Pascual Gemma, Rodríguez Marta, Corrales Celia, Turégano Fernando, García-Honduvilla Natalio, Bellón Juan M, Buján Julia
Department of Medical Specialities, Faculty of Medicine, University of Alcala, E-28871 Alcalá de Henares, Madrid, Spain.
Cryobiology. 2004 Feb;48(1):62-71. doi: 10.1016/j.cryobiol.2003.12.004.
The endothelial loss provoked by the methods of vascular cryopreservation used at most human vessel banks is one of the main factors leading to the failure of grafting procedures performed using cryopreserved vessel substitutes. This study evaluates the effects of the storage temperature and thawing protocol on the endothelial cell loss suffered by cryopreserved vessels, and optimises the thawing temperature and protocol for cryopreserving arterial grafts in terms of that producing least endothelial loss. Segments of the common iliac artery of the minipig (n = 20) were frozen at a temperature reduction rate of 1 degrees C/min in a biological freezer. After storing the arterial fragments for 30 days, study groups were established according to the storage temperature (-80, -145 or -196 degrees C) and subsequent thawing procedure (slow or rapid thawing). Fresh vessel segments served as the control group. Once thawed, the specimens were examined by light, transmission, and scanning electron microscopy. The covered endothelial surface was determined by image analysis. Data for the different groups were compared by one way ANOVA. When cryopreservation at each of the storage temperatures was followed by slow thawing, the endothelial cells showed improved morphological features and viability over those of specimens subjected to rapid thawing. Rapidly thawed endothelial cells showed irreversible ultrastructural damage such as mitochondrial dilation and rupture, reticular fragmentation, and peripheral nuclear condensation. In contrast, slow thawing gave rise to changes compatible with reversible damage in a large proportion of the endothelial cells: general swelling, reticular dilation, mitochondrial swelling, and nuclear chromatin condensation. Gradually thawed cryopreserved arteries showed a lower proportion of damaged cells identified by the TUNEL method compared to the corresponding rapidly thawed specimens (p < 0.05, for all temperatures). In all the groups in which vessels underwent rapid thawing (except at -145 degrees C), significant differences (p < 0.05) in endothelial cover values were recorded with respect to control groups. Storage of cryopreserved vessels at -80 degrees C followed by rapid thawing led to greatest endothelial cell loss (61.36+/-9.06% covered endothelial surface), while a temperature of -145 degrees C followed by slow thawing was best at preserving the endothelium of the vessel wall (89.38+/-16.67% surface cover). In conclusion, storage at a temperature of -145 degrees C in nitrogen vapour followed by gradual automated thawing seems to be the best way of preserving the endothelial surface of the arterial cryograft. This method gives rise to best endothelial cell viability and cover values, with obvious benefits for subsequent grafting.
大多数人体血管库所采用的血管冷冻保存方法引发的内皮细胞损失，是导致使用冷冻保存血管替代物进行移植手术失败的主要因素之一。本研究评估了储存温度和解冻方案对冷冻保存血管内皮细胞损失的影响，并就产生最少内皮细胞损失的情况，优化了冷冻保存动脉移植物的解冻温度和解冻方案。将小型猪（n = 20）的髂总动脉段在生物冷冻箱中以每分钟1摄氏度的降温速率冷冻。在将动脉片段储存30天后，根据储存温度（-80、-145或-196摄氏度）和随后的解冻程序（慢速或快速解冻）设立研究组。新鲜血管段作为对照组。解冻后，通过光学显微镜、透射电子显微镜和扫描电子显微镜对标本进行检查。通过图像分析确定覆盖的内皮表面。不同组的数据通过单因素方差分析进行比较。当在每个储存温度下进行冷冻保存后采用慢速解冻时，内皮细胞在形态特征和活力方面比快速解冻的标本有所改善。快速解冻的内皮细胞显示出不可逆的超微结构损伤，如线粒体扩张和破裂、网状结构破碎以及细胞核周边凝聚。相比之下，慢速解冻在很大比例的内皮细胞中引起了与可逆损伤相符的变化：普遍肿胀、网状结构扩张、线粒体肿胀和核染色质凝聚。与相应的快速解冻标本相比，逐步解冻的冷冻保存动脉经TUNEL法鉴定的受损细胞比例更低（所有温度下p < 0.05）。在所有血管进行快速解冻的组中（-145摄氏度除外），与对照组相比，内皮覆盖值存在显著差异（p < 0.05）。在-80摄氏度下冷冻保存血管后快速解冻导致内皮细胞损失最大（内皮覆盖表面为61.36±9.06%），而-145摄氏度下慢速解冻在保存血管壁内皮方面效果最佳（表面覆盖为89.38±16.67%）。总之，在氮蒸气中于-145摄氏度下储存，随后进行逐步自动解冻，似乎是保存动脉冷冻移植物内皮表面的最佳方法。该方法能产生最佳的内皮细胞活力和覆盖值，对后续移植具有明显益处。
