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

Reinier J. de Vries, Shannon N. Tessier, Peony D. Banik, Sonal Nagpal, Stephanie E.J. Cronin, Sinan Ozer, Ehab O.A. Hafiz, Thomas M. van Gulik, Martin L. Yarmush, James F. Markmann, Mehmet Toner, Heidi Yeh, Korkut Uygun

Research output: Contribution to journalArticlepeer-review

25 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)2024-2040
Number of pages17
JournalNature Protocols
Issue number6
StatePublished - Jun 1 2020
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)


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