*Purpose: Normothermic ex vivo machine perfusion of liver grafts (EVLP) is emerging as a superior preservation technology to cold storage. Normothermic EVLP transforms a metabolically dormant, continuously injured organ into a functioning one and improves the function and outcome of marginal liver transplants. Mechanisms underlying the improved restoration of livers subjected to EVLP prior to transplant are under study. We tested the hypothesis that subjecting livers to EVLP would decrease their innate immune activation and ability to active donor-reactive responses following reperfusion.

*Methods: Since the donor-reactive response is largely induced by graft production and release of exosomes, we tested graft production of exosomes in the perfusate during the 4-6 hours of EVLP. Perfusate was obtained at the beginning and every two hours after initiation of EVLP for 10 livers conditioned for transplant. Exosomes were isolated from the perfusates by a four centrifugation cycle and quantitated by ZetaView.

*Results: For each of the 10 livers, graft exosomes were released as a bolus within 2 hours of EVLP initiation and then were no longer detectable in the perfusate. The exosomes were able to stimulate the proliferation of CFSE-labelled CD4 and CD8 T cells when added to peripheral blood mononuclear cell cultures for 3 days and then analyzed by flow cytometry for CFSE dilution as an indicator of T cell proliferation. Biopsies were procured from livers subjected to EVLP and control unconditioned livers just prior to placement into the recipient abdomen (implantation biopsy) and another at 1-2 hours after revascularization, just prior to abdomen closure (reperfusion biopsy). Biopsy RNA was isolated and subjected to Nano String analysis using the Human Pan Cancer CodeSet. Reperfusion biopsies (n = 5) of liver transplants subjected to EVLP had marked down regulation of gene expression associated with pathways involved in complement activation and complement regulation, inflammasome activation, and chemokine receptor signaling.

*Conclusions: Overall, these results indicate that normothermic EVLP prior to transplant reduces the inflammatory response to reperfusion during transplant and suggests that the graft allogenicity may also be decreased through release of immunogenic exosomes during the perfusion.

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