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Subzero non-frozen preservation of human livers in the supercooled state


Preservation of human organs at subzero temperatures has been an elusive goal for decades. The major complication hindering successful subzero preservation is the formation of ice at temperatures below freezing. Supercooling, or subzero non-freezing, preservation completely avoids ice formation at subzero temperatures. We previously showed that rat livers can be viably preserved three times longer by supercooling as compared to hypothermic preservation at +4 °C. Scalability of supercooling preservation to human organs was intrinsically limited because of volume-dependent stochastic ice formation at subzero temperatures. However, we recently adapted the rat preservation approach so it could be applied to larger organs. Here, we describe a supercooling protocol that averts freezing of human livers by minimizing air–liquid interfaces as favorable sites of ice nucleation and uses preconditioning with cryoprotective agents to depress the freezing point of the liver tissue. Human livers are homogeneously preconditioned during multiple machine perfusion stages at different temperatures. Including preparation, the protocol takes 31 h to complete. Using this protocol, human livers can be stored free of ice at –4 °C, which substantially extends the ex vivo life of the organ. To our knowledge, this is the first detailed protocol describing how to perform subzero preservation of human organs.

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Fig. 1: The supercooling protocol.
Fig. 2: Human liver perfusion system.
Fig. 3: Schematic design of the flow diverter.
Fig. 4: Liver graft after preparation for supercooling preservation.
Fig. 5: Key ex vivo viability parameters during pre- and post-supercooling SNMP.

Data availability

The authors declare that the data supporting the findings of this study are available within the paper and its Supplementary Information files. Any additional data, if needed, will be provided upon request.


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Funding from the US National Institutes of Health (R01DK096075, R01DK107875, R01DK114506 and R21EB023031) and the Department of Defense RTRP W81XWH-17-1-0680 and DHP SBIR H151-013-0141 is gratefully acknowledged. We thank Sylvatica Biotech, Inc., for collaboration and support through the NIH (R21EB023031) and the Department of Defense (DHP SBIR H151-013-0141). This work was partially supported by the Office of Assistant Secretary of Defense for Health Affairs, through the Reconstructive Transplant Research Program, Technology Development Award (under Award No. W81XWH-17-1-0680). The US Army Medical Research Acquisition Activity, 820 Chandler Street, Fort Detrick, MD 21702-5014 is the awarding and administering acquisition office. Opinions, interpretations, conclusions and recommendations are those of the author and are not necessarily endorsed by the Department of Defense. R.J.V. acknowledges support from the Tosteson Fellowship awarded by the Executive Committee on Research at the Massachusetts General Hospital and a stipend from the Michael van Vloten Fund for Surgical Research. S.N.T. acknowledges support from NIH K99 HL143149. We thank M. Karabacak, Y. M. Yu and F. Lin at the Mass Spectrometry Core Facility (Shriners Hospital for Children, Boston, Massachusetts) for assistance with adenylate quantification. We thank L. Burlage, A. Matton, B. Bruinsma and C. Pendexter for experimental assistance. Finally, appreciation is extended to LiveON NY, and we are especially grateful for our collaboration with New England Donor Services and their generous support that enables research with human donor organs.

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Authors and Affiliations



R.J.d.V., S.N.T. and K.U. conceived and designed the supercooling protocol for human livers; R.J.d.V., S.N.T. and S.O. designed and constructed the perfusion and supercooling system; R.J.d.V., S.N.T., P.D.B., S.N., S.E.J.C. and S.O. performed supercooling experiments and acquired experimental data; R.J.d.V., S.N.T., E.O.A.H., H.Y., M.L.Y., J.F.M., M.T. and K.U. analyzed and interpreted data; R.J.d.V. wrote the manuscript; R.J.d.V., S.N.T., E.O.A.H., T.M.v.G., M.L.Y., J.F.M., M.T., H.Y. and K.U. participated in critical revision of the manuscript for intellectual content; R.J.d.V., S.N.T. and K.U. performed statistical analyses. All authors contributed to the preparation of the manuscript.

Corresponding author

Correspondence to Korkut Uygun.

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Competing interests

M.T., M.L.Y., R.J.d.V., K.U. and S.N.T. have provisional patent applications relevant to this study. K.U. has a financial interest in Organ Solutions, a company focused on developing organ preservation technology. The authors’ interests are managed by Massachusetts General Hospital and Partners HealthCare in accordance with their conflict of interest policies.

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Peer review information Nature Protocols thanks Cyril Moers and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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de Vries, R. J. et al. Nat. Biotechnol. 37, 1131–1136 (2019):

Integrated supplementary information

Supplementary Figure 1 Design of custom glass perfusion chamber.

The perfusion chamber is custom made according to these drawings. Although the drawings are on scale, the dimensions are not critical and may vary as the part is hand-made from blown glass.

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de Vries, R.J., Tessier, S.N., Banik, P.D. et al. Subzero non-frozen preservation of human livers in the supercooled state. Nat Protoc 15, 2024–2040 (2020).

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