RESUMEN
Different methods of corneal cryopreservation have been introduced, those employing intracellular cryoprotectants such as Me2SO or glycerol being the most widely favored. We investigated the influence of several freeze-thaw trauma variables on the survival of porcine endothelial monolayers when employing the extracellular cryoprotective agent dextran. We first examined the effects of various dextran concentrations and then, having ascertained the optimal concentration, further investigated the influence of fetal calf serum (FCS) concentration in the cryopreservation medium, the cooling rate, the thawing temperature, and the length of the preincubation in the freezing medium prior to cryopreservation. The numerical densities of endothelial cells were determined at dissection in hypoosmotic balanced salt solution and after organ culture by staining with alizarin red S and trypan blue. Morphological evaluation was not performed directly after thawing but after a subsequent organ culture at 37 degrees C to detect latent cell damage after freeze-thaw trauma. Our data revealed that corneas cryopreserved in minimal essential medium containing 10% dextran but lacking FCS, preincubated for 3 h, frozen at a cooling rate of 1 degrees C/min, and thawed at 37 degrees C incurred the lowest cell losses (22.4%, SD +/- 3.8). We conclude that dextran is an effective cryoprotectant for freezing of porcine corneas. However, variations between species in the results of cryopreservation require further investigation of an in vivo animal model and studies with human corneas before its clinical use can be recommended.
Asunto(s)
Córnea , Criopreservación/métodos , Crioprotectores , Dextranos , Conservación de Tejido/métodos , Animales , Bovinos , Supervivencia Celular , Medios de Cultivo , Endotelio Corneal/citología , Técnicas In Vitro , PorcinosRESUMEN
PURPOSE: To investigate the impact of transportation, simulated under laboratory conditions, on the corneal endothelium preserved by Optisol-GS, Likorol-DX, Likorol, and MK-Medium. METHODS: Three hundred twenty corneas from freshly slaughtered pigs were stored in Optisol-GS. Likorol-DX, Likorol. and MK-Medium for 1, 3, 6, or 10 days. After short-term preservation, the transportation was simulated on a laboratory shaker at 4 degrees C with an acceleration of 0-100 km/h in 16 seconds for 5 hours. Mate corneas served as the control. Corneal endothelial cell density was determined at the time of dissection, directly before the experiment, and after subsequent organ culture. RESULTS: A significant cell loss induced by transportation simulation was not observed in any experimental group. Preservation in MK-Medium starting at 3 days of short-term preservation resulted in a significant cell loss. Storage for up to 6 days in Optisol-GS, Likorol-DX, and Likorol did not result in a significant decrease in cell density. CONCLUSION: Short-term preserved corneas can be routinely distributed without a reevaluation, if the tissue is preserved for a shorter time as recommended by the manufacturers.