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Cryopreservation of Whole Rat Livers by Vitrification and Nanowarming

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Abstract

Liver cryopreservation has the potential to enable indefinite organ banking. This study investigated vitrification—the ice-free cryopreservation of livers in a glass-like state—as a promising alternative to conventional cryopreservation, which uniformly fails due to damage from ice formation or cracking. Our unique “nanowarming” technology, which involves perfusing biospecimens with cryoprotective agents (CPAs) and silica-coated iron oxide nanoparticles (sIONPs) and then, after vitrification, exciting the nanoparticles via radiofrequency waves, enables rewarming of vitrified specimens fast enough to avoid ice formation and uniformly enough to prevent cracking from thermal stresses, thereby addressing the two main failures of conventional cryopreservation. This study demonstrates the ability to load rat livers with both CPA and sIONPs by vascular perfusion, cool them rapidly to an ice-free vitrified state, and rapidly and homogenously rewarm them. While there was some elevation of liver enzymes (Alanine Aminotransferase) and impaired indocyanine green (ICG) excretion, the nanowarmed livers were viable, maintained normal tissue architecture, had preserved vascular endothelium, and demonstrated hepatocyte and organ-level function, including production of bile and hepatocyte uptake of ICG during normothermic reperfusion. These findings suggest that cryopreservation of whole livers via vitrification and nanowarming has the potential to achieve organ banking for transplant and other biomedical applications.

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Abbreviations

ALT:

Alanine aminotransferase

AMF:

Alternating magnetic field

CRF:

Controlled rate freezer

CPA:

Cryoprotective agent

CCR:

Critical cooling rate

CWR:

Critical warming rate

DCD:

Donation after cardiac death

ECD:

Extended criteria donors

EC:

Euro-Collins

EM:

Electromagnetic

EG:

Ethylene glycol

HU:

Hounsfield units

ICG:

Indocyanine green

LDH:

Lactate dehydrogenase

µCT:

Micro-computed tomography

RF:

Radiofrequency

sIONP:

Silica coated iron oxide nanoparticles

T g :

Glass transition temperature

T m :

Melting temperature

UW:

University of Wisconsin

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Acknowledgments

This work was funded by NIH (HL135046, DK117425, DK131209, DK119043, and DK126551) and NSF EEC 1941543. JSR acknowledges the support of the Schulze Diabetes Institute. We acknowledge the assistance of Dr. Mark Sanders of the University of Minnesota Imaging Center for assistance in freeze-substitution experiments.

Data Availability

The raw data required to reproduce these findings are available to download from Mendeley https://doi.org/10.17632/9nsz5chp79.1.

Conflict of interest

The authors (Sharma, Lee, Etheridge, Bischof, Finger) declare patents issued and pending related to the described methodology.

Author information

Author notes

  1. Anirudh Sharma and Charles Y. Lee have contributed equally to this work.

  2. John C. Bischof and Erik B. Finger have contributed equally to this work as co-senior authors.

Authors and Affiliations

  1. Department of Mechanical Engineering, University of Minnesota, Minneapolis, MN, 55455, USA

    Anirudh Sharma, Zonghu Han, Zhe Gao, Michael L. Etheridge & John C. Bischof

  2. Department of Mechanical Engineering and Engineering Science, University of North Carolina, Charlotte, NC, 28223, USA

    Charles Y. Lee

  3. Center for Biomedical Engineering and Science, University of North Carolina, Charlotte, NC, 28223, USA

    Charles Y. Lee & Mark G. Clemens

  4. Department of Surgery, University of Minnesota, Minneapolis, MN, 55455, USA

    Bat-Erdene Namsrai, Diane Tobolt, Joseph Sushil Rao & Erik B. Finger

  5. Schulze Diabetes Institute, University of Minnesota, Minneapolis, MN, 55455, USA

    Joseph Sushil Rao

  6. Department of Radiology, Center for Magnetic Resonance Research, University of Minnesota, Minneapolis, MN, 55455, USA

    Michael Garwood

  7. Department of Biological Sciences, University of North Carolina, Charlotte, NC, 28223, USA

    Mark G. Clemens

  8. Division of Solid Organ Transplantation, University of Minnesota, 420 Delaware St. S.E., MMC 195, Minneapolis, MN, 55455, USA

    Erik B. Finger

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  1. Anirudh Sharma
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Sharma, A., Lee, C.Y., Namsrai, BE. et al. Cryopreservation of Whole Rat Livers by Vitrification and Nanowarming. Ann Biomed Eng 51, 566–577 (2023). https://doi.org/10.1007/s10439-022-03064-2

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