On the last day of March, surgeons at Massachusetts General Hospital began an operation that they hoped might lead to a permanent change in how kidneys are transplanted in people.
That morning’s patient was not a person. It was a pig, lying anesthetized on a table. The pig was missing one kidney and needed an implant.
While kidneys typically must be transplanted within 24 to 36 hours, the kidney going into the pig had been removed 10 days before, frozen and then thawed early that morning.
Never before had anyone transplanted a frozen organ into a large animal. There was so much that could go wrong.
“I think there is about a 50 percent chance that it will work,” Korkut Uygun, a professor of surgery and a leader of the team, said before the surgery. Dr. Uygun is on the scientific advisory board of Sylvatica Biotech Inc., a company that is developing freezing methods to preserve organs.
But the promise from freezing and storing organs is great.
There is a severe and ongoing shortage of kidneys for transplants — more than 92,000 people are on waiting lists. One reason is that the window of 24 to 36 hours is so brief that it limits the number of recipients who are good matches.
How much better it might be to have a bank of stored, frozen organs so an organ transplant could be almost like an elective surgery.
That, at least, has been the decades-long dream of transplant surgeons.
But the attempts of medical researchers to freeze organs were thwarted at every turn. In many cases, ice crystals formed and destroyed the organs. Other times, the substance meant to stop the crystals from forming, a cryoprotectant, was toxic and killed cells. Or the frozen organ became so brittle it cracked.
Then, said John Bischof, a cryobiology researcher at the University of Minnesota who was not involved with the pig kidney project, even when the freezing seemed to go well, there was the problem of thawing the organ.
When they froze an organ, scientists tried to make sure that any ice crystals that formed were so tiny they did not damage the organ. But those crystals had a tendency to grow as the organ warmed, slashing delicate cells.
“You have to outrun the ice crystals as they grow,” Dr. Bischof said.
“The essential insight was: You can’t go fast enough in the middle of an organ if all you do is warm it at the edges,” he said. “If heating starts only on the outside of the frozen organ, the temperature differences from the edge to the center of in the organ can lead to stress that fractures the organ like an ice cube that cracks when you put it in your drink.”
He added, “You have to heat uniformly, from the inside.”
His colleague, Dr. Erik Finger, a transplant surgeon also at the University of Minnesota who was also not involved in the Mass General experiment, said that while the freezing had to take place slowly to prevent ice damage, rewarming would have to go fast, 10 to 100 times faster than the cooling process.
Investigators tinkered with their systems, eventually learning to successfully freeze, thaw and transplant rat kidneys.
But bigger animals introduced new problems.
“For four decades, rewarming was the issue,” Dr. Finger said. “But as you increase the size of the organ, cooling becomes an issue.” Suddenly, the cryoprotectants that worked with tiny rat organs were no longer sufficient.
At Massachusetts General, researchers tried a different approach. It began with Shannon Tessier, a postdoctoral fellow in Dr. Uygun’s lab and now an associate professor of surgery at Harvard Medical School who is on an advisory board for Sylvatica Biotech and has a patent application related to the method used in the March surgery. Some years ago, she was studying Canadian wood frogs.
When the weather turns cold, the frog’s metabolism changes, allowing it to freeze itself. All its cellular processes stop. Its heart stops. It is essentially dead.
The frog is so brittle that lab workers have be very gentle. “You can break off its arm if you are not careful,” said McLean Taggart, a technician in the lab.
“Shannon came into the lab and said, ‘Is it possible to translate this to human organs?’” Mr. Taggart said.
That led to work to learn how the frog goes into its deep freeze. Just before it hibernates, the frog starts producing large amounts of glucose. The glucose accumulates inside cells, where it reduces the freezing point of water, preventing ice from forming.
But a frog is an amphibian. Would something like that method work on a warm-blooded mammal, or its organs?
It turns out that it does. A mammal, the arctic squirrel, supercools itself when the temperature drops by using a similar method. Its cells reach a temperature below the freezing point of water — chilled, but not enough for ice to form. Its metabolism slows so much it does not have to eat.
Like the researchers before them, the group at Mass General started with rat livers and tried to mimic the process. They decided to work with recently removed but still live organs using the same process as the wood frog -— chilling them enough to stop metabolic processes, but not enough to risk the formation of large ice crystals.
They began by infusing an artificial glucose that can’t be metabolized. The sugar accumulates in cells, but because it is unusable, the cells enter a form of suspended animation, their metabolic processes paused.
At the same time, the investigators add a diluted antifreeze — propylene glycol — which replaces water left in the cells. The result is that very little ice forms inside cells, which is where damage from organ freezing occurs.
Their storage solution is a mixture of the dilute propylene glycol and artificial sugar, plus Snomax, the substance used to make artificial snow on ski slopes. Snomax creates tiny uniform ice crystals, which helps ensure that the ice that forms does not cause damage.
To thaw the organs, the group reverses the process, putting the livers in a warm solution containing propylene glycol and the artificial glucose and gradually diluting the chemicals until they are gone.
It took about five years of trial and error to get the process right, the researchers said.
The next step was to move up to larger mammal species. They would try to freeze and thaw pig kidneys.
Their ultimate goal was ambitious — they would like to make banks of frozen pig kidneys that were genetically modified to be used in human patients.
Other transplant surgeons at Dr. Uygun’s hospital are starting to experiment with genetically modified pig kidneys. They have transplanted them into several human patients, with mixed outcomes. On Friday, a patient whose kidney had lasted longest so far — 130 days — had to have it removed because her body rejected it.
No one knew if the method used by Dr. Uygun and his colleagues would succeed.
“The protocol was optimized for livers,” Dr. Uygun said. “We didn’t think it would work.”
But it did.
The team tested the method, freezing and thawing 30 of pig kidneys, making sure the organs remained healthy after the freezing process. They learned they could keep the kidneys frozen for up to a month with no obvious damage.
But would a previously frozen kidney function if it was transplanted into a pig?
In the test in March, the kidney had remained frozen for 10 days and was to be transplanted back into the pig from which it had been taken.
At 3 a.m. the team started thawing the kidney, a process that took two hours.
At 9 a.m., Dr. Alban Longchamp and Dr. Tatsuo Kawai, transplant surgeons at Mass General, opened the pig’s abdomen and prepared the animal for the surgery.
At 10:30, they sewed the kidney in.
The whitish gray organ quickly turned pink as blood flowed into it.
Finally, success: Before they sewed up the pig, the researchers watched as the transplanted kidney produced pee.
