Improvement of the Cold Storage of Isolated Human Hepatocytes
Improvement of the Cold Storage of Isolated Human Hepatocytes
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DOI：
10.3727/096368911x580509
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发表时间：
2012-01-01
影响因子：
3.3
通讯作者：
Rauen, Ursula
中科院分区：
文献类型：
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作者：
Pless, Gesine;Sauer, Igor M.;Rauen, Ursula
临床应用和不同研究领域（如细胞移植、生物人工肝支持和药理学测试）需要越来越多的人肝细胞。这一需求要求为分离的人肝细胞提供足够的储存选择。由于冷冻保存导致严重的冷冻损伤，目前在2-8 ℃下在为储存实体器官而开发的保存溶液中进行短期储存。然而，除了减缓细胞代谢外，寒冷还诱导细胞损伤，这在许多细胞类型中是铁依赖性的，并且不被当前的储存溶液抵消。在这项研究中，我们的目的是表征人类肝细胞的储存损伤，并为这些细胞的冷藏开发定制的解决方案。从部分肝切除获得的材料中分离人肝细胞，接种在单层培养物中，经过一段预培养期后，在经典和新溶液中低温储存，然后在细胞培养基中复温。人肝细胞显示冷诱导损伤，在威斯康星州大学溶液或细胞培养基中冷藏1周和复温3小时后，导致>80%的细胞死亡（LDH释放）。加入铁螯合剂去铁胺和LK 614可显著减轻冷损伤。基于新的器官保存溶液Custodiol-N的改良溶液的实验表明，富离子变体优于贫离子变体，富氯溶液优于贫氯溶液，钾作为主要阳离子上级钠，pH 7.0上级pH 7.4。在如此优化的溶液中冷藏2周后的LDH释放低于20%，大大改善了人肝细胞的冷藏。通过评估肝细胞线粒体膜电位和功能参数（刃天青减少、胰高血糖素刺激的葡萄糖释放）证实了结果，因此建议使用定制的肝细胞储存溶液冷藏这些细胞。
Increasing amounts of human hepatocytes are needed for clinical applications and different fields of research, such as cell transplantation, bioartificial liver support, and pharmacological testing. This demand calls for adequate storage options for isolated human liver cells. As cryopreservation results in severe cryoinjury, short-term storage is currently performed at 2-8 degrees C in preservation solutions developed for the storage of solid organs. However, besides slowing down cell metabolism, cold also induces cell injury, which is, in many cell types, iron dependent and not counteracted by current storage solutions. In this study, we aimed to characterize storage injury to human hepatocytes and develop a customized solution for cold storage of these cells. Human hepatocytes were isolated from material obtained from partial liver resections, seeded in monolayer cultures, and, after a preculture period, stored in the cold in classical and new solutions followed by rewarming in cell culture medium. Human hepatocytes displayed cold-induced injury, resulting in >80% cell death (LDH release) after 1 week of cold storage in University of Wisconsin solution or cell culture medium and 3 h of rewarming. Cold-induced injury could be significantly reduced by the addition of the iron chelators deferoxamine and LK 614. Experiments with modified solutions based on the new organ preservation solution Custodiol-N showed that ion-rich variants were better than ion-poor variants, chloride-rich solutions better than chloride-poor solutions, potassium as main cation superior to sodium, and pH 7.0 superior to pH 7.4. LDH release after 2 weeks of cold storage in the thus optimized solution was below 20%, greatly improving cold storage of human hepatocytes. The results were confirmed by the assessment of hepatocellular mitochondrial membrane potential and functional parameters (resazurin reduction, glucagon-stimulated glucose liberation) and thus suggest the use of a customized hepatocyte storage solution for the cold storage of these cells.