Morphological study of cryopreserved human corneal endothelium

The aim of the present paper is to describe the morphological changes that occur in human corneal endothelium as an immediate consequence of corneal cryopreservation. Therefore, 16 human donor corneas were cryopreserved with an original procedure at a 1 degrees C/min cooling rate in a freezing solution cryoprotected with 7% dimethylsulphoxide until a final temperature of -100 degrees C was reached. After storage of the corneas in liquid nitrogen for periods ranging from 1 to 96 days (mean: 34.31 days), the corneas were thawed in a water bath at +37 degrees C. Eight additional control corneas were processed without cryopreservation. Morphological assessment of the endothelial layer was performed by scanning electron microscopy and trypan blue and alizarin red S vital staining. Results showed cryoinduced damage at variable degrees in all cryopreserved corneas. They were classified into three groups according to the intensity and extension of the cryoinduced damage: group I (n = 10): corneas with minor endothelial alterations consisting in the presence of microholes in the posterior cell membrane; group II (n = 1): corneas with generalized disruption of endothelial intercellular junctions and intact cell membranes; group III (n = 5): corneas with severe endothelial damage consisting of massive cell necrosis and complete alteration of the morphological pattern of the endothelium. All control corneas had intact endothelial layers. Cryoinduced damage cannot be completely avoided with the cryopreservation protocol tested. The high interindividual variability of the results observed is not related to the storage time of the cornea in liquid nitrogen.