Marsh D C, Hjelmhaug J A, Vreugdenhil P K, Kerr J A, Rice M J, Belzer F O, Southard J H
Department of Surgery, University of Wisconsin, Madison 53792.
Hepatology. 1991 Mar;13(3):500-8.
Hepatocyte suspensions provide a rapid method to determine how hypothermic storage affects liver cell metabolism and viability. Using these studies, improved methods of hypothermic liver preservation for transplantation may be developed. In this study, rat hepatocytes were cold-stored for up to 7 days in University of Wisconsin liver preservation solution. At the end of each day of storage hepatocytes were resuspended in Krebs-Henseleit buffer or tissue-culture medium (Liebovitz-15; Fischer's; modified Fischer's, which was similar to Fischer's but with glycine and cysteine added; or Waymouth's medium). Hepatocyte viability was assessed by rewarming and oxygenating the suspensions and measuring the percentage of leakage of lactate dehydrogenase from the cells, the cellular concentration of potassium and the stimulation of respiration by succinate, all measures of plasma membrane integrity. Additionally, concentrations of ATP and glutathione after rewarming and reoxygenation in the various resuspension media were measured. Hepatocyte permeability to lactate dehydrogenase did not increase during cold storage of 1 to 7 days (7.2% +/- 2% leakage), indicating that most of the hepatocytes remained viable during cold storage. However, when rewarmed, loss of viability (leakage of lactate dehydrogenase) was dependent on the composition of the resuspension media. In Krebs-Henseleit buffer, viability was reduced after 2 and 3 days of storage (lactate dehydrogenase leakage on rewarming = 70% to 90%). Leakage of lactate dehydrogenase was reduced significantly after resuspension in tissue-culture media. After 6 days of storage, lactate dehydrogenase leakage from hepatocytes stored in Liebovitz- 15 or modified Fischer's was only about 30%.(ABSTRACT TRUNCATED AT 250 WORDS)
肝细胞悬液提供了一种快速方法，用于确定低温保存如何影响肝细胞代谢和活力。通过这些研究，或许可以开发出改进的用于移植的低温肝脏保存方法。在本研究中，大鼠肝细胞在威斯康星大学肝脏保存液中冷藏长达7天。在储存的每一天结束时，将肝细胞重悬于 Krebs-Henseleit 缓冲液或组织培养基（莱博维茨-15培养基；费舍尔培养基；改良费舍尔培养基，其与费舍尔培养基相似，但添加了甘氨酸和半胱氨酸；或韦茅斯培养基）中。通过对悬液进行复温和充氧，并测量乳酸脱氢酶从细胞中的泄漏百分比、细胞内钾离子浓度以及琥珀酸对呼吸的刺激作用（所有这些都是质膜完整性的指标）来评估肝细胞活力。此外，还测量了在各种重悬培养基中复温和复氧后ATP 和谷胱甘肽的浓度。在1至7天的冷藏期间，肝细胞对乳酸脱氢酶的通透性没有增加（泄漏率为7.2%±2%），这表明大多数肝细胞在冷藏期间仍保持活力。然而，复温时，活力丧失（乳酸脱氢酶泄漏）取决于重悬培养基的成分。在 Krebs-Henseleit 缓冲液中，储存2天和3天后活力降低（复温时乳酸脱氢酶泄漏率为70%至90%）。重悬于组织培养基后，乳酸脱氢酶的泄漏显著减少。储存6天后，储存在莱博维茨-15培养基或改良费舍尔培养基中的肝细胞的乳酸脱氢酶泄漏率仅约为30%。（摘要截断于250字）
