Abrahamse Salomon L, van Runnard Heimel Pieter, Hartman Robin J, Chamuleau Rob A F M, van Gulik Thomas M
Departments of Surgery (Surgical Laboratory), Academic Medical Center, The University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands.
Cell Transplant. 2003;12(1):59-68. doi: 10.3727/000000003783985160.
Donor cells can be preserved in University of Wisconsin (UW), histidine-tryptophan-ketoglutarate (HTK), or Celsior solution. However, differences in efficacy and mode of action in preventing hypothermia-induced cell injury have not been unequivocally clarified. Therefore, we investigated and compared necrotic and apoptotic cell death of freshly isolated primary porcine hepatocytes after hypothermic preservation in UW, HTK, and Celsior solutions and subsequent normothermic culturing. Hepatocytes were isolated from porcine livers, divided in fractions, and hypothermically (4 degrees C) stored in phosphate-buffered saline (PBS), UW, HTK, or Celsior solution. Cell necrosis and apoptosis were assessed after 24- and 48-h hypothermic storage and after 24-h normothermic culturing following the hypothermic preservation periods. Necrosis was assessed by trypan blue exclusion, lactate dehydrogenase (LDH) release, and mitochondrial 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reduction. Apoptosis was assessed by the induction of histone-associated DNA fragments and cellular caspase-3 activity. Trypan blue exclusion, LDH release, and MTT reduction of hypothermically preserved hepatocytes showed a decrease in cell viability of more than 50% during the first 24 h of hypothermic preservation. Cell viability was further decreased after 48-h preservation. DNA fragmentation was slightly enhanced in hepatocytes after preservation in all solutions, but caspase-3 activity was not significantly increased in these cells. Normothermic culturing of hypothermically preserved cells further decreased cell viability as assessed by LDH release and MTT reduction. Normothermic culturing of hypothermically preserved hepatocytes induced DNA fragmentation, but caspase-3 activity was not hanced in these cells. Trypan blue exclusion, LDH leakage, and MTT reduction demonstrated the highest cell viability after storage in Celsior, and DNA fragmentation was the lowest in cells that had been stored in PBS and UW solutions. None of the preservation solutions tested in this study was capable of adequately preventing cell death of isolated porcine hepatocytes after 24-h hypothermic preservation and subsequent 24-h normothermic culturing. Culturing of isolated and hypothermically preserved hepatocytes induces DNA fragmentation, but does not lead to caspase-3 activation. With respect to necrosis and DNA fragmentation of hypothermically preserved cells, UW and Celsior were superior to PBS and HTK solutions in this model of isolated porcine hepatocyte preservation.
供体细胞可保存在威斯康星大学（UW）溶液、组氨酸-色氨酸-酮戊二酸（HTK）溶液或赛尔西奥（Celsior）溶液中。然而，在预防低温诱导的细胞损伤方面，其功效和作用方式的差异尚未得到明确阐明。因此，我们研究并比较了新鲜分离的原代猪肝细胞在UW、HTK和Celsior溶液中低温保存及随后常温培养后的坏死和凋亡细胞死亡情况。从猪肝脏中分离出肝细胞，分成几份，在磷酸盐缓冲盐水（PBS）、UW、HTK或Celsior溶液中低温（4℃）保存。在低温保存24小时和48小时后以及低温保存期后的常温培养24小时后，评估细胞坏死和凋亡情况。通过台盼蓝排斥试验、乳酸脱氢酶（LDH）释放和线粒体3-[4,5-二甲基噻唑-2-基]-2,5-二苯基四氮唑溴盐（MTT）还原试验评估坏死情况。通过组蛋白相关DNA片段的诱导和细胞半胱天冬酶-3活性评估凋亡情况。低温保存的肝细胞的台盼蓝排斥试验、LDH释放和MTT还原试验显示，在低温保存的前24小时内细胞活力下降超过50%。保存48小时后细胞活力进一步下降。在所有溶液中保存后的肝细胞中，DNA片段化略有增强，但这些细胞中的半胱天冬酶-3活性没有显著增加。通过LDH释放和MTT还原试验评估，低温保存细胞的常温培养进一步降低了细胞活力。低温保存的肝细胞的常温培养诱导了DNA片段化，但这些细胞中的半胱天冬酶-3活性没有增强。台盼蓝排斥试验、LDH泄漏和MTT还原试验表明，在Celsior溶液中保存后细胞活力最高，而在PBS和UW溶液中保存的细胞中DNA片段化最低。在本研究中测试的任何一种保存溶液都不能充分预防分离的猪肝细胞在低温保存24小时及随后常温培养24小时后的细胞死亡。分离的低温保存肝细胞的培养诱导了DNA片段化，但不会导致半胱天冬酶-3激活。就低温保存细胞的坏死和DNA片段化而言，在这种分离的猪肝细胞保存模型中，UW和Celsior溶液优于PBS和HTK溶液。
