Generation of an In Vitro Wound Healing Model Based on Stratified Human Corneal Epithelial Cells

Abstract

Purpose: Many species of animals and culture models have been used to study corneal wound healing. Despite their benefits and limitations, no models that resemble the physiological response to injury have been developed using human stratified epithelia. The aim of this study was to develop a reproducible in vitro wound healing model using stratified human corneal-limbal epithelial (HCLE) cells.

Methods: Immortalized HCLE cells were grown to confluence in serum-free medium and switched to 10% serum to promote differentiation and stratification. Afterwards, different injury models involving mechanical, thermal and chemical insults were performed. Photographs were taken with a phase-contrast microscope and analyzed using ImageJ software. The area, perimeter, Ferret’s diameter and circularity of the wounds were measured and compared among the different models. In addition, a rose bengal uptake assay was carried out to confirm the presence of barrier function in stratified cells outside the wounded area.

Results: Wounds ranging 0.4-3.3 mm² area and 3.3-7.8 mm perimeter were performed applying different insults in all models. Ferret’s diameter values ranged between 0.9 and 2.3 mm, whereas circularity varied between 40% and 83%. A mechanical model using a 2mm-trephine showed the best results in terms of reproducibility and circularity levels (83+12%). Mechanical injury models based on plastic and metal tips resulted in circularity values between 40 and 64%, showing a great level of disparity in area, perimeter and Ferret’s diameter. Thermal (liquid nitrogen) and chemical (0.5M NaOH or 20% ethanol) based models resulted in worst circularity levels compared to mechanical injury models alone. Rose bengal uptake assay demonstrated the existence of a barrier function outside the wound in all models evaluated.

Conclusions: Mechanical injuries using a trephine showed optimal levels of reproducibility compared to other models of injury in stratified corneal cultures. This model could potentially be used to evaluate in vitro the pathophysiology of human wound healing and the efficacy of topical drugs in promoting healing responses.