Fractures in cryopreserved elastic arteries.

The aim of this study was to define the conditions under which macroscopic fractures occur in vascular tissue during cryopreservation and to develop a practical cryopreservation method that prevents fracturing. The common carotid artery of the rabbit was subjected to a cryopreservation process that has been optimised for retention of in vitro function and cytological structure. This involves the stepwise addition and subsequent removal of dimethyl sulfoxide using a calculated protocol that avoids osmotic injury and minimises toxic action, controlled cooling, storage at -180 degrees C, and rapid warming. Seventy-five percent of such arteries were grossly fractured. The cooling and warming conditions were systematically varied to determine when in the cooling/storage/warming process the fractures occurred. Differential scanning calorimetry was then used to identify any corresponding thermal events. It was found that the fractures occurred as the temperature range -150 to -100 degrees C was traversed during the warming phase of the process. The glass transition temperature of a maximally freeze-concentrated solution of the cryoprotectant used was found to be -123 degrees C. Reducing the warming rate between the storage temperature (-180 degrees C) and -100 degrees C to < 50 degrees C/min prevented the fractures. Subsequent thawing could then be carried out rapidly in a 37 degrees C water bath without risk of fracture. We suggest that the fractures probably result from the thermal stresses created by rapid warming of the vitreous material that is produced by freeze-concentration of the aqueous phase. Relatively slow warming to -100 degrees C, at which temperature the vitreous material has softened, reduces these stresses and avoids the fractures.