General Surgery 2U, Liver Transplantation Center, AOU Città della Salute e della Scienza di Torino, University of Turin, Turin, Italy.
2i3T, Società per La Gestione Dell'incubatore Di Imprese e Per Il Trasferimento Tecnologico Dell'Università degli Studi di Torino, Scarl., Molecular Biotechnology Center (MBC), Turin, Italy.
Transplantation. 2018 May;102(5):e205-e210. doi: 10.1097/TP.0000000000002123.
The gold standard for organ preservation before transplantation is static cold storage, which is unable to fully protect suboptimal livers from ischemia/reperfusion injury. An emerging alternative is normothermic machine perfusion (NMP), which permits organ reconditioning. Here, we aimed to explore the feasibility of a pharmacological intervention on isolated rat livers by using a combination of NMP and human liver stem cells-derived extracellular vesicles (HLSC-EV).
We established an ex vivo murine model of NMP capable to maintain liver function despite an ongoing hypoxic injury induced by hemodilution. Livers were perfused for 4 hours without (control group, n = 10) or with HLSC-EV (treated group, n = 9). Bile production was quantified; perfusate samples were collected hourly to measure metabolic (pH, pO2, pCO2) and cytolysis parameters (AST, alanine aminotransferase, lactate dehydrogenase). At the end of perfusion, we assessed HLSC-EV engraftment by immunofluorescence, tissue injury by histology, apoptosis by terminal deoxynucleotidyl transferase dUTP nick-end labeling assay, tissue hypoxia-inducible factor 1-α, and transforming growth factor-beta 1 RNA expression by quantitative reverse transcription-polymerase chain reaction.
During hypoxic NMP, livers were able to maintain homeostasis and produce bile. In the treated group, AST (P = 0.018) and lactate dehydrogenase (P = 0.032) levels were significantly lower than those of the control group at 3 hours of perfusion, and AST levels persisted lower at 4 hours (P = 0.003). By the end of NMP, HLSC-EV had been uptaken by hepatocytes, and EV treatment significantly reduced histological damage (P = 0.030), apoptosis (P = 0.049), and RNA overexpression of hypoxia-inducible factor 1-α (P < 0.0001) and transforming growth factor-beta 1 (P = 0.014).
HLSC-EV treatment, even in a short-duration model, was feasible and effectively reduced liver injury during hypoxic NMP.
器官移植前的金标准是静态冷保存，但它无法完全保护不理想的肝脏免受缺血/再灌注损伤。一种新兴的替代方法是常温机器灌注（NMP），它可以使器官再适应。在这里，我们旨在通过使用 NMP 和人肝干细胞衍生的细胞外囊泡（HLSC-EV）的组合，探索对离体大鼠肝脏进行药理学干预的可行性。
我们建立了一种体外鼠 NMP 模型，即使在由血液稀释引起的持续缺氧损伤下，该模型也能够维持肝功能。肝脏在没有（对照组，n=10）或有 HLSC-EV（治疗组，n=9）的情况下灌注 4 小时。定量胆汁生成；每小时收集一次灌注液样本，以测量代谢（pH 值、pO2、pCO2）和细胞溶解参数（AST、丙氨酸氨基转移酶、乳酸脱氢酶）。在灌注结束时，我们通过免疫荧光评估 HLSC-EV 移植，通过组织学评估组织损伤，通过末端脱氧核苷酸转移酶 dUTP 末端标记测定法评估细胞凋亡，通过定量逆转录-聚合酶链反应评估组织缺氧诱导因子 1-α 和转化生长因子-β 1 RNA 表达。
在缺氧 NMP 期间，肝脏能够维持内稳态并产生胆汁。在治疗组中，与对照组相比，在灌注 3 小时时 AST（P=0.018）和乳酸脱氢酶（P=0.032）水平显著降低，在 4 小时时 AST 水平持续降低（P=0.003）。在 NMP 结束时，HLSC-EV 已被肝细胞摄取，EV 治疗可显著减轻组织损伤（P=0.030）、细胞凋亡（P=0.049）以及缺氧诱导因子 1-α（P＜0.0001）和转化生长因子-β 1（P=0.014）的 RNA 过表达。
即使在短时间模型中，HLSC-EV 治疗也是可行的，可有效减轻缺氧 NMP 期间的肝脏损伤。
