液氮保存对人主动脉瓣组织细胞活力的影响

Influence of cryopreservation on tissue viability of human aortic valve homografts

目的 主要观察液氮 (- 196℃ )保存 1～ 2 4mo不同时间段的瓣膜组织细胞活力及组织培养细胞生长情况的变化 ,为临床适用的人同种主动脉瓣的适宜保存期限提供实验依据 .方法 选取 (18～ 35 )岁健康成年男性脑死亡 30 min内取材的主动脉瓣膜 2 0个 .依据保存时间分为 10组 , 组为新鲜对照组 ,取材后直接供实验用 , ～ 组为冷冻保存组 ,分别为冷冻保存 1,3,6 ,9,12 ,15 ,18,2 1,2 4m o的瓣膜 ,每组 2个瓣膜共 6个瓣叶 .冷冻保存组各组瓣膜缓慢降温 (1℃·min- 1 ) ,液氮中保存 ,实验时进行快速复温 .瓣叶剪为细小组织块 ,一半置入培养瓶中做组织培养 ,相差显微镜下观察其组织细胞生长情况 ;另一半组织块用胰酶消化 ,光镜下做细胞计数并计算其细胞活力 .结果 组的组织细胞活力最高(93.8% ) , ～ 组细胞活力均较 组明显减低 (6 7.3% vs93.8% P<0 .0 5 ) ; , , 组细胞活力较 ～ 组瓣膜各组明显减低 (5 2 .4% vs76 .5 % P<0 .0 5 ) . 组组织细胞 1wk镜下可见 ,生长较快 ,4wk时镜下细胞数较多 ; ～ 组组织细胞 2 wk镜下可见 ,生长较 组稍缓慢 ,4wk时镜下细胞数减少 ; , , 组细胞 3wk镜下可见 ,生长较慢 ,4wk时镜下细胞数较 组明显减少 .结论 液氮保存对人同种瓣膜组织细胞活力有显著影响 。

AIM To observe the cellular viability and cell proliferative capacity of human aortic valves cryopreserved in liquid nitrogen at -196℃. METHODS Twenty valves, procured from 18 to 35 years old healthy adult hearts within 30 min after the determination of brain death, were divided into 10 groups. Group Ⅰ, the control group, is of fresh valves. Valves from Group Ⅱ to Group Ⅹ were those preserved in liquid nitrogen for 1, 3, 6, 9, 12, 15, 18, 21, 24 mo respectively. The fresh valves were examined immediately after procurement. All the cryopreserved valves were thawed in 37℃ saline pool. Each specimen was cut into pieces of 1 mm×1 mm×1 mm. Half of the tissues were reduced with 2 5 g·L -1 trypsin and dyed with trypan blue, then the number of viable and nonviable cells were counted. The remaining tissues were incubated in RPMI1640 medium containing 100 mL·L -1 fetal calf serum at 37℃ to observe the capacity of cell proliferation on the 7th and 28th day. RESULTS Fresh valves seemed to have the highest cell survival rate of all the groups (93.8%). In cryopreservation groups, there was a tendency that the longer the duration of preservation was, the lower the survival rate was. Valves from Group Ⅱ to Group Ⅹ showed a significant decrease in cell survival rate compared with those from Group Ⅰ(67.3% vs 93 8% P <0 05). Valves from Group Ⅷ, Ⅸ and Ⅹ showed a significant decrease in cell survival rate compared with those from Group Ⅰ to Group Ⅶ (52.4% vs 76.5% P <0.05). Cells in Group Ⅰ grew out of the tissue pieces during the first week and fully covered the bottom of culture container during the fourth week. In the middle of the second week, however, cells of Group Ⅱ to Group Ⅶ started to appear at the edge of the tissues and occupied the most part of the container bottom. In Group Ⅷ, Ⅸ and Ⅹ, cells were scarcely seen during the first week and the cell number was markedly decreased during the fourth week compared with that of the fresh valves. CONCLUSION Cryopreservation in liquid nitrogen has significant effects on the cellular viability and cell proliferative capacity of human aortic valve tissues. Valves that are preserved for 18 or more months have lower cellular survival rates and show a dramatic decrease in cellular capacity of proliferation. According to the results of our experiments, the durability of valves cryopreserved 18 or more months would decrease correspondingly.