Rendal Vázquez M Esther, Díaz Román T M, Rodríguez Cabarcos M, Zavanella Botta C, Domenech García N, González Cuesta M, Sánchez Dopico M J, Pértega Díaz S, Andión Núñez C
Unit of Cryobiology, Complejo Hospitalario Juan Canalejo, Carretera del Pasaje s/n, Hospital Teresa Herrera, 15006 La Coruna, Spain.
Cell Tissue Bank. 2008 Jun;9(2):101-7. doi: 10.1007/s10561-008-9063-6. Epub 2008 Mar 5.
To analyse the influence of cold ischemic time (CIT) (2-24 h) and of cryopreservation (liquid phase) on the viability of the valvular fibroblasts and in the presence of apoptosis. Cardiac valves from 10 pigs were evaluated by anatomo-pathological study of the wall, muscle and leaflet. At the same time, the presence of cellular death due to apoptosis was investigated in two ways; directly on tissue by Apodetec system and by two-colour flow cytometry assay analyzing a suspension of fibroblast from valve leaflets using Anexina V and propidium iodure (PI). We established three groups of samples to compare different experimental conditions: 2 h of ischemia (group 1), 24 h of ischemia (group 2), and a programme of cryopreservation (-1 degrees C/min) after 2 h of ischemia, followed by storage in liquid nitrogen during a week and thawing was performed (group 3). The analysis of viabilities showed slight differences between all three groups. The results indicated CIT of 24 h undergoing more structural affectation than CIT of 2 h. Flow cytometry analysis did not show important differences between groups; however cryopreserved samples (group 3) slightly less viability and a higher percentage of death by apoptosis than group 1 and 2 using flow cytometry. Apoptosis was confirmed on tissue from all valves but mainly in samples of group 2 and group 3. In summary, the viability of the valves in the case of ischemic times of 2 h, 24 h or after cryopreservation/thawing differs slightly. The death of the cells is mainly mediated by necrosis and not by apoptosis.
分析冷缺血时间（CIT）（2 - 24小时）和冷冻保存（液相）对瓣膜成纤维细胞活力以及细胞凋亡情况的影响。对10头猪的心脏瓣膜进行了壁、肌肉和瓣叶的解剖病理学研究。同时，通过两种方式研究因细胞凋亡导致的细胞死亡情况；一种是使用Apodetec系统直接对组织进行检测，另一种是通过双色流式细胞术分析瓣膜瓣叶成纤维细胞悬浮液，使用膜联蛋白V和碘化丙啶（PI）进行检测。我们设立了三组样本以比较不同的实验条件：2小时缺血组（第1组）、24小时缺血组（第2组）以及2小时缺血后进行冷冻保存程序（-1℃/分钟），随后在液氮中储存一周并进行解冻的组（第3组）。活力分析显示三组之间存在细微差异。结果表明，24小时的冷缺血时间比2小时的冷缺血时间对结构的影响更大。流式细胞术分析未显示各组之间存在重要差异；然而，使用流式细胞术检测时，冷冻保存的样本（第3组）活力略低，凋亡导致的死亡百分比高于第1组和第2组。在所有瓣膜组织中均证实存在细胞凋亡，但主要发生在第2组和第3组的样本中。总之，在缺血2小时、24小时或冷冻保存/解冻后的瓣膜活力略有不同。细胞死亡主要由坏死介导，而非凋亡。
