There are currently ~100,000 patients on the organ transplant waiting list in the US, a number that far exceeds the supply of available organs, and that continues to grow ~5% each year. The most promising solutions, bioartificial tissue and organ construction and donor organ reengineering methodologies, are both ultimately limited by biopreservation technologies, as any tissue engineered products prepared in a laboratory will have to be stored for a period of time until utilization. The current gold standard for whole organ preservation is cold storage on ice for up to 72 hours, during which time the organ continuously deteriorates. A superior biopreservation method that extends the tissue storage time beyond current limitations is yet to be developed. Such a method would provide a crucial enabling technology for tissue and organ preservation, tissue and organ transport, and tissue and organ transplantation. The objective of this study is to extend the viable preservation time of hepatic tissues by sub-zero non- freezing (SZNF) storage in a supercooled preservation medium. The central hypothesis of this study relies on two phenomena: 1) that 3-O-methyl-glucose (3OMG) lowers achievable stable SZNF temperature without major toxic side effects, and that 2) rewarming by normothermic perfusion reduces reperfusion damage. Our hypothesis has been formulated based on our preliminary findings establishing 3OMG as a minimally toxic cryoprotectant for hepatocytes, and establishing that normothermic perfusion can significantly reverse the damaging effects of ischemia. The rationale of the study is that if supercooled preservation can be achieved while avoiding antifreeze toxicity, then organ metabolism can be further slowed thereby reducing anoxic/ischemic damage to minimal levels. Establishment of a sub-zero nonfreezing preservation technology will be a welcome innovation to the field. The work described herein will help develop this enabling technology of supercooled storage, and also establish quantitative standards for evaluating the liver and bioartificial organ viability following preservation. While we focus on the liver, we expect that the protocols established here will also serve as the basis for subzero nonfreezing preservation of other tissue engineered products, such as artificial organ substitutes and seeded scaffold constructs.

Public Health Relevance

There are currently 97,000 patients on the transplant waiting list, and the number increases by ~5% every year. A critical bottleneck in making more donor organs as well tissue engineering alternatives available to the public is the limited preservation duration. The objective of this study is to extend the viable preservation time of organs and bioartificial alternatives by enabling extended storage at sub-freezing temperatures without ice formation. The results of this study are expected to directly improve public health by increasing donor organ availability and making more transplantations possible.

National Institute of Health (NIH)
National Institute of Biomedical Imaging and Bioengineering (NIBIB)
Research Project (R01)
Project #
Application #
Study Section
Special Emphasis Panel (ZEB1-OSR-D (M1))
Program Officer
Hunziker, Rosemarie
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Massachusetts General Hospital
United States
Zip Code
Geerts, Sharon; Ozer, Sinan; Jaramillo, Maria et al. (2016) Nondestructive Methods for Monitoring Cell Removal During Rat Liver Decellularization. Tissue Eng Part C Methods 22:671-8
Bruinsma, Bote G; Sridharan, Gautham V; Weeder, Pepijn D et al. (2016) Metabolic profiling during ex vivo machine perfusion of the human liver. Sci Rep 6:22415
Puts, C F; Berendsen, T A; Bruinsma, B G et al. (2015) Polyethylene glycol protects primary hepatocytes during supercooling preservation. Cryobiology 71:125-9
Bruinsma, Bote G; Avruch, James H; Weeder, Pepijn D et al. (2015) Functional human liver preservation and recovery by means of subnormothermic machine perfusion. J Vis Exp :
Bruinsma, Bote G; Berendsen, Tim A; Izamis, Maria-Louisa et al. (2015) Supercooling preservation and transplantation of the rat liver. Nat Protoc 10:484-94
Bruinsma, Bote G; Yarmush, Martin L; Uygun, Korkut (2014) Organomatics and organometrics: Novel platforms for long-term whole-organ culture. Technology (Singap World Sci) 2:13
op den Dries, Sanna; Westerkamp, Andrie C; Karimian, Negin et al. (2014) Injury to peribiliary glands and vascular plexus before liver transplantation predicts formation of non-anastomotic biliary strictures. J Hepatol 60:1172-9
Bruinsma, B G; Yeh, H; Ozer, S et al. (2014) Subnormothermic machine perfusion for ex vivo preservation and recovery of the human liver for transplantation. Am J Transplant 14:1400-9
Nativ, Nir I; Yarmush, Gabriel; So, Ashley et al. (2014) Elevated sensitivity of macrosteatotic hepatocytes to hypoxia/reoxygenation stress is reversed by a novel defatting protocol. Liver Transpl 20:1000-11
Berendsen, Tim A; Bruinsma, Bote G; Puts, Catheleyne F et al. (2014) Supercooling enables long-term transplantation survival following 4 days of liver preservation. Nat Med 20:790-3

Showing the most recent 10 out of 22 publications