A new in vitro wound healing assay for the evaluation of the effects of new materials and ocular drugs on stratified human corneal epithelium

Session Details

Session Title: Special Cases II

Session Date/Time: Monday 07/09/2015 | 17:00-18:30

Paper Time: 17:58

Venue: Room 11

First Author: : M.Gonzalez-Andrades USA

Co Author(s): :    A. Cruzat   J. Mauris   L. Alonso-Pastor   C. Dohlman   P. Argueso        

Abstract Details

Purpose:

The effects of developmental drugs and new materials on corneal epithelium have been evaluated using different models. The purpose of this study was to generate an in vitro wound healing model for the evaluation of the effects of new materials and ocular drugs, based on stratified human corneal-limbal epithelial (HCLE) cells that resembles the native corneal epithelium.

Setting:

Cornea Service, Department of Ophthalmology, Massachusetts Eye & Ear Infirmary. Schepens Eye Research Institute, Harvard Medical School. Changing Places Research Group, Media Lab, Massachusetts Institute of Technology.

Methods:

Immortalized HCLE cells were grown to confluence in serum-free medium and switched to 10% serum to promote differentiation and stratification. Afterwards, a mechanical injury with a trephine, combined with epithelial debridement, was performed. Cells were grown in culture media DMEM/F12 supplemented with EGF, with or without serum. The effect of serum on the wound healing response was evaluated using phase-contrast microscopy and a barrier function (rose bengal uptake) assay. Moreover, a double label of occludin and F-actin was performed to evaluate the tight junctions and the actin cytoskeleton.

Results:

Mechanical wounds using a trephine were reproducible. In the absence of serum, none of the wounds were able to completely re-epithelialize at 72 hours. However, 83+/-16% of the wounds completely healed at 72 hours in the group that received serum-containing media. Of those wounds that completely healed, 76+/-9% showed stratification in more than 50% of the re-epithelialized area. A heterogeneous cellular distribution of actin was observed immediately after wounding, followed by re-arrangement and formation of perpendicular actin bundles in the leading edge during migration. Presence of tight junctions was demonstrated in the superficial layers following re-epithelialization, suggesting the formation of a proper epithelial barrier.

Conclusions:

Trephine injuries to cultured stratified human corneal epithelial cells show optimal wound closure reproducibility. Further, serum promotes re-epithelialization in vitro, suggesting that this model could potentially be used to evaluate the effects of drugs and materials in the corneal epithelium. Funding: NIH/NEI-EY024031.

Financial Interest:

NONE