Pig kidneys that were cooled to -150 °C and stored in a freezer for months have been successfully rewarmed and shown to work again for the first time, say scientists. They hope to replicate the findings with human organs.
The kidneys—which are similar in size to human ones—were left in a glass-like state, in one case for over a year. Once they had been rewarmed, tested in machines, and transplanted into pigs, the kidneys regained their color and made urine—key signs they were working.
“Ten years ago … I wouldn’t say I had high hopes it was actually going to work,” says Erik Finger, a transplant surgeon at the University of Minnesota who co-led the work. “After we did the last transplant, I walked out of the room and said, ‘This is a big deal.’” The research was shared at the preprint server bioRxiv and has not yet been peer-reviewed.
The kidneys aren’t perfect, and the researchers have yet to demonstrate that they can sustain life over the long term. But the results represent a milestone achievement, say others in the field.
“Thank God that someone’s actually managed to do it in one organ and prove that it works,” says Peter Kilbride, a cryobiologist and cofounder of Kyros Kairos, a company focused on improving funding for cryopreservation research, who was not involved in the research. “For years, people have been trying and failing to get to this step. Finally, we’ve got this breakthrough.”
“Their work is amazing and will enable us to truly, indefinitely bank organs,” says Korkut Uygun, a biomedical engineer and professor of surgery at Harvard Medical School, who was also not involved in the research. “It is a revolutionary time for transplantation.”
Buying time
Every year, tens of thousands of organ transplants take place in the US alone. These procedures are thought to have saved 2 million years of life in the last 25 years. But there’s plenty of room for improvement.
One major challenge is the lack of available organs. Even when an organ becomes available, it can be difficult to get it to the right recipient within 24 to 36 hours, which is the current limit for preserving human organs.
People who receive organ transplants must also take drugs that suppress their immune systems for the rest of their lives. These drugs are meant to stop their bodies from rejecting the new organs. But they come with side effects and increase the risk of infections and cancer.
The ability to bank organs for long periods of time holds the promise to address all these challenges. Doctors would have more time to match organs to recipients. They’d be able to limit the risk of organ rejection, too—potentially by preparing recipients, and their immune systems, beforehand.
“With real organ banking … instead of the patients waiting for the organ, it’s the organs that are waiting for the patients,” says Finger.
Scientists have been able to cool cells to ultra-low temperatures through a process called vitrification, which essentially turns them to something like glass. This procedure is already used to store eggs, sperm, and embryos. Vitrifying whole organs is much harder.
Some problems can be avoided with cryoprotectants—chemicals that work like antifreeze and prevent damaging ice crystals from forming. But these chemicals tend to be incredibly toxic. It’s also difficult to rapidly and uniformly cool large tissues, and just as hard to evenly rewarm an organ all the way through.
Working with John Bischof, a bioengineer at the University of Minnesota, Finger and colleagues have come up with a solution. Once an organ has been loaded with a cryoprotectant, they infuse it with iron oxide nanoparticles. These nanoparticles don’t do anything to the cells. But when it’s time to rewarm the organ, they can be activated with an electromagnetic field.
When the radiofrequency of that field switches between two states, the particles also flip, realigning themselves with the field. That causes them to release heat throughout the organ. Adjusting the power of the field allows the team to control the rate of heating so that it gets faster once the organ reaches -120°C.
The researchers successfully vitrified rat kidneys using this approach in work published in 2023. Back then, they were able to rewarm kidneys that had been kept in a freezer for up to 100 days before transplanting them into rats.
Since then, the team has been trying to achieve the same success with bigger organs. Pig kidneys are thought to be a good model for human ones—they’re about the same size and look similar. Rat kidneys weigh around one gram; pig kidneys are around 100.
It hasn’t been easy. The cryoprotectant used for vitrifying rat kidneys didn’t work as well in the pig organs. “We had to rediscover and modify the cocktail,” says Bischof. “That’s why it’s taken us three years.”
Deep-freezing kidneys
The first step in the procedure is to remove a kidney from a donor pig. Once it’s out, the kidney is flushed with a commonly used organ preservation solution to remove the blood, bagged, and temporarily laid on a bed of ice in a cooler so it can be taken over to the engineering lab.
There, team members unpack the kidney and hook it up to a device they’ve designed to control the organ’s temperature and pressure. Then they start to load it up with the cocktail of cryoprotectants, slowly enough to ensure an even spread through the organ but quickly enough to minimize any toxic effects. It takes around two hours.
The next step is to infuse the organ with the iron oxide nanoparticles, which are pumped through the organ for around seven to eight minutes, says Finger. Then it’s time to cool the organ by pumping cold nitrogen vapor into a chamber housing it.
It’s a delicate process, and the team must ensure that the organ is cooling all the way through. At the same time, they must lower the kidney’s temperature as quickly as possible to prevent ice from forming. “Oftentimes there are multiple people doing different things to make sure that things don’t go awry,” says Bischof.
Once the organ reaches the vitrified state at around -120 °C, it “behaves like a glass,” says Finger. “At that point, you can stay stable for long periods of time—we think indefinitely.” The team then pauses the cooling process to ensure that the organ is glassy throughout. Otherwise, it could crack.
After the pause, if everything goes well, the kidney is ultimately cooled to -150 °C. Once it’s in the freezer, it can stay there for days, weeks, or even months. The team has kept a small number of kidneys banked for over a year.
Pink right up
The real test of these kidneys comes when they’re taken out of the freezer, rewarmed, and flushed of their chemicals and nanoparticles. Can they support life? So far, the team has tested some of them in a couple of different ways.
In one set of experiments, the researchers put rewarmed kidneys on perfusion devices—systems that are already used in transplant surgeries both to provide organs with nutrients and to help doctors assess them while they’re out of the body.
In another set of experiments, Finger and his colleagues transplanted rewarmed kidneys into pigs and monitored them for two hours. In both experiments, the rewarmed kidneys seemed to recover, says Finger. “They pink right up,” he says.
The team see blood flow into the rewarmed, transplanted kidney, allowing it to “pink up.”COURTESY OF THE RESEARCHERS
There are lots of ways to measure the function of a kidney, but one key test is whether or not the organ can make urine. By this measure, the rewarmed kidneys did well, although they produced around two-thirds of the amount that would normally be expected. “They produce urine … within a minute or so,” says Finger. “We know that they work.”
Most of the rewarmed kidneys had been banked for days or weeks. But one had been in the freezer for just over a year. When Finger and his colleagues rewarmed it and put it on a perfusion machine, they found that it produced urine “immediately.” “It actually looked better than many of the [other] kidneys,” says Finger. “We’re comfortable now saying we can store them for a year and rewarm them with excellent function.”
“It’s a very big deal to go a year,” says Kevin Myer, president and CEO of LifeGift, an organ procurement organization based in Texas. Being able to store organs for that length of time would transform organ transplantation. He envisions banks of organs that could be used on demand in emergency cases where people can’t wait, such as for some drug overdoses or gunshot wounds.
Final hurdles
But the procedure isn’t ready for prime time just yet. As well as producing less urine than expected, the rewarmed kidneys also showed signs of injury. The damage seems to be caused by the cryoprotectant chemicals, say the researchers.
“This is one big breakthrough that solves half the problem,” as Kilbride puts it.
The team has shown that the rewarmed kidneys can regain blood flow, which is “very important,” says Greg Fahy, a cryobiologist who is considered a pioneer in the field. But he’s worried about the extent of the damage to the organs. “We can’t tell from these experiments whether these kidneys might have supported life or not.”
But Fahy, who is chief scientific officer at the biotech companies Intervene Immune and 21st Century Medicine (where he is also executive director), acknowledges that it’s not clear how these kidneys might fare once they’re inside a living animal for the long term.
Finger also hopes to find ways to repair the damage seen in the kidneys. Other research has shown that the health of transplant organs can improve while they’re on perfusion machines. And damaged kidneys might undergo some level of self-repair once they’re in a body. At any rate, human kidneys may cope better with the process than pig kidneys, says Finger.
The next step will be to test the kidneys in pigs over longer periods of time. The team, along with many other researchers in the field, will continue the search for less toxic cryoprotectants, too. Finger, Bischof, and their colleagues are also planning experiments with human kidneys.
Finger is optimistic. “We’re going to see that there’s injury, and it’s not perfect … but now we’ve got to the point where it works,” he says. “These are big, historic milestones in our field, and we’re excited about it.”
