How to deep freeze an entire organ—and bring it back to life | Science

How to deep freeze an entire organ—and bring it back to life | Science

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.”

Joseph Sushil Rao prepares microscope and surgical tools
Joseph Sushil Rao preforms surgery on a live rat. Seen  here is the rats kidney.
Joseph Sushil Rao, a transplant surgeon–in–training at the University of Minnesota, prepares to remove a kidney from a laboratory rat (first image) before the organ is preserved at extremely low temperatures. The complex plumbing of a kidney requires delicate handling by Rao during all stages of a transplant (second image). Evan Taylor Studios assisted by Tom Okins

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.

Stopping the biological clock

Advances in using extreme cold to slow biological processes could touch everything from donated organs to fresh produce. A University of Minnesota team has developed one approach, dubbed “nanowarming,” which thaws an organ evenly to avoid damage from ice.

graphic showing stages of nanowarming
C. Bickel/Science

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.”

Zonghu Han connects a rat kidney to a system that distributes  nanoparticles throughout the tissue for vitrification
University of Minnesota postdoctoral researcher Zonghu Han connects a rat kidney to tubes that pump antifreeze chemicals called cryoprotectants and tiny iron oxide particles into the organ.Evan Taylor Studios assisted by Tom Okins

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.”

engineer uses cyro gloves to remove a device holding a rat kidney
Zonghu Han uses protective gloves while removing a container holding a rat kidney, cooled to –150°C. The extreme temperature and cryoprotectants put the organ into a vitrified state, in which liquid in the organ forms a smooth, glasslike substance rather than sharp ice crystals.Evan Taylor Studios assisted by Tom Okins

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.

engineer removes a bag holding a rat kidney. The kidney was infused and submersed in dark nanoparticles
Zonghu Han removes a plastic bag containing a vitrified kidney from a device that warmed it, using a fast-changing magnetic field to heat the iron particles inside the organ.Evan Taylor Studios assisted by Tom Okins

In a Nature Communications paper in early June, Bischof's team reported putting five rat kidneys through this treatment and reimplanting them. All of the recipient animals lived a month before they were killed to study their condition. Now, the researchers have graduated to pig kidneys, closer to the size of a human kidney. Bischof declined to discuss details of the unpublished pig work. “There's no physical reason that we're aware of why this [warming procedure] won't work” in larger organs, he says.

Although nanowarming, as Bischof calls it, is his tool of choice for the kidneys, it requires costly machinery and one-at-a-time treatment. In May, Smithsonian Institution marine biologist Mary Hagedorn was at a laboratory outside Tampa, Florida, testing a simpler approach developed by the Bischof lab: a fine metal mesh engineered to quickly transmit temperature—both cold and heat. She is trying it on batches of coral larvae. If it works and can be scaled up, Hagedorn thinks this process could be a critical piece of her campaign to bank dozens of coral species in the coming decade, before increasingly hot and polluted oceans spell their end.

The mesh has already proved successful on fruit fly larvae in Minnesota, and with two species of mushroom coral in Hawaii and Australia. In Florida, Hagedorn and colleagues were trying it on Diploria labyrinthiformis, a kind of brain coral whose larvae are more than 100 times bigger than those of mushroom coral. In the first few attempts, rewarmed larvae were falling apart. Each larval size, Hagedorn was learning, needs its own version of the treatment. “We're struggling a little bit to get this to work,” she says.

WHILE SCIENTISTS such as Bischof and Hagedorn wrestle with vitrification, others are seeking an easier route by avoiding ultralow temperatures that require large infusions of cryoprotectant and make rewarming so challenging.

At Harvard University and MGH, scientists are taking cues from nature to push tissues below freezing while holding back the ice. The wood frog Rana sylvatica is a champion of this realm. Found in much of North America, including the frigid Canadian Arctic, it can spring to life after spending months with as much as two-thirds of its body frozen at temperatures as low as –16°C.

wood frog (Rana sylvatica) frozen under controlled laboratory conditions
The wood frog (Rana sylvatica) can survive for months in a partially frozen state in places like the Canadian Arctic. Strategies used by the frog to endure such extremes are inspiring efforts to better preserve human tissue and organs.J.M. Storey/Carleton University

As winter arrives, a cascade of physiologic changes prepares the frog to survive freezing, says Shannon Tessier, an MGH biomedical researcher who had studied it and other animals that hibernate at near freezing. Its liver churns out glucose that acts as antifreeze inside tissues. Antioxidant levels inside tissues increase, protecting against damage caused by sudden changes in the amount of oxygen in cells. Special proteins in the frog's bloodstream act as seeds for ice crystals, steering ice growth to begin in the more durable vasculature and not in other, more delicate tissues. Such changes “are the thematic things that we want to pull forward to human organs and tissues,” she says.

In Boston, a team of scientists including Tessier mimicked the frogs by flooding human livers with a synthetic sugar that, unlike natural glucose, can't be metabolized into toxic byproducts. In 2019, they announced the approach had enabled them to store human livers at –4°C for 27 hours, more than double the standard life span of donated livers.

More recently, the team combined this synthetic sugar with an infusion of Snomax, an ingredient normally used as a seed for snow in snowmaking machines at ski resorts. Like the proteins in the wood frog's blood, Snomax slowed ice formation and concentrated it in the blood vessels of rat livers, enabling them to be stored partially frozen for up to 5 days at –15°C, then thawed with limited damage. Five days is far short of the virtually limitless storage time for a vitrified organ, but still useful, says Toner, who is working with Tessier. The extra time could, for example, allow a patient waiting for an organ to receive a bone marrow transplant, a possible measure to coax the immune system to accept the new organ.

On the other side of the country, Boris Rubinsky had the idea that higher pressures might help him supercool organs without damage. In the early 2000s, Rubinsky, a biomedical engineer at the University of California, Berkeley, began to cool objects inside sealed metal containers. As water inside approached freezing it expanded, raising the pressure. The higher pressure, he discovered, limited the formation of ice. “In all modesty, it's a revolutionary approach to preservation,” he says.

In April, the team cooled a pig heart to –4°C for 21 hours in one of their pressure chambers, then warmed and implanted it into another pig where it began to beat on its own. In addition to avoiding the challenges that can come with full vitrification, Rubinsky finds his strategy enables him to use less cryoprotectant than other methods require, reducing toxic side effects.

Because it is simple and relatively low-tech, the approach could also be put to work to preserve food without the ice damage that can degrade today's frozen vegetables and meats. The metal storage chambers fit in standard commercial freezers. And because the method works at warmer temperatures than those often used for storing frozen food, Rubinsky and colleagues project widespread use could cut global energy consumption by more than 6 billion kilowatt-hours per year—equivalent to Latvia's total annual electricity demand.

Cristina Bilbao-Sainz, a food technologist at a U.S. Department of Agriculture (USDA) lab in Albany, California, started to work on the approach after Rubinsky gave a presentation to the center in 2016. “It was a simple idea, but so novel,” Bilbao-Sainz says. “We totally saw that this could have a future.”

The first attempt was discouraging. Raspberries, a delicate fruit, turned to mush when thawed. Then Bilbao-Sainz tried tomatoes. Twenty grape tomatoes sat for 1 month in a sealed container filled with sugar water at –2.5°C. They emerged looking like they had just been picked, she says.

Bilbao-Sainz has also had good results with spinach, cherries, and potatoes. Now, she is trying blueberries. In December 2022, the USDA lab started to work with an unnamed company interested in using the technology. “It could have a big impact,” she says.

engineer carefully removes a preserved rat kidney from a cryo freezer
Zonghu Han removes a vitrified rat kidney from a storage freezer. Kidneys have been stored like this for up to 100 days before being successfully transplanted into a rat.Evan Taylor Studios assisted by Tom Okins

THE ADVANCES are coming swiftly enough to make those Hollywood scripts seem less outlandish. Some scientists raise the possibility of stockpiling vitrified human organs grown in genetically engineered pigs for future transplants. Tessier is working to keep whole animals—tiny zebrafish larvae—in suspended animation. So far, she has partially frozen them at –10°C for 3 days. When thawed, half of the fish survived and kept growing. Switch out people for fish and improve the success rate, and a science-fiction staple could eventually become a reality. “You can think about even long-term space travel,” Tessier says. Hello, 2001: A Space Odyssey.

But that's a far cry from today. As if to illustrate the gap between vision and reality, the UMN demonstration goes awry at one of the last steps. As the black nanoparticles are flushed from the rat kidney, water condenses on the chilled equipment, causing a device to malfunction. It pumps a solution commonly used to preserve organs during transplants into the kidney at out-of-control pressures.

Han and Bischof stare dejectedly at the damaged organ. Before any of this technology makes its way into operating rooms, it will not only have to scale up to human size, but will also need to pass muster with safety regulators such as the U.S. Food and Drug Administration. “All of this engineering that we're doing in the lab has to be made failsafe,” Bischof says as he points toward the kidney. “This is just an example of some of the things that can go wrong.”