Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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

Abstract

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.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it

$39.95

Prices may be subject to local taxes which are calculated during checkout

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.

References

  1. Giwa, S. et al. The promise of organ and tissue preservation to transform medicine. Nat. Biotechnol. 35, 530–542 (2017).

    Article  CAS  Google Scholar 

  2. Buying time for transplants. Nat. Biotechnol. 35, 801 (2017).

  3. de Vries, R. J., Yarmush, M. & Uygun, K. Systems engineering the organ preservation process for transplantation. Curr. Opin. Biotechnol. 58, 192–201 (2019).

    Article  Google Scholar 

  4. Bruinsma, B. G. & Uygun, K. Subzero organ preservation: the dawn of a new ice age? Curr. Opin. Organ Transpl. 22, 281–286 (2017).

    Article  Google Scholar 

  5. Pan, E. T. et al. Cold ischemia time is an important risk factor for post-liver transplant prolonged length of stay. Liver Transpl. 24, 762–768 (2018).

    Article  Google Scholar 

  6. Usta, O. B. et al. Supercooling as a viable non-freezing cell preservation method of rat hepatocytes. PLoS ONE 8, e69334 (2013).

    Article  CAS  Google Scholar 

  7. Berendsen, T. A. et al. Supercooling enables long-term transplantation survival following 4 days of liver preservation. Nat. Med. 20, 790–793 (2014).

    Article  CAS  Google Scholar 

  8. Bruinsma, B. G. et al. Supercooling preservation and transplantation of the rat liver. Nat. Protoc. 10, 484–494 (2015).

    Article  CAS  Google Scholar 

  9. Huang, H., Yarmush, M. L. & Usta, O. B. Long-term deep-supercooling of large-volume water and red cell suspensions via surface sealing with immiscible liquids. Nat. Commun. 9, 3201 (2018).

    Article  Google Scholar 

  10. de Vries, R. J. et al. Supercooling extends preservation time of human livers. Nat. Biotechnol. 37, 1131–1136 (2019).

  11. Fuller, B. J., Petrenko, A. & Guibert, E. Human organs come out of the deep cold. Nat. Biotechnol. 37, 1127–1128 (2019).

    Article  CAS  Google Scholar 

  12. Guarrera, J. V. et al. Hypothermic machine preservation facilitates successful transplantation of “orphan” extended criteria donor livers: machine preservation of ECD livers. Am. J. Transpl. 15, 161–169 (2015).

    Article  CAS  Google Scholar 

  13. Muller, X. et al. Can hypothermic oxygenated perfusion (HOPE) rescue futile DCD liver grafts? HPB 21, 1156–1165 (2019).

    Article  CAS  Google Scholar 

  14. Schlegel, A. et al. Outcomes of DCD liver transplantation using organs treated by hypothermic oxygenated perfusion before implantation. J. Hepatol. 70, 50–57 (2019).

    Article  CAS  Google Scholar 

  15. Mergental, H. et al. Transplantation of declined liver allografts following normothermic ex-situ evaluation. Am. J. Transplant. 16, 3235–3245 (2016).

    Article  CAS  Google Scholar 

  16. Ravikumar, R. et al. Liver transplantation after ex vivo normothermic machine preservation: a phase 1 (first-in-man) clinical trial. Am. J. Transplant. 16, 1779–1787 (2016).

    Article  CAS  Google Scholar 

  17. Bral, M. et al. Preliminary single-center Canadian experience of human normothermic ex vivo liver perfusion: results of a clinical trial. Am. J. Transplant. 17, 1071–1080 (2017).

    Article  CAS  Google Scholar 

  18. Watson, C. J. E. et al. Observations on the ex situ perfusion of livers for transplantation. Am. J. Transplant. 18, 2005–2020 (2018).

  19. Nasralla, D. et al. A randomized trial of normothermic preservation in liver transplantation. Nature 557, 50–56 (2018).

    Article  CAS  Google Scholar 

  20. Hoyer, D. P. et al. Controlled oxygenated rewarming of cold stored livers prior to transplantation: first clinical application of a new concept. Transplantation 100, 147–152 (2016).

    Article  CAS  Google Scholar 

  21. de Vries, Y. et al. Pretransplant sequential hypo- and normothermic machine perfusion of suboptimal livers donated after circulatory death using a hemoglobin-based oxygen carrier perfusion solution. Am. J. Transplant. 19, 1202–1211 (2019).

    Article  Google Scholar 

  22. Bruinsma, B. G. et al. Subnormothermic machine perfusion for ex vivo preservation and recovery of the human liver for transplantation: subnormothermic machine perfusion of human livers. Am. J. Transpl. 14, 1400–1409 (2014).

    Article  CAS  Google Scholar 

  23. Bruinsma, B. G. et al. Metabolic profiling during ex vivo machine perfusion of the human liver. Sci. Rep. 6, 22415 (2016).

    Article  CAS  Google Scholar 

  24. Sridharan, G. V. et al. Metabolomic modularity analysis (MMA) to quantify human liver perfusion dynamics. Metabolites 7, 58 (2017).

    Article  Google Scholar 

  25. Karangwa, S. A. et al. Machine perfusion of donor livers for transplantation: a proposal for standardized nomenclature and reporting guidelines. Am. J. Transplant. 16, 2932–2942 (2016).

    Article  CAS  Google Scholar 

  26. Baust, J. G., Gao, D. & Baust, J. M. Cryopreservation: an emerging paradigm change. Organogenesis 5, 90–96 (2009).

    Article  Google Scholar 

  27. Pegg, D. E. Principles of cryopreservation. Methods Mol. Biol. 368, 39–57 (2007).

    Article  CAS  Google Scholar 

  28. Finger, E. B. & Bischof, J. C. Cryopreservation by vitrification: a promising approach for transplant organ banking. Curr. Opin. Organ Transplant. 23, 353–360 (2018).

    Article  CAS  Google Scholar 

  29. Fahy, G. M., Wowk, B. & Wu, J. Cryopreservation of complex systems: the missing link in the regenerative medicine supply chain. Rejuvenation Res 9, 279–291 (2006).

    Article  CAS  Google Scholar 

  30. Manuchehrabadi, N. et al. Improved tissue cryopreservation using inductive heating of magnetic nanoparticles. Sci. Transl. Med. 9, eaah4586 (2017).

  31. Storey, K. B. & Storey, J. M. Molecular biology of freezing tolerance. Compr. Physiol. 3, 1283–1308 (2013).

  32. Dutheil, D., Underhaug Gjerde, A., Petit-Paris, I., Mauco, G. & Holmsen, H. Polyethylene glycols interact with membrane glycerophospholipids: is this part of their mechanism for hypothermic graft protection? J. Chem. Biol. 2, 39–49 (2009).

    Article  Google Scholar 

  33. de Vries, R. et al. Extending the human liver preservation time for transplantation by supercooling. Transplantation 102, S396 (2018).

    Article  Google Scholar 

  34. Vajdová, K., Graf, R. & Clavien, P.-A. ATP-supplies in the cold-preserved liver: a long-neglected factor of organ viability. Hepatology 36, 1543–1552 (2002).

    Article  Google Scholar 

  35. Higashi, H., Takenaka, K., Fukuzawa, K., Yoshida, Y. & Sugimachi, K. Restoration of ATP contents in the transplanted liver closely relates to graft viability in dogs. Eur. Surg. Res. 21, 76–82 (1989).

    Article  CAS  Google Scholar 

  36. Bruinsma, B. G. et al. Peritransplant energy changes and their correlation to outcome after human liver transplantation. Transplantation 101, 1637–1644 (2017).

    Article  Google Scholar 

  37. Lanir, A. et al. Hepatic transplantation survival: correlation with adenine nucleotide level in donor liver. Hepatology 8, 471–475 (1988).

    Article  CAS  Google Scholar 

  38. Kamiike, W. et al. Adenine nucleotide metabolism and its relation to organ viability in human liver transplantation. Transplantation 45, 138–143 (1988).

    Article  CAS  Google Scholar 

  39. op den Dries, S. et al. Ex vivo normothermic machine perfusion and viability testing of discarded human donor livers: normothermic perfusion of human livers. Am. J. Transpl. 13, 1327–1335 (2013).

    Article  CAS  Google Scholar 

  40. Sutton, M. E. et al. Criteria for viability assessment of discarded human donor livers during ex vivo normothermic machine perfusion. PloS ONE 9, e110642 (2014).

    Article  Google Scholar 

  41. Reiling, J. et al. Urea production during normothermic machine perfusion: price of success? Liver Transpl. 21, 700–703 (2015).

    Article  Google Scholar 

Download references

Acknowledgements

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.

Author information

Authors and Affiliations

Authors

Contributions

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.

Ethics declarations

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.

Additional information

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

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key reference using this protocol

de Vries, R. J. et al. Nat. Biotechnol. 37, 1131–1136 (2019): https://doi.org/10.1038/s41587-019-0223-y

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.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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). https://doi.org/10.1038/s41596-020-0319-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41596-020-0319-3

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research