Morphological Study of Cryopreserved Human Corneal Endothelium

Abstract

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 °C/min cooling rate in a freezing solution cryoprotected with 7% dimethylsulphoxide until a final temperature of –100 °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 °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.

Keratocyte injury in human corneas cryopreserved under standard conditions

“…Our results confirm data published by other authors who have described the structural disruption of corneal endothelial cells and other changes after cryopreservation (Wusteman 1997;Canals 1999). In addition to this, we have used TEM to quantify the relative contribution of apoptosis versus necrosis in keratocyte cell death.…”

Hyperpolarization of the plasma membrane potential provokes reorganization of the actin cytoskeleton and increases the stability of adherens junctions in bovine corneal endothelial cells in culture

“…Moreover, the disruption of the intercellular adhesion of endothelial layers limits the lifespan of cold-stored organs for transplant [Carles et al, 1994;Eberl et al, 1999]. In particular, the lifespan of cold-stored eye bank corneas is limited by the gradual loss of endothelial integrity [Armitage, 2008;Day et al, 2008], in part due to alterations of the actin cytoskeleton and of the intercellular junctions [Canals et al, 1999;Hsu et al, 1999].…”

Cryoprotection of porcine cornea: a scanning electron microscopy study

“…Studies have been reported on cryoprotection of porcine cornea in dextran and Me 2 SO of concentrations higher than those used in our experimental system (Canals et al, 1996;Wusterman et al, 1999). The human cornea has been shown to successfully recover from freezing cryoprotected in Me 2 SO (Canals et al, 1999), Me 2 SO, formamide and 1,2-propanediol , Me 2 SO, glycerol and 2,3-butanediol . Although the results of these experiments have been satisfactory, the damage cannot be completely avoided using these cryoprotectors.…”