The overall objective is to help to alleviate the dual problem facing transplantation medicine, of shortage of donor tissues (and their maximal processing), and the relatively short storage time. We will develop novel, improved protocols for preparation of corneal, limbal and conjunctival cells for potential grafting, and will investigate alternative approaches to prolong the ‘shelf-life’ of cells and tissue intended for grafting.

The project will be organised as four workpackages: (1) Endothelial cells for grafting: Cells from peripheral corneal endothelium as far as the trabecular meshwork or (Cells from peripheral corneal endothelium and from its close vicinity (insert area) will be characterised, and the isolation and transfer method will be optimised to achieve a graft with high density of cells for experimental transplantation on human eye. (2) Restoration of damaged endothelium: The aim is to investigate the reparation and self-regenerative potential (if any) of endothelium, and to encourage/induce it by chemical stimulation (treatment) in three-scale human model. (3) Limbal and conjunctival cells: Limbal and conjunctival epithelial cells will be expanded ex vivo with the aim to prepare them for grafting. The methods for goblet cells isolation and cultivation will be introduced. The methodology for preparation and transplantation of long-term stored allogenic limbal segments with decreased immunogenicity, but preserved stemness will be prepared for pre-clinical study. Cells and tissues engineered for grafting will by vitrified, i.e. frozen without ice formation and consequent cell destruction, their viability will be tested after thawing. This possibility of a long-term storage will drastically improve transplantation options. (4) Testing DNA integrity: DNA and chromosomal damage will be measured in prepared cells, including cells after devitrification (thawed) to monitor viability as a guide to increase the safety of tissues intended for grafting. DNA damage screening methods will be used to test for DNA damage on the ocular surface in various ocular pathologies.