Establishment of a novel in vitro model of stratified epithelial wound healing with barrier function

Abstract

The repair of wounds through collective movement of epithelial cells is a fundamental process in multicellular organisms. In stratified epithelia such as the cornea and skin, healing occurs in three steps that include a latent, migratory, and reconstruction phases. Several simple and inexpensive assays have been developed to study the biology of cell migration in vitro. However, these assays are mostly based on monolayer systems that fail to reproduce the differentiation processes associated to multilayered systems. Here, we describe a straightforward in vitro wound assay to evaluate the healing and restoration of barrier function in stratified human corneal epithelial cells. In this assay, circular punch injuries lead to the collective migration of the epithelium as coherent sheets. The closure of the wound was associated with the restoration of the transcellular barrier and the re-establishment of apical intercellular junctions. Altogether, this new model of wound healing provides an important research tool to study the mechanisms leading to barrier function in stratified epithelia and may facilitate the development of future therapeutic applications.

Probing dynamic processes in the eye at multiple spatial and temporal scales with multimodal full field OCT

“…Our figures are therefore of a similar order of magnitude, although lower, possibly as we were imaging ex-vivo samples at 25°C instead of in vivo cornea at 37°C and imaged close to an almost closed wound. Indeed, our measured migration rates are closer to those reported for similar in vitro reepithelialization cell migration studies in rabbit (10 − 16 µm.h −1 [34]). Our algorithm was validated by comparing with manual tracking of 200 voxels in cells on successive images.…”

Effects of gamma radiation sterilization on the structural and biological properties of decellularized corneal xenografts

“…At confluence, live/dead staining was performed with a staining kit (Life Technologies Corporation, Oregon, USA), where cells were double-stained by calceinacetoxymethyl (Calcein AM) and ethidium homodimer-1 (EthD-1). On live culture, to promote stratification after confluence, the media was changed to DMEM/Ham's F-12 media (Corning, VA, USA) supplemented with 10% newborn calf serum and 10 ng/ml EGF, and cultured for 7 days, as reported previously [49]. Afterwards, phase contrast images were taken with 10x objectives using an inverted microscope (Nikon Eclipse TS100, Nikon Instruments Inc.; Melville, NY).…”

Three-Dimensional Human Cell Culture Models to Study the Pathophysiology of the Anterior Eye

“…Thus, the development of 3D models that allow for the study of corneal wound healing would provide important advantages. Gonzalez-Andrades et al established an in vitro model of the cornea with a barrier function that is useful for studying wound healing [ 112 ]. With the same purpose of assessing corneal wound healing, McKay et al reported the development of a coculture system of human corneal epithelial cells and corneal stromal fibroblasts that secrete their own extracellular matrix [ 113 ].…”