Development and Optimization of an Ex Vivo Model of Corneal Epithelium Damage with 1-Heptanol: Investigating the Influence of Donor Clinical Parameters and MSC-sEV Treatment on Healing Capacity.

Abstract

Purpose: To develop and characterize a reproducible human corneal epithelial wound-healing model using 1-heptanol, and to investigate the healing potential of Bone Marrow-derived Mesenchymal Stromal Cell small Extracellular Vesicles (MSC-sEV) and the influence of donor characteristics on epithelial healing.

Methods: Eighty-eight (n=88) human corneoscleral tissues unsuitable for transplantation were employed. Corneal epithelial damage was induced with 1-heptanol and monitored every 24 hours up to 96 hours using fluorescein and trypan blue staining. Histological assessment was performed on untreated and damaged tissues. Damaged areas were measured with FIJI software, and healing rates were calculated. MSC-sEV were isolated with size exclusion chromatography and characterized for their size, morphology and biomarkers. Their impact on healing was assessed in both in vitro scratch assays on cultured human corneal epithelial cells and on ex vivo 1-heptanol-damaged corneas.

Results: Histological analysis revealed detached corneal epithelium in the central area, while other layers remained unaffected. Healing rate peaked at 48 hours post-damage. Trypan blue and Fluorescein staining correlated and the former highlighted a higher initial healing rate than the latter. Diabetic and heart-beating brain-deceased donors showed impaired healing rates. MSC-sEV (79.8 nm, spherical bilayer, positive for TSG101, CD9, CD63, and CD81) significantly improved epithelial wound healing in both in vitro and ex vivo models.

Conclusion: 1-heptanol effectively induces reproducible corneal epithelial damage, and the ex vivo organ-cultured human cornea heals the epithelium within 96 hours. Diabetes and donation from heart-beating brain-deceased donors reduce healing capacity. MSC-sEV boost epithelial repair in damaged corneas.