1 Dhawan Lab at Institute of Liver Studies, King's College London School of Medicine at King's College Hospital, London, United Kingdom.
2 Department of Pediatrics, King Chulalongkorn Memorial Hospital, Bangkok, Thailand.
Cell Transplant. 2017 Aug;26(8):1341-1354. doi: 10.1177/0963689717720050.
Intraperitoneal transplantation of hepatocyte microbeads is an attractive option for the management of acute liver failure. Encapsulation of hepatocytes in alginate microbeads supports their function and prevents immune attack of the cells. Establishment of banked cryopreserved hepatocyte microbeads is important for emergency use. The aim of this study was to develop an optimized protocol for cryopreservation of hepatocyte microbeads for clinical transplantation using modified freezing solutions. Four freezing solutions with potential for clinical application were investigated. Human and rat hepatocytes cryopreserved with University of Wisconsin (UW)/10% dimethyl sulfoxide (DMSO)/5% (300 mM) glucose and CryoStor CS10 showed better postthawing cell viability, attachment, and hepatocyte functions than with histidine-tryptophan-ketoglutarate/10% DMSO/5% glucose and Bambanker. The 2 freezing solutions that gave better results were studied with human and rat hepatocytes microbeads. Similar effects on cryopreserved microbead morphology (external and ultrastructural), viability, and hepatocyte-functions post thawing were observed over 7 d in culture. UW/DMSO/glucose, as a basal freezing medium, was used to investigate the additional effects of cytoprotectants: a pan-caspase inhibitor (benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone [ZVAD]), an antioxidant (desferoxamine [DFO]), and a buffering and mechanical protectant (human serum albumin [HSA]) on RMBs. ZVAD (60 µM) had a beneficial effect on cell viability that was greater than with DFO (1 mM), HSA (2%), and basal freezing medium alone. Improvements in the ultrastructure of encapsulated hepatocytes and a lower degree of cell apoptosis were observed with all 3 cytoprotectants, with ZVAD tending to provide the greatest effect. Cytochrome P450 activity was significantly higher in the 3 cytoprotectant groups than with fresh microbeads. In conclusion, developing an optimized cryopreservation protocol by adding cytoprotectants such as ZVAD could improve the outcome of cryopreserved hepatocyte microbeads for future clinical use.
细胞微球经腹腔移植是治疗急性肝衰竭的一种很有吸引力的选择。藻酸盐微球包裹的肝细胞可以支持其功能并防止细胞的免疫攻击。建立可储存的低温保存的肝细胞微球对于紧急使用非常重要。本研究旨在开发一种使用改良的冷冻溶液对肝细胞微球进行临床移植的优化冷冻保存方案。研究了四种具有临床应用潜力的冷冻溶液。与Histidine-Tryptophan-Ketoglutarate/10% DMSO/5%葡萄糖和 Bambanker 相比，用 UW/10%DMSO/5%（300mM）葡萄糖和 CryoStor CS10 冷冻保存的人源和大鼠源肝细胞的冷冻后细胞活力、附着和肝细胞功能更好。对这两种效果更好的冷冻溶液进行了研究，结果发现，在培养的 7 天内，对冷冻保存的微球形态（外部和超微结构）、活力和肝细胞功能均有类似的影响。UW/DMSO/葡萄糖作为基础冷冻培养基，研究了细胞保护剂的附加作用：一种全胱天冬酶抑制剂（苯甲氧基羰基-Val-Ala-dl-Asp-氟甲基酮[ZVAD]）、一种抗氧化剂（去铁胺[DFO]）和一种缓冲和机械保护剂（人血清白蛋白[HSA]）对 RMB 的影响。ZVAD（60μM）对细胞活力的有益作用大于 DFO（1mM）、HSA（2%）和基础冷冻培养基单独使用。所有 3 种细胞保护剂都观察到了对包封的肝细胞超微结构的改善和细胞凋亡程度的降低，ZVAD 效果最好。与新鲜微球相比，3 种细胞保护剂组的细胞色素 P450 活性显著升高。总之，通过添加细胞保护剂（如 ZVAD）来开发优化的冷冻保存方案可以提高低温保存的肝细胞微球在未来临床应用中的效果。
