Abstract
The current standard for liver preservation involves cooling of the organ on ice (0–4 °C). Although it is successful for shorter durations, this method of preservation does not allow long-term storage of the liver. The gradual loss of hepatic viability during preservation puts pressure on organ sharing and allocation, may limit the use of suboptimal grafts and necessitates rushed transplantation to achieve desirable post-transplantation outcomes. In an attempt to improve and prolong liver viability during storage, alternative preservation methods are under investigation. For instance, ex vivo machine perfusion systems aim to sustain and even improve viability by supporting hepatic function at warm temperatures, rather than simply slowing down deterioration by cooling. Here we describe a novel subzero preservation technique that combines ex vivo machine perfusion with cryoprotectants to facilitate long-term supercooled preservation. The technique improves the preservation of rat livers to prolong storage times as much as threefold, which is validated by successful long-term recipient survival after orthotopic transplantation. This protocol describes how to load rat livers with cryoprotectants to prevent both intracellular and extracellular ice formation and to protect against hypothermic injury. Cryoprotectants are loaded ex vivo using subnormothermic machine perfusion (SNMP), after which livers can be cooled to −6 °C without freezing and kept viable for up to 96 h. Cooling to a supercooled state is controlled, followed by 3 h of SNMP recovery and orthotopic liver transplantation.
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Acknowledgements
Funding from the US National Institutes of Health (R00DK080942, R01DK096075 and R01EB008678) and the Shriners Hospitals for Children is gratefully acknowledged.
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B.G.B., T.A.B., M.-L.I., M.L.Y. and K.U. optimized and developed the rat liver supercooling technique. T.A.B. and B.G.B. performed the rat transplantations and gathered data from the supercooling and the static cold storage control experiments. K.U. oversaw the analysis and final presentation of the data. B.G.B. and T.A.B. contributed to the manuscript, and B.G.B., T.A.B., M.-L.I., H.Y., M.L.Y. and K.U. contributed to the review.
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K.U. and M.L.Y. are inventors on a pending international patent application that involves some of the perfusion technology used in this work (WO/2011/002926); T.A.B., M.L.Y. and K.U. are inventors on a pending international patent application that describes the supercooling protocol used in this work (WO/2011/35223); and B.G.B. and K.U. have a provisional patent application related to this work that describes scale-up of the perfusion protocol to human livers. K.U. has a financial interest in Organ Solutions LLC, a company focused on developing organ preservation technology. K.U.'s interests are managed by the Massachusetts General Hospital and Partners HealthCare in accordance with their conflict-of-interest policies.
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Bruinsma, B., Berendsen, T., Izamis, ML. et al. Supercooling preservation and transplantation of the rat liver. Nat Protoc 10, 484–494 (2015). https://doi.org/10.1038/nprot.2015.011
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DOI: https://doi.org/10.1038/nprot.2015.011
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