Supercooling extends preservation time of human livers

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Abstract

The inability to preserve vascular organs beyond several hours contributes to the scarcity of organs for transplantation1,2. Standard hypothermic preservation at +4 °C (refs. 1,3) limits liver preservation to less than 12 h. Our group previously showed that supercooled ice-free storage at –6 °C can extend viable preservation of rat livers4,5 However, scaling supercooling preservation to human organs is intrinsically limited because of volume-dependent stochastic ice formation. Here, we describe an improved supercooling protocol that averts freezing of human livers by minimizing favorable sites of ice nucleation and homogeneous preconditioning with protective agents during machine perfusion. We show that human livers can be stored at –4 °C with supercooling followed by subnormothermic machine perfusion, effectively extending the ex vivo life of the organ by 27 h. We show that viability of livers before and after supercooling is unchanged, and that after supercooling livers can withstand the stress of simulated transplantation by ex vivo normothermic reperfusion with blood.

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Fig. 1: Outline of research design.
Fig. 2: Key ex vivo viability parameters and histology during pre- and post-supercooling SNMP.
Fig. 3: Key ex vivo viability parameters during simulated transplantation by normothermic blood reperfusion.

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 reasonable request.

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Acknowledgements

Funding from the US National Institutes of Health (NIH) (grant nos. R01DK096075, R01DK107875 and R01DK114506) and the Department of Defense (contract no. RTRP W81XWH-17-1-0680) are gratefully acknowledged. We thank Sylvatica Biotech, Inc. and are grateful for support through the NIH (grant no. R21EB023031) and the US Army MRDC (contract no. W81XWH-16-C-0067). R.J.V. acknowledges a stipend from the Michael van Vloten Fund for Surgical Research. S.N.T. acknowledges support from NIH grant no. K99 HL143149. We thank M. Karabacak, Y.M. Yu and F. Lin at the Mass Spectrometry Core Facility (Shriners Hospital for Children, Boston, MA) for assistance with adenylate quantification. We thank L. Burlage, A. Matton, B. Bruinsma and C. Pendexter for experimental assistance. Finally, appreciation is extended to LiveOnNY, and we are especially grateful for our collaboration with New England Donor Services and their generous support that enables research with human donor organs. The views, opinions and/or findings contained in this manuscript are those of the authors and should not be construed as an official position, policy or decision of any of the institutions that supported the research, unless so designated by other documentation.

Author information

R.J.V., S.N.T. and K.U. conceived and designed the study. R.J.V., P.D.B., S.N., S.E.J.C. and S.O. performed data acquisition. R.J.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.V., S.N.T. and S.O. designed and constructed the perfusion and supercooling system. R.J.V., S.N.T. and K.U. wrote the manuscript. R.J.V., S.N.T., E.O.A.H., T.M.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.V., S.N.T. and K.U. performed statistical analysis. All authors contributed to the preparation of the manuscript.

Correspondence to Korkut Uygun.

Ethics declarations

Competing interests

The authors declare competing financial interests. M.T., M.L.Y., R.J.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. Authors’ interests are managed by MGH and Partners HealthCare in accordance with their conflict of interest policies.

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Integrated supplementary information

Supplementary Figure 1 First attempt at supercooling a human liver with the rat protocol.

Left: A liver after convetntional hypothermic preservation (HP) in University of Winsconsin solution (UW). Right: A liver frozen at –6ºC after ealier failed attempts to supercool, shown after recovery from the chiller.

Supplementary Figure 2 Melting point depression of the liver grafts.

The symbols of the dot plot overlay correspond to the unique independent biological replicates (n = 5) and match between Figs. 2 and 3; Supplementary Figs. 2, 4 and 5; and Supplementary Table 2. Left: melting point of the human livers preserved University of Wisconsin hypothermic preservation (HP) solution. Right: melting point of the same human livers preserved in our supercooling (SC) preservation solution. Star denotes statistical significance (p = 0.001, paired two-tailed student’s t tests).

Supplementary Figure 3 Photos of the livers during the supercooling protocol.

a) Liver during the cooling phase of subnormothermic machine perfusion (SNMP). (b) Liver submerged in the chiller basin during ice-free subzero supercooled storage. (c) Liver during reperfusion.

Supplementary Figure 4 Important ex vivo viability parameters and histology during pre- and post-supercooling subnormothermic machine perfusion.

(a) Tissue adenylate triphoshate (ATP) and adenylate monophosphate (AMP) ratio. Blue denotes pre-supercooling and green denotes post-supercooling throughout the figure. The symbols of the dot plot overlay correspond to the unique independent biological replicates (n = 5 throughout the figure) and match between Figs. 2 and 3; Supplementary Figs. 2, 4 and 5; and Supplementary Table 2. (b) Tissue adenylate triphosphate (ATP) and adenylate diphosphate (ADP) ratio. (c) Lactate clearance derived from in and outflow measurements. (d) Lactate concentration (top) and pH (below) of the arterial inflow. (e) Perfusate potassium concentrations. (f) Urea concentrations in the perfusate. Stars denote statistical significance (p < 0.05, repeated measures two-way ANOVA followed by the Sidak multiple comparisons test). Boxes: median and IQR. Whiskers: min and max. Error bars of line graphs: mean ± SEM.

Supplementary Figure 5 Important ex vivo viability parameters during simulated transplantation by normothermic blood reperfusion.

(a) Tissue adenylate triphosphate (ATP) and adenylate monophosphate (AMP) ratio. The symbols of the dot plot overlay correspond to the unique independent biological replicates (n = 3 throughout the figure) and match between Figs. 2 and 3; Supplementary Figs. 2, 4 and 5; and Supplementary Table 2. (b) Tissue adenylate triphosphate (ATP) and adenylate diphosphate (ADP) ratio. (c) Oxygen uptake. (d) Plasma urea concentrations. (e) Lactate clearance derived from in and outflow measurements. (f) Plasma potassium concentrations. Error bars: mean ± SEM.

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