Lehle Karla, Hoenicka Markus, Jacobs Volker R, Schmid Franz X, Birnbaum Dietrich E
Clinic of Cardiothoracic Surgery, University of Regensburg, Franz-Josef-Strauss-Allee 11, 93042 Regensburg, Germany.
Cryobiology. 2005 Apr;50(2):154-61. doi: 10.1016/j.cryobiol.2004.12.006.
To investigate the influence of cryopreservation on endothelial cell growth, morphology, and function human umbilical vein endothelial cells (HUVECs) were frozen following a standard protocol. Cell suspensions were exposed to 10% dimethyl sulfoxide in a high-potassium solution, cooled to -80 degrees C at 1 degrees C/min and stored in liquid nitrogen for 7-36 days. Samples were thawed in a 37 degrees C water bath and the cryoprotectant was removed by serial dilution. The growth of cell suspensions was assayed by culturing 7300 cells/cm2 for 3-5 days in order to determine the cell multiplication factor. Fresh and cryopreserved/thawed cells were analyzed for their growth, and their anti-inflammatory and anti-coagulant function by using cellular ELISA. Cryopreservation resulted in a retrieval of 66 +/- 5% and a viability of 79 +/- 3%. Cryopreserved/thawed and fresh cells showed identical doubling times and identical cell counts in the confluent monolayers. However, the lag phase of thawed HUVECs was approximately 36 h longer, resulting in significant differences in the cell multiplication factor at 3 and 5 days after seeding. After expansion to a sufficient cell count the lag phases were identical. Fresh and cryopreserved/thawed cells showed comparable anti-inflammatory and anti-coagulant activity, as judged by the basal and TNF-induced VCAM-1, ICAM-1, E-selectin, and thrombomodulin expression. Cryopreserved/thawed and recultivated endothelial cells are suitable for endothelialization of autologous allograft veins. Such tissue-engineered grafts will offer the necessary clinical safety for those patients who lack autologous material.
为研究冷冻保存对内皮细胞生长、形态和功能的影响，按照标准方案对人脐静脉内皮细胞（HUVECs）进行冷冻。细胞悬液在高钾溶液中暴露于10%二甲基亚砜，以1℃/分钟的速度冷却至-80℃，并在液氮中保存7至36天。样品在37℃水浴中解冻，通过连续稀释去除冷冻保护剂。通过以7300个细胞/cm²培养3至5天来测定细胞悬液的生长，以确定细胞增殖因子。通过细胞ELISA分析新鲜的和冷冻保存/解冻后的细胞的生长情况及其抗炎和抗凝功能。冷冻保存后细胞回收率为66±5%，活力为79±3%。冷冻保存/解冻后的细胞和新鲜细胞在汇合单层中的倍增时间和细胞计数相同。然而，解冻后的HUVECs的延迟期大约长36小时，导致接种后3天和5天的细胞增殖因子存在显著差异。在扩增至足够的细胞数量后，延迟期相同。根据基础和肿瘤坏死因子诱导的血管细胞黏附分子-1（VCAM-1）、细胞间黏附分子-1（ICAM-1）、E-选择素和血栓调节蛋白的表达判断，新鲜的和冷冻保存/解冻后的细胞具有相当的抗炎和抗凝活性。冷冻保存/解冻后再培养的内皮细胞适用于自体同种异体移植静脉的内皮化。这种组织工程移植物将为那些缺乏自体材料的患者提供必要的临床安全性。
