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

Abstract

AbstractPurposeStudying in vitro cell migration of endothelial sheets is challenging since capacity for cell migration needs to be maintained while the tissue must remain fixed on a rigid substrate. Here, a 3D culture technique is designed to maintain cellular viability and reduce tissue handling in order to analyze in vitro endothelial cell migration from corneal grafts.MethodsTwelve Quarter‐Descemet membrane endothelial keratoplasty (Q‐DMEK) grafts obtained from four corneas with intact and viable endothelial cells but ineligible for transplantation were included for explant culture. Graft quadrants were embedded in temperature‐reversible hydrogel and cultured over 2–3 weeks. Cell movement was assessed by light microscopy at fixed standardized time intervals. After gel removal, immunohistochemistry analysis was performed to characterize the phenotype of the migrating cells. Cell viability was assessed by Trypan Blue and Calcein‐AM.ResultsAll 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 in the endothelial cell monolayers expanded from the radial graft edges. The endothelial cells maintained the expression patterns of phenotypical markers.ConclusionsTemperature‐reversible hydrogel proved to be a very useful matrix for studying in vitro endothelial cell migration from explant grafts and allowed for subsequent immunohistochemistry analysis after gel removal. Studying in vitro endothelial cell migration from customized endothelial grafts should result in a better understanding of cellular migration mechanisms which play an important role in achieving faster corneal clearance in patients after corneal transplantation.