Purpose The aim of this study was to establish and optimize a new and reproducible epithelial wound healing model on human corneas. This assay was used to study the kinetics of epithelial regeneration following a chemical injury.

Methods Thirty (n=30) human corneas unsuitable for transplant were used for the experiments. Corneas were cultured in Storagix medium (FBOV) at 31°C. Epithelial integrity before the beginning of the experiments (pre-wound) was assessed using the vital dyes trypan blue (TB, TB-S 0.25%, AL.CHI.MI.A. srl) and sodium fluorescein (Fluo). 1-heptanol soaked paper disks (6 mm) were applied in the centre of the corneas for 1’ to trigger a chemical damage at the epithelial layer. Afterwards, sodium fluorescein and TB stainings were repeated to quantify the damaged area and to monitor healing progression. The damaged area (mm2) was calculated for each time point with Fiji software. Wound healing rate (HR, mm2/die) was calculated for both Fluo (HRF) and TB (HRTB) measurements using the previously described formula:

Arithmetical averages (HRFAVG and HRTBAVG) of HRs were calculated and correlated by Pearson correlation coefficient with the following donor’s parameters: age, sex, post-mortem time (PMT, time between death and tissue procurement), stromal defects, septicaemia, body temperature, diabetes.

Results The execution of the heptanol wounding is highly reproducible, as highlighted by Fluo and TB staining. The average time for full recovery from wounding was 3,8 ± 0,41 days for Fluo and 3,5 ± 0,63 days for TB. Fluo and TB stainings are interchangeable as they significantly correlate (Pearson correlation coefficient = 0.630; p>0.05). A negative linear correlation was observed between HR and PMT (HRFAVG: corrected R2: 0.243, p = 0.003; HRTBAVG: corrected R2: 0,132, p = 0.028), but not with the other donors’ parameters.

Conclusion Our wound/healing model might be of great interest for studies of epithelial regeneration kinetics and validation of drugs for the treatment of ocular defects. The inverse correlation between PMT and HR provides valuable insights for scientists investigating the regenerative properties of the corneal epithelium, as well as for eye bank personnel aiming to preserve the regenerative potential of corneal epithelium.