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Summary The purpose of this study was to summarize the possibilities of valve replacement with special emphasis on aortic allografts. A special preservation protocol for canine aortic allografts was developed. Based on the current literature the viability of 12 aortic valve allografts obtained from euthanized dogs was assessed on procurement, after preparation and after storage using different storage or freezing media. The media used for storage were balanced ion solution (Thomaejonin), the feeding medium Medium 199 and the potassium-rich TRIS-buffer CP-Tes solution. The freezing media were DMSO and ethylene glycol. These were compared at three different concentration levels. In order to prevent osmotic trauma, two of them were optimized by using a mathematical method that respects certain limits of intracellular water volumes. As the indicator of viability the valve endothelium was used; this is considered to be the most sensitive component in terms of traumatic or deficient conditions. We used an acridine orange and ethidium bromide based fluorescence microscopy method, which allows intact and damaged cells to be differentiated. We were able to show that the described procurement technique allows valve endothelium integrity of 99.81% to be achieved. Some of the specimens revealed single cell death, which may in part be related to physiologic cell regeneration. The euthanization appears not to have an effect on the allograft quality. As four identically treated allografts revealed some pre-existing damage we recommend viability testing of adjacent valve leaflets. Excellent results were obtained if cold storage with either feeding medium or CP-Tes solution was performed within a period of up to 72 hours postmortem. The loss of viability in allografts stored in ion balanced solution was marginal, which contrasts with reported results in rat aortic allografts. There were no clearly defined differences between the results achieved with the two different freezing media. In contrast, the mode of application had significant implications for cell survival in both of them. The mathematical adjustment of dilution and supplementation resulted in an optimized outcome in comparison to ad-hoc dilutions and supplementations. This demonstrates impressive prevention of osmotic cell damage during preparation for cryopreservation using the method described.