Clinical Application of Cryopreserved Allograft Valves and Establishment of Cryopreserved Tissue Bank

随着冷冻保存技术在除日本以外的许多发达国家的出现，人类同种异体瓣膜越来越多地被用作半月瓣的替代品。为临床应用同种异体瓣膜，我们介绍了组织冷冻保存系统。同种异体瓣膜是从身体和肾移植供者那里获得的，并征得他们的同意。同种异体瓣膜以灭菌方式收集，用抗生素溶液灭菌24小时后超低温保存。冷冻保存的同种异体瓣膜在含有10%二甲基亚砜的营养液中用程序冷冻机冷冻，然后保存在液氮中(-196ﾟC)。为了临床使用同种异体瓣膜，我们在保存前、抗生素灭菌后和冷冻保存30d时对异体瓣膜的细菌生长和细胞活性进行了评估。我们用流式细胞仪检测了移植瓣的细胞活性，结果表明，如果热缺血时间小于520分钟(8.7小时)，深低温保存的同种异体瓣成纤维细胞保存完好(超过70%)。经无菌鉴定和细胞活性测定，对18例主动脉瓣病变患者行深低温保存的同种异体主动脉瓣植入术，效果良好。在我们的临床研究中，从运动血流动力学的角度来看，同种异体主动脉瓣是一种理想的半月瓣替代品，尤其是对于主动脉根部较小的患者。从成人供体获取的冷冻保存的同种异体肺瓣也被成功地植入了患有肺闭锁的儿童RV-PA管道中的缩小尺寸的同种异体肺移植物。

Human allograft valves have increasingly been used as a semilunar valve substitute with the advent of cryopreservation techniques in many developed countries except Japan. We introduced the tissue cryopreservation system for the purpose of clinical application of allograft valves in our hospital. Allograft valves were procured from the cadavers and donors of renal transplantation with a consent. Allograft valves were collected under the sterilized manner and were cryopreserved after sterilization with antibiotic solution for 24 hours. The cryopreserved allograft valves were frozen by a program freezer in the nutrient medium containing 10% dimethylsulfoxide, and subsequently stored in liquid nitrogen (-196ﾟC). For the clinical use of the allograft valve, we assessed bacterial growth and cell viability of the allograft valve before storage, after antibiotic sterilization and at 30 days of cryopreservation. We evaluated cell viability of the allograft valve by flowcytometry, that revealed that fibroblasts of the cryopreserved allograft valve were well preserved (over 70%) if warm ischemic time was less than 520 minutes (8.7 hours). Following germ-free confirmation and cell viability assessment, cryopreserved aortic allograft valves were implanted in 18 patients having aortic valve disease with good results. In our clinical investigation, the aortic allograft valve is an ideal substitute for the semilunar valve from the exercise hemodynamic aspect, particulary in patients with a small aortic root. Cryopreserved pulmonary allograft valves harvested from the adult donor were also successfully implanted as a size-reduced pulmonary allograft for an RV-PA conduit in children with pulmonary atresia.