Improving endothelial cell migration by novel in vitro 3D culture of corneal grafts

Abstract

Purpose : Studying in vitro cell migration of endothelial sheets is challenging since capacity for cell migration needs to be maintained while at the same time the tissue must remain fixed on a rigid substrate. In this study, a 3D culture technique is designed to maintain cellular viability and to reduce tissue handling in order to analyze in vitro endothelial cell migration from a corneal graft.

Methods : Twelve Quarter-Descemet membrane endothelial keratoplasty (Q-DMEK) grafts obtained from 4 corneas with intact and viable endothelial cells but ineligible for transplantation were included in this study for explant culture. Graft quadrants were embedded in a three-dimensional culture system using a temperature-reversible hydrogel and cultured over 2-3 weeks in a humidified atmosphere at 37°C and 5% CO₂. Cell movement was assessed by light microscopy at fixed standardized time intervals. After the end of culture the gel was removed and immunohistochemistry analysis was performed to characterize the phenotype of the cells that had migrated from the Q-DMEK. Cell viability was assessed by Trypan Blue staining (0.04%) and Calcein-AM staining.

Results : All grafts embedded in the 3D hydrogel system maintained cell viability and displayed a capacity for cell migration. The migration pattern was asymmetric as endothelial cells moved as a sheet only from the radial cut graft edges, while no activity was seen in the peripheral graft area. Immunostaining performed on the samples after removing the gel showed the presence of proliferation and migration markers (Nanog, Nestin, Ki67, CD73) in the endothelial cell monolayers expanded from the radial graft edges. The endothelial cells maintained the expression patterns of phenotypical markers (ZO-1, Na⁺/K⁺ -ATPase, and vimentin).

Conclusions : Temperature-reversible hydrogel proved to be a very useful matrix for studying in vitro corneal endothelial cell migration from explant grafts and allowed for subsequent immunohistochemistry analysis after gel removal. Studying in vitro endothelial cell migration from custom-sized endothelial grafts such as Q-DMEK grafts should result in a better understanding of cellular migration mechanisms which may play an important role in achieving faster corneal clearance in patients after corneal transplantation.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.