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Rudolf F. Guthoff, Marine Hovakimyan, Theresa Ramoth, Marian Löbler, Klaus-Peter Schmitz, Oliver Stachs; Evaluation Of Protective Effects Of Trehalose On Desiccated Epithelial Cells In Three Dimensional Reconstructed Human Corneal Epithelium. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1843.
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Dry eye is a multifactorial disease of tears and ocular surface that results in symptoms of discomfort, modification of the tear film composition, and tear film instability. The main treatment modality is the application of tear substitutes, containing hyuluronan, cellulose derivates or artificial polymers.Trehalose is a naturally occurring disaccharide that has been proposed to be a protective agent in dry eye condition. Using an established three-dimensional model based on reconstituted human corneal epithelium (3D-HCE), this study investigated in vitro the efficacy of trehalose to protect corneal epithelial cells from damage by desiccation.
The normal morphology of 3D-HCE was examined by in vivo confocal laser-scanning microscopy (CLSM, Heidelberg Retina Tomograph/Rostock Cornea Module).The 3D-HCE was 45 minutes desiccated at room temperature with or without pre-incubation with trehalose. After an overnight incubation for rehydratation, the evaluation of the protective role of trehalose was conducted using different in vitro cell viability assays (Cell Quanti Blue, Live Dead) and confocal immunofluorescence analysis.
3D-HCE tissue revealed similar features with human cornea at histological level, demonstrating several cellular layers with substructured morphology. After desiccation the percentage of living cells was 32 ± 27% in 3D-HCE tissue without trehalose pre-incubation, as shown by Cell Quanti Blue assay. In contrast, the pre-incubated 3D-HCE revealed 98 ± 4 % of living cells. Consistently, in the Live-Dead the desiccated 3D-HCE without trehalose pre-incubation revealed enormous high number of dead cells. Also the confocal immunofluorescence analysis revealed much better preservation of tight junctions in trehalose-treated tissue.
We could demonstrate the protective role of trehalose by using reconstructed corneal epithelium (3D-HCE), which mimics human corneal epithelium and has the potential to become a valuable model in ophthalmic research.For the first time, in vivo CLSM could be used for characterisation of 3D cell culture systems at histological level. If adapted for the use in cell culture conditions, in vivo CLSM can be successfully applied to characterize the HCE tissue and monitor it over time.
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