage in cell culture and the murine dry eye model. The present study evaluates the protective role of trehalose in reconstructed human corneal epithelium (3D-HCE) during desiccation.

Materials and methods: The morphology of 3D-HCE was examined using in vivo an ex vivo confocal laser-scanning microscopy (CLSM). The 3D-HCE was desiccated with or without pre-treatment with trehalose. Evaluation of protective role of trehalose was conducted using different in vitro cell viability assays and CLSM. Tissue thickness for each condition was determined by optical coherence tomography (OCT).

Results: 3D-HCE tissue revealed similar features with human cornea at histological level. After desiccation the percentage of living cells was only 32% in 3D-HCE tissue without pre-incubation and 98% in trehalose-pre-incubated tissue, as shown by a cell viability assay. These findings were confirmed by using a Live-Dead assay. Also, the confocal immunofluorescence analysis revealed much better preservation of tight junctions in trehalose-pre-treated tissue.

Conclusions: CLSM and an in vitro cell viability assays could be successfully used for the characterization of 3D-HCE tissue. We demonstrated the protective role of trehalose using reconstructed corneal epithelium (3D-HCE), which mimics HCE and has the potential to become a valuable model in ophthalmic research.

KEYWORDS::

• Cornea
• Dry eye
• Epithelium
• Trehalose
• 3D-culture system

ACKNOWLEDGMENTS

The authors would like to sincerely thank R. Kala for performing OCT and G. Karsten for excellent technical assistance.

Declaration of interest: The study was supported by the DFG (Transregio 37, Micro- and Nanosystems in Medicine – Reconstruction of Biological Functions).