Pegg D E, Wusteman M C, Boylan S
Biology Department, University of York, United Kingdom.
Cryobiology. 1997 Mar;34(2):183-92. doi: 10.1006/cryo.1996.1997.
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.
本研究的目的是确定在冷冻保存过程中血管组织发生宏观破裂的条件，并开发一种防止破裂的实用冷冻保存方法。对兔颈总动脉进行了冷冻保存处理，该处理已针对体外功能和细胞学结构的保留进行了优化。这包括使用计算好的方案逐步添加和随后去除二甲基亚砜，该方案可避免渗透损伤并将毒性作用降至最低，控制冷却、在 -180℃ 储存以及快速复温。75% 的此类动脉出现了肉眼可见的破裂。系统地改变冷却和复温条件，以确定在冷却/储存/复温过程中的何时发生破裂。然后使用差示扫描量热法来识别任何相应的热事件。结果发现，在该过程的复温阶段，当温度范围在 -150 至 -100℃ 之间时发生了破裂。所使用的冷冻保护剂的最大冷冻浓缩溶液的玻璃化转变温度为 -123℃。将储存温度（-180℃）和 -100℃ 之间的复温速率降低至 <50℃/分钟可防止破裂。随后可以在 37℃ 水浴中快速解冻而无破裂风险。我们认为，破裂可能是由于水相冷冻浓缩产生的玻璃态物质快速复温所产生的热应力导致的。相对缓慢地复温至 -100℃，此时玻璃态物质已软化，可降低这些应力并避免破裂。
