A new supercooling technique that triples the time livers can be preserved for transplant could make more livers available for patients and increase the usability of donated organs for developing regenerative therapies.1 Harvard Medical School and spinout Organ Solutions LLC are extending the studies from rats to larger species and scaling up the method in preparation for an FDA submission next year.

Korkut Uygun, the lead scientist on the study, founded Organ Solutions to help drive the studies and commercialize the technology. Uygun is an assistant professor of surgery at Harvard Medical School.

Liver availability is the main roadblock limiting the number of transplants each year. Thus, finding a way to prolong liver viability could expand the geographical region the organs can be sent to and hence the number of eligible recipients.

In addition, human hepatocytes from livers not healthy enough for transplant are needed for developing alternatives to transplantation, but those cells are also in short supply.

"The shortage of human livers is probably our biggest challenge in clinical liver transplantation and cell therapies for liver disease, such as bioartificial liver," said Scott Nyberg, who is a professor of surgery and director of the Artificial Liver Program at the Mayo Clinic. He is also founder of Liver Cell Therapies Inc., which is developing a bioartificial liver.

"Current organ storage technologies have become the major bottleneck for organ transplantation today and the tissue-engineered replacements of tomorrow. We are hoping this method will provide an important step in moving from practical but limited current storage techniques to functional organ preservation," said Uygun.

Although there is a centralized national network to link organ donors with patients, the distance between donor and recipient is an important factor in assigning organs. According to Paul Magnin, interim CEO of Organ Solutions, matches are typically made within small geographic regions such as the six states of New England.

Staying cool

When organs are recovered from donors, they are stored in a cold organ-preservation solution and transported to the recipient hospital. Although cryopreservation has been attempted as a way to extend organ viability, the extreme temperatures involved cause too much tissue damage to organs intended for transplantation. Machine perfusion, which involves ex vivo artificial circulation, is used routinely for kidneys and short-term organ recovery after injury.

Livers for transplantation can be preserved for 12-24 hours after recovery. Uygun and colleagues came up with the idea that by combining supercooling with machine perfusion, they could overcome problems associated with cryopreservation and extend the time livers can be preserved prior to transplant.

Supercooling is a method for tissue preservation that involves maintaining the tissue at subzero temperatures without allowing it to freeze. By supercooling the tissue, the team wanted to slow down metabolism as far as possible without causing damage. However, there were three problems associated with supercooling that the researchers needed to preempt: ice formation, irreversible injury to plasma membranes, and oxidative damage from the cold temperature and the subsequent warming when the tissue is revived.

The researchers modified the standard preservation solution to protect against those effects. Because polyethylene glycol polymers protect plasma membranes in epithelial cells and glucose can protect internal membranes, 35 kDa polyethylene glycol and the nonmetabolizable glucose derivative 3-O-methyl-d-glucose were added to the solution.

The team turned to machine perfusion to reduce ischemic damage because it can minimize hypothermic endothelial injury, help reinitialize metabolic activity, replenish ATP and prime the vasculature for reperfusion.

Earlier studies from Uygun's lab showed that function can be preserved in livers deemed unsuitable for transplantation if the organs are cooled to subphysiological temperatures before initiating ex vivo circulation, in a process dubbed subnormothermic machine perfusion (SNMP).2

The team thus created a protocol for testing the method in rats that involved stepwise lowering of the temperature and perfusion with the modified preservation solution (see "Liver preservation protocol").

All rats that received livers preserved for three days using the combination protocol survived for three months after transplant with no detectable signs of organ failure. By contrast, all rats that received livers preserved with the standard protocol for three days died within two days of transplant.

When livers were preserved for 4 days in the combined protocol, 58% of the recipient rats survived for 3 months. Rats that survived 30 days with 4-day preserved livers had greater hepatic resistance-resistance to circulation during the recovery phase-than animals that did not survive that long. In addition, bile production was increased in the survivors. The reasons for the link between positive transplant outcome and increased hepatic resistance and bile production are not clear.

Uygun said that hepatic resistance and bile production during recovery could be used as markers to distinguish transplantable from nontransplantable organs. "ATP is also a good marker," he said. "We are looking for even more practical markers-perhaps oxygen uptake."

Because the scientists tested numerous permutations of their methodology, Uygun was confident that every component of the protocol contributed to the prolonged preservation. "The preservative was necessary, SNMP was necessary and supercooling was necessary," he said.

The work was published in Nature Medicine. Scientists from the University Medical Center Utrecht and Rutgers University also contributed to the study.

"The lower temperature attained reduces the rate of energy consumption and prolongs the maximum storage time compared to 'normal' cold storage at ice temperature. The ability to preserve organs for three days would improve the logistics of liver transplantation. For example, liver transplantation might become a less urgent procedure. Alternatively, it would enable livers to be transported for greater distances to the best recipient," said Peter Friend, a professor of transplantation at the University of Oxford.

"However," he added, "the technology still results in an organ that is becoming progressively energy depleted, and the direct effect of cooling on cell membranes would, if anything, be more severe. Therefore, it is not yet clear if this technology would improve the ability to transplant successfully the very marginal organs for which conventional cold preservation is inadequate."

In the short term, Uygun's team hopes to adapt the method for marginal livers. In the longer term, the team hopes to increase the number of healthy livers that are available. "We would like to abolish waiting lists," said Uygun.

Uygun told SciBX that his team is addressing two goals in parallel: scaling up the procedure for ex vivo application to human livers and applying the method to a pig transplant model.

"With the two, we think we'll be ready for an FDA application in a year or so if all goes well," said Uygun. "First, we will scale up to human livers to ensure the supercooling protocol works, and then we'll move to pigs for transplant," he told SciBX.

Because there are potential complications that are not well modeled in the rat, the team wants to test the system on larger animals. One such complication is the amount of free water in livers larger than those of rats. Uygun said that there is a higher likelihood of ice formation during preservation that can damage larger livers. He believes the team has not reached the limit of the method and that the technique can be further optimized for the larger organs if necessary.

Bioartificial livers

The team also plans to use the supercooling SNMP technology to increase the number of human hepatocytes that can be used both in research and for developing bioartificial livers-bioreactors that perform the functions of a normal liver.

"Livers have an amazing ability to regenerate," said Nyberg. Bioartificial livers can provide temporary support to allow liver regeneration and avoid a liver transplant altogether or bridge a patient to a successful transplant. He added, "Most patients with acute liver failure are healthy and in the prime of life, so the impact of successful bioartificial liver therapy can be quite significant."

"If it is unburdened from the load of cleaning the blood for a while, there is a lot of evidence that the liver can heal itself," said Magnin.

According to Nyberg, acute liver failure occurs in about 2,500 patients in the U.S. each year. Another 100,000 patients in the U.S. develop acute-on-chronic liver failure, for example, from conditions such as cirrhosis. Global numbers are 50- to 100-fold greater.

He said that the major limitations impeding the development of human hepatocyte-based bioartificial livers are the availability and functionality of hepatocytes obtained either from cell lines or human stem cells.

Magnin told SciBX that Organ Solutions could provide human hepatocytes for scientists developing such technologies.

"Akin to dialysis for the kidney, we could provide cartridges containing human liver cells for a bioartificial liver that can allow the liver to recover," he said. For example, he said, "patients could use the bioartificial liver for six months, replacing the cartridge every two weeks. Certain patients could then dispose of the artificial liver, return to their native liver and live a healthy life."

There are no FDA-approved bioartificial liver devices. At least two companies are developing bioartificial livers. Vital Therapies Inc. has ELAD, a bioartificial liver based on a human cell line, in Phase III trials to treat acute liver failure. Liver Cell Therapies is preparing a spheroid reservoir bioartificial liver (SRBAL) for a Phase I trial. SRBAL is based on primary hepatocytes and contains a semipermeable membrane that separates the cells from the patient's circulation as an immune barrier and an added safety feature.

Partners HealthCare has filed three patent applications covering supercooled preservation, machine perfusion recovery and quality assessment of the livers prior to transplant. The IP will be licensed to Organ Solutions.

Donner, A. SciBX 7(31); doi:10.1038/scibx.2014.915
Published online Aug. 14, 2014

REFERENCES

1.   Berendsen, T.A. et al. Nat. Med.; published online June 29, 2014; doi:10.1038/nm.3588
Contact:
Korkut Uygun, Harvard Medical School, Boston, Mass.
e-mail: uygun.korkut@mgh.harvard.edu
Contact:
Martin L. Yarmush, same affiliation as above
e-mail: myarmush@mgh.harvard.edu

2.   Bruinsma, B.G. et al. Am. J. Transplant. 14, 1400-1409 (2014)

COMPANIES AND INSTITUTIONS MENTIONED

Food and Drug Administration, Silver Spring, Md.

Harvard Medical School, Boston, Mass.

Liver Cell Therapies Inc., Rochester, Minn.

Mayo Clinic, Rochester, Minn.

Organ Solutions LLC, Wilmington, Del.

Partners HealthCare, Boston, Mass.

Rutgers University, Piscataway, N.J.

University Medical Center Utrecht, Utrecht, the Netherlands

University of Oxford, Oxford, U.K.

Vital Therapies Inc. (NASDAQ:VTL), San Diego, Calif.