再次液氮冷冻保存对猪主动脉瓣膜细胞活性及组织结构的影响

Effects of recryopreservation on cellular viability and structure of the porcine aortic valve

目的 了解再次冷冻保存对主动脉瓣膜细胞活性及组织结构的影响 ,探讨液氮冷冻保存的主动脉瓣解冻后再次冷冻保存使用的可行性。 方法 将猪主动脉瓣叶在抗菌处理后按随机数字表法分成三组 ,每组 6个瓣叶 ,组 作对照 ,组 、组 控制降温速率降至 - 80℃后在液氮中保存 ,1个月后融化解冻。组 解冻并在室温下放置 15分钟后更换保存液 ,再次降温至 - 80℃后放入液氮中保存 ,2个月后再融化解冻。采用 XTT比色法测定各组瓣膜细胞活性 ,用免疫荧光组织化学染色、光学显微镜、透射电子显微镜行组织学检测。 结果 组 冷冻保存后瓣膜细胞活性下降到组 的 6 3.97% ,组织结构一定程度受损 ;组 瓣膜细胞活性下降至组 的 38.6 0 % ,组织结构损害也进一步加重。 结论 液氮冷冻保存的猪主动脉瓣一经解冻融化 ,不宜再次冷冻保存使用。

Objective To investigate the effects of recryopres e rvation on cellular viability and structure integrity of the aortic valve and th e feasibility of recryopreservation of once-thawed cryopreserved homograft valv es. Methods Porcine aortic valve leaflets were divided into three group s after sterilization(each group 6 valve leaflets). Group Ⅰ act as control, gr oup Ⅱ and group Ⅲ were frozen to -80 ℃ by enclosing into Cryo 1℃ freezing container and placing in -80℃ refrigera tor for 4 hours and stored in liquid nitrogen. One month after cryopreservation, the leaflets were thawed rapidly in a warm bath at 40℃. Group Ⅲ was left at r oom temperature for 15 minutes and replaced preserving nutrient medium, then r e-cryopreserved, and thawed after two months. The viability and histology of the porcine aortic valves were examined by XTT-tetrazolium colorimetric assay, indirect immunofluorescence histochemical stain, transmission electron microscop y and light microscopy. Results The valve viability of group Ⅱ decreased to 63.97% of group Ⅰ after thawing, group Ⅲ decreased to 38.60% o f group Ⅰ after rethawing. After first thawing, the endothelial cells had a par tial loss, the alignment of the fibers was damaged, the fibroblasts did show som e degree of ultrastructural evidence of injury and suppressed monoclonal binding to vimentin, overt dissolution of chondroitin sulfate simultaneous with a relat ive sparing of fibronectin in valve matrix was evidenced by immunohistochemical stain. These findings were significantly aggravated after rethawing except for f ibronectin. Conclusion It is not appropriate to recryopreserve the once-cryopreserved and thawed porcine aortic valve homografts for use.