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Extracellular Vesicles from Human Liver Stem Cells Reduce Injury in an Ex Vivo Normothermic Hypoxic Rat Liver Perfusion Model

Rigo, Federica, MD1; De Stefano, Nicola, BMSc1; Navarro-Tableros, Victor, MD, PhD2; David, Ezio, MD3; Rizza, Giorgia, MD1; Catalano, Giorgia, MD1; Gilbo, Nicholas, MD1; Maione, Francesca, MD1; Gonella, Federica, MD1; Roggio, Dorotea, MSc1; Martini, Silvia, MD4; Patrono, Damiano, MD, PhD1; Salizzoni, Mauro, MD1; Camussi, Giovanni, MD5; Romagnoli, Renato, MD1

doi: 10.1097/TP.0000000000002123
Original Basic Science—Liver

Background 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).

Methods 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.

Results 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).

Conclusions HLSC-EV treatment, even in a short-duration model, was feasible and effectively reduced liver injury during hypoxic NMP.

The authors investigate the feasibility and efficacy of a pharmacological intervention on isolated rat livers by using a combination of normorthermic machine perfusion and human liver stem-cell-derived extracellular vesicles.

1 General Surgery 2U, Liver Transplantation Center, AOU Città della Salute e della Scienza di Torino, University of Turin, Turin, Italy.

2 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.

3 Pathology Unit, Molinette Hospital, AOU Città della Salute e della Scienza di Torino, Turin, Italy.

4 Gastrohepatology Unit, Molinette Hospital, AOU Città della Salute e della Scienza di Torino, Turin, Italy.

5 Department of Medical Sciences, University of Turin, Turin, Italy.

Received 5 August 2017. Revision received 27 December 2017.

Accepted 27 December 2017.

F.R., N.D.S., and V.N.-T. contributed equally to this work.

G.C. and R.R. are senior authors and contributed equally to this work.

Ricerca Locale Ex 60%, University of Turin—year 2015 and year 2016.

Conflict of interest: G.C. is named as inventor in patents related to the regenerative effects of HL SC-derived extracellular vesicles. All other authors declare no conflict of interest.

F.R., N.D.S., and V.N. performed the experiments, analyzed the data, and wrote the article. E.D. and D.R. contributed in histopathological and biomolecular analyses. G.R., G.C., N.G., F.M., F.G., S.M., and D.P. participated in surgical procedures and data analysis. M.S., G.C., and R.R. gave intellectual input for study design, analyzed the data, and revised the article.

Correspondence: Renato Romagnoli, MD, General Surgery, 2U-Liver Transplantation Center, AOU Città della Salute e della Scienza di Torino, Molinette Hospital, Department of Surgical Sciences, University of Turin, Corso Dogliotti 14, 10126 Torino, Italy. (renato.romagnoli@unito.it).

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