Corneal endothelial cells from old donors: differentiation, senescence, proliferative capacities and optimized culture conditions

AbstractPurpose The intrinsically low proliferative capacity of human corneal endothelial cells (CECs) and their rapid senescence in in vitro culture constitute major limitations for cell bioengineering destined to treat endothelial dysfunctions. In Europe, the mean donor age of 70 adds a supplementary difficulty to the complex equation. Aim: to clarify whether old donor corneas could be however used for cell expansionMethods Morphology and differentiation of central and peripheral CECs were analyzed by immunostaining of ion transport‐related proteins, progenitor and neuronal markers in corneas retrieved in donor >50 years. The proliferative capacity of CECs from organ‐cultured corneas was quantified by EdU proliferation assay. The differences of central vs peripheral CECs were further characterized in vitro by studying their migration, proliferation and differentiation in serum‐containing medium.Results Central CECs highly expressed specific ion transport‐related proteins, and had a high senescence level. On the contrary, CECs located in the periphery or extreme periphery were less specialized, expressed progenitors and neuronal markers and were more prone to endothelial‐mesenchymal transformation in serum‐containing medium. With a new culture sequence that limited proliferation and maintained differentiation, corneas from donors >70 yo, could successfully be used to reconstitute a monolayer of stable endothelial phenotype with ECD of 2000 cells/mm2 but require pooling of corneas.Conclusion This work could allow to “recycle” the 20% of corneas usually discarded by eye banks because of age‐related ECD decline