MINNEAPOLIS—The rat kidney on the operating table in front of Joseph Sushil Rao looked like it had been through hell. Which it had—a very cold one.
Normally a deep pink, this thumbnail-size organ was blanched a corpselike gray. In the past 6 hours, it had been plucked from the abdomen of a white lab rat, pumped full of a black fluid, stuck in a freezer cooled to –150°C, and zapped by a powerful magnet.
Now, in a cramped, windowless room on the 11th floor of the University of Minnesota’s (UMN’s) Malcolm Moos Health Sciences Tower, Rao lifted the kidney from a small plastic box and gently laid it inside the open abdomen of another white rat. Peering through a microscope, the transplant surgeon–in–training deftly spliced the kidney’s artery and vein into the rat’s abdominal blood vessels using a thread half the thickness of a human hair.
When he finally removed the tiny clips pinching off the blood supply from the aorta, the kidney blushed pink, a good first sign. Then he waited. Forty-five minutes later, a golden drop of urine emerged from the ureter that would normally feed from the kidney to the bladder.
Just before midnight, Rao snapped a close-up photo with his iPhone, proof that the kidney was working. He sent the photo and an ecstatic email to his two bosses, transplant surgeon Erik Finger and biomedical engineer John Bischof, titled “First successful transplant of vitrified, nanowarmed rat kidney.”
“I’m out of words,” he wrote. “This is a proud moment for us all. It was not easy. But, it paid off.”
That moment in April 2022 was one in a series of recent breakthroughs in the quest to effectively stop biological time. After decades of frustration and halting progress, scientists in the past 10 years have made major advances using extreme cold to slow or even halt the decay that is the usual fate of all living things. They’ve developed new ways to reduce the toxicity of chemical antifreeze treatments, minimize the formation of destructive ice, and thaw objects rapidly and evenly. Since 2018, labs have frozen and then revived bits of coral, fruit fly larva, zebrafish embryos, and rat kidneys. They have also applied gentler techniques to cool everything from tomatoes to entire pig livers to just below freezing without ice formation, keeping them virtually fresh for days or weeks.
Medical uses, particularly organ transplants, are a key driver for today’s work. Scientists hope to eventually create cryopreserved banks of tissues such as skin, entire organs, or even limbs, easing shortages and giving doctors time to better prepare recipients for transplants. But the advances in preservation also extend to specks of human tissue used to screen pharmaceuticals, species on the brink of extinction, fruit flies studied by geneticists, produce bound for grocery stores, and fish embryos stored for aquaculture. Mehmet Toner, a bioengineer at Massachusetts General Hospital (MGH) and one of the leaders in the field of cryopreservation, likens the vision of stored living tissue available on demand to a more familiar cornucopia. “I call it,” he says, “the Amazon of living things.”
SOMEONE RAISED on Hollywood movies might think the technology to freeze and revive entire organisms is right around the corner. Star Wars’s Han Solo is trapped in “carbonite” and resuscitated. Tom Cruise gets turned into a human popsicle in a dystopian prison in Minority Report. Captain America is entombed in Arctic ice in a Marvel movie and rewarmed nearly 70 years later for a sequel.
Reality is far less simple. The largest living thing routinely stored at temperatures well below zero and brought back to life is the size of a grain of table salt: a human embryo. Try that with an entire person using today’s technology and the result would be a lifeless body filled with toxic chemicals, says cryobiologist Greg Fahy. “You would be in sorry shape.”
Fahy was one of a pair of scientists whose 1985 Nature paper revealed a chemical process that allowed mouse embryos to be stored at nearly –200°C. Their technique addressed the major barrier to freezing living tissue: ice.
When water freezes, it can wreak havoc inside tissue. The water molecules go from a tightly packed, amorphous fluid to a rigid lattice. Ice crystals tear through cells like knives. Salts in cell fluids get concentrated at toxic levels in the tissue parts that freeze last. Anyone who has frozen and thawed a strawberry has seen the result: a mushy, discolored version of what came off the plant.
Getting tissue below the freezing point while minimizing ice is crucial. (That’s why cryobiologists don’t like to say they “freeze” tissue.) For the mouse embryo, Fahy and his colleague at the American Red Cross, William Rall, first soaked the little ball of cells in a chemical cocktail that leached out much of the water, replacing it with chemicals similar to the antifreeze in a car’s radiator. These cryoprotectants, as they are known, dilute the water molecules in a viscous fluid that discourages ice crystal formation.
Then they cooled the embryo, kept in a slender plastic straw, using –196°C liquid nitrogen. Between the rapid cooling and the cryoprotectant, ice didn’t have time to form. Rather than line up in a tidy crystalline pattern, water molecules were stuck in a random mass like a rigid liquid, a process known as vitrification. The result was a hard, smooth, glasslike substance without the problematic properties of ice. To rewarm the embryo, Rall stirred the straw in 0°C water.
The mouse embryo work paved the way for banking similar-size human embryos, transforming fertility treatment. But what works for a tiny embryo of about 100 cells doesn’t size up easily to whole organs. It’s hard to get cryoprotectant to soak evenly into a bigger piece of tissue. The center can take longer to solidify, which fosters ice formation. Pumping in more cryoprotectant to counter ice can be damaging because the chemicals are toxic.
Rewarming poses its own problems. If an object warms too slowly, ice crystals can materialize as the tissue approaches the freezing point. If it doesn’t warm uniformly, stresses caused by uneven expansion or contraction can crack the object like an ice cube dropped in a glass of water.
In 2002, Fahy stepped up his work in mouse embryos to rabbit kidneys. He got as far as implanting a previously vitrified organ into an animal. The rabbit survived nearly 7 weeks. But it was sickly. A necropsy revealed that although the kidney was functional enough to keep the animal alive, much of it was damaged.
Fahy has been chipping away at the problem ever since, testing different chemical mixtures and cooling and warming protocols. “It turned out to be harder than I assumed,” says Fahy, who is now executive director of 21st Century Medicine, a private cryopreservation research company. “I think all of this will pay off, but we’re not quite there yet.”
THERE’S GOOD REASON to persist. The rapid decay of organs is one of the biggest problems bedeviling organ transplants for people. From the moment a human heart or lung is disconnected from a donor, doctors have 4 to 6 hours to get it hooked up to a new patient’s blood supply before it is irretrievably damaged. For a liver, the window is 8 to 12 hours. For a kidney it’s about 1 day.
The rush creates burdens for the medical system and for patients. Surgeons are called to the hospital in the middle of the night. Transplant recipients have a foreign organ plugged into their body without time for treatments that would help their immune system acclimate. More than 60% of donated hearts and lungs never make it to a recipient in time. Fewer than 10% of people who need organ transplants actually get them, the World Health Organization estimates.
Cryopreservation holds out the possibility that organs could be stored for days, weeks, or even years before they are implanted. That could save organs from getting tossed after a few hours and would enable doctors to find organs more easily when needed or choose those that are a closer immunologic match to recipients.
“It could touch so many aspects of biomedicine, truly change the way that we can treat health,” says Sebastian Giwa, an economist and former hedge fund manager who founded the nonprofit Organ Preservation Alliance in 2012.
Giwa has helped launch several cryopreservation-related companies. One, GaiaLife, is experimenting with vitrifying ovaries. The goal is to remove the egg-bearing organs from people before they undergo ovary-damaging medical treatment such as chemotherapy, then reimplant them after the treatment is over. So far researchers working with the company have reimplanted vitrified ovaries into five sheep; in four of the animals the ovaries produced progesterone, a sign they were working, says Alison Ting, a reproductive biologist and the company’s chief scientific officer. Ting declined to describe the details of the company’s methods but says the progress “gives me the optimism to say that the first in human could be sooner than 5 years.”
BY VITRIFYING animal organs, Fahy demonstrated a key first step, Bischof says. “The problem was he couldn’t rewarm them.”
Finding ways to warm vitrified tissue quickly and evenly has been the focus of Bischof’s lab. In the past few years, his team has tried everything from lasers to heat-conducting mesh. With larger objects, such as rat kidneys, they have made progress with a powerful magnetic field coupled with iron nanoparticles.
On an unseasonably hot day in April, Zonghu Han, a UMN mechanical engineering postdoctoral researcher, connected a slender plastic tube to a rat kidney resting on a bed of gauze. He made a few keystrokes on a computer and a black fluid began to flow into the organ. The color came from the iron nanoparticles suspended in cryoprotectant. When the organ turned a glossy ebony from the infusion, Han slipped it into a small plastic bag, and lowered it into a nearby freezer cooled to –148°C.
The clock for the kidney’s survival had been ticking for more than 3 hours, since Rao, the transplant surgeon, had removed it from a rat in a reenactment of that 2022 breakthrough surgery. Now, as the kidney’s temperature plummeted inside the freezer, the biological processes gradually destroying the organ ground to a halt. “We have stored [a rat kidney] up to 100 days before transplantation,” Han says. “It’s safe in there indefinitely.”
In this case the kidney got just 45 minutes. Han opened the lid in a billow of vapor and lifted out a tiny, rigid packet containing the vitrified organ. He placed the packet inside a small metal cup attached to a cream-colored metal box. When he pressed a button, the box generated a magnetic field around the cup that flipped the positive and negative poles 360,000 times every second. That fluctuation heated the iron particles and thawed the kidney in 90 seconds.
“That’s our secret sauce,” Bischof says of the process as he watches.
