“I’ve been doing this for 25 years now, and still, whenever we freeze the frogs...

Then Jon Costanzo adjusts the temperature of the terrarium, and the air around the frog begins to warm. Within 30 minutes, the amphibian’s skin noticeably softens, taking on its characteristic sheen. Three hours later, one eye blinks. Then the lungs shudder to life, and the frog’s sides heave. Six hours from the start of warming, one leg twitches, then the other. Finally, as the timer chimes 10 hours, the frog hops once, twice, and burrows out of sight into the moss.

A small brown frog squats motionless in a den of green moss. It inhales no breath, has no heartbeat, yet it is not dead. Rock hard and icy to the touch, this speckled North American wood frog is frozen alive at –2?°C, and has been so for the last 24 hours.

For most animals on the planet, prolonged exposure to temperatures below freezing means death. But for the wood frog (Rana sylvatica), and for an unlikely collection of other organisms ranging from insects to plants to fish, surviving the cold is a routine part of life. The Alaskan Upis beetle survives at –60?°C in the wild and down to –100?°C in a laboratory. Species of Arctic fish swim fluidly through –2?°C water, and snow fleas hop atop snow banks at –7?°C. These animals all have tricks either to survive freezing, called freeze tolerance, or to lower their internal freezing temperature so they don’t freeze at all, called freeze avoidance.

Organ cryopreservation would transform medicine the way refrigeration transformed the food industry.

However, cooling a tissue that is not adapted to tolerate or avoid freezing—as cryobiologists seek to do with human organs—is a whole different ball game. One can expect irreversible and widespread damage from the formation of ice crystals at temperatures below 0?°C: cells shrivel and collapse, extracellular matrices rip apart, blood vessels disintegrate.

But researchers aren’t giving up. Organ cryopreservation, if possible, would transform medicine the way refrigeration transformed the food industry. Currently, human organs harvested for transplant are not frozen—they are kept in cold storage, which prevents deterioration for a few hours at the most. Human hearts, for example, can be preserved for only 4 to 5 hours. But if scientists could learn the tricks of the trade from nature, and add days, weeks, or even years to the lifetime of an organ, hospitals could bank frozen organs for transplant as needed.

Though it won’t be easy, many believe that organ cryopreservation should be possible. Indeed, thanks to chemical cryoprotectants and sophisticated freezers, scientists and companies already have techniques to freeze sperm, eggs, embryos, and pools of cells such as blood or stem cells. And in the last decade, successful preservation of some solid tissues has offered hope that the long-neglected field is not dead in the water.

In 2005, heart surgeons in Israel used an antifreeze protein from fish to preserve rat hearts at −1.3 ?C for 21 hours, then successfully transplanted them into recipient rats, where the hearts pumped away for 24 hours prior to dissection for analysis. In 2003, a California company similarly preserved a rabbit kidney at below-freezing temperatures and then thawed and transplanted it into a recipient rabbit, which remained healthy with that kidney alone for more than a month. And since 2000, researchers at the US Department of Agriculture have been cryopreserving the embryos of tropical flies for years at a time, then thawing them with ease and watching them hatch and live normal lives. (See “Glass Menagerie” sidebar below.)

Many of the field’s successes were spawned by the in-depth study of organisms like the wood frog, and cryobiologists argue that somewhere in nature lies the answer to prolonging—perhaps indefinitely—the shelf life of human organs. “There’s a good chance that if we study natural adaptation—how other cells and tissues have been able to tolerate the challenges of freezing—that may provide at least some ideas of directions in which to look,” says Richard Lee, who runs the Laboratory for Ecophysiological Cryobiology with Costanzo at Miami University.



© JOELLE BOLT

Frostbite

In the early 1960s, a graduate student at Stanford University named Art DeVries observed fish swimming in the icy waters around Antarctica and asked a simple question: If the water in the Antarctic Ocean is –1.9?°C (it’s salty enough to stay several degrees below the 0?°C freezing point of fresh water), and fish have a freezing point around –1?°C, why aren’t the fish frozen solid?

DeVries collected some fish, and from them isolated the answer: a simple protein with an unusual repeating structure that allows it to bind to ice crystals, thus preventing them from growing into larger crystals. Known as antifreeze proteins (AFPs), the compounds effectively lower the freezing point of a solution. (See illustration below.) Because the blood of Antarctic fish is chock full of AFPs, their freezing temperature is actually about –2.5?°C—just low enough to avoid turning into frozen fish sticks.

AFPs have been isolated from the blood and tissues of fishes, snow fleas, beetles, caterpillars, and other organisms. These proteins have been used in numerous experiments in agriculture, in hopes of lowering the freezing temperature of crops, but applying the same technique to mammalian cells has proven tricky, as mammals are adapted to maintain high body temperatures. “We’re trying to figure out how to make our tissues do something really bizarre: tolerate freezing,” says Lee.

In at least one case—the transplanted rat hearts—scientists have achieved that bizarre feat. In 2005, a team of researchers led by Boris Rubinsky, an ice physics expert at the University of California, Berkeley, and Jacob Lavee, director of the Heart Transplant Unit at Sheba Medical Center in Israel, removed rat hearts and preserved them in a solution of sterile water and fish AFPs. The hearts were cooled in solution to –1.3?°C for up to 24 hours, then warmed, rinsed, and transplanted into the abdomen of recipient rats as auxiliary organs—not to pump blood, but to test the hearts’ viability. After more than 200 rat transplantation experiments, the researchers confidently decreed that the procedure worked. The use of AFPs improved heart preservation time from 4 hours at 5?°C to 21 hours at –1.3?°C.1 Buoyed by the success, Lavee preserved the heart of a 220-pound pig for 19 hours at –1.1?°C, transplanted it into another pig, and “the heart worked beautifully,” he says. To this day, Lavee retains “almost no doubt” that the procedure could work with human hearts.

Unfortunately, what looked like a new start for organ preservation turned out to be a dead end. Lavee and Rubinsky failed to find funding to continue the work, and the research ground to a halt. “Apparently, organ transplantation is not a big enough market for pharma or any company to invest the money needed to do animal and human experiments,” says Lavee.

COURTESY OF GREG FAHY Glass Menagerie Over the last 70 years, the technique for freezing human sperm and embryos, a mainstay of fertility clinics, has not differed much from how frogs freeze in Ohio—add a glut of glycerol and lower the temperature slowly. But today, clinics and hospitals are turning to a technique that no known organism experiences in nature—transforming tissues to glass. Vitrification is the rapid cooling of a substance to a glass state, achieved by pumping enough cryoprotectants into cells or tissues, and cooling them fast enough, so that they transform into an ice-free glass. Through vitrification, scientists have successfully completed two of the most complex examples of cryopreservation to date: a 40,000-cell fly embryo and a rabbit kidney. Led by entomologist Roger Leopold, a team of researchers at the US Department of Agriculture’s Agricultural Research Service (ARS) Laboratory in Fargo, North Dakota, has spent the last 20 years developing a vitrification protocol to freeze six different strains of screwworm fly embryos, each composed of an estimated 40,000 differentiated and organized cells. The project is part of a successful program that has eradicated the screwworm fly, a livestock parasite, from the US and Central America by releasing sterile flies to collapse native populations. Freezing screwworm embryos eliminates the need to continuously rear fly colonies and ensures the continuation of specific strains of the flies for future research. Leopold’s cryopreservation procedure—which involves soaking the embryos in a cryoprotectant bath of ethylene glycol, polyethylene glycol, and trehalose, then vitrifying them in liquid nitrogen—is run by a robot that preserves up to 5,000 embryos in 40 minutes. So far, the team has kept flies “on glass” at −196 °C for more than 7 years and successfully brought them back to life. The ability to freeze 40,000-cell embryos gives Leopold “some hope that we could cryopreserve something other than an 8-cell embryo in mammals,” he says. Greg Fahy shares that hope. In 2002, Fahy and colleagues at 21st Century Medicine, a California-based cryopreservation research company, successfully vitrified a rabbit kidney at −130 °C for 20 minutes, then warmed it and transplanted it into a recipient rabbit that lived for 48 days before being euthanized for research purposes (Organogenesis, 5:167-75, 2009). Fahy has been unable to replicate the success, but is still optimistic that vitrification of whole mammalian organs is just around the corner. “I don’t see any reason why it can’t happen,” says Fahy. “We have a proof of principle: we transplanted a rabbit kidney and had it survive.” But vitrification is not without challenges, Fahy admits, the greatest of which is the high concentration of cryoprotectants needed to prevent ice formation. The toxicity of such concentrations can be more damaging than ice itself, says the University of Notre Dame’s Jack Duman, yet it could be possible to add antifreeze proteins to help lower the necessary concentration. That combination of strategies, however, has yet to be explored.

But other teams working to use antifreeze proteins in cryopreservation forge ahead. South Carolina-based Cell & Tissue Systems, a cryopreservation and tissue-storage company, is currently using insect AFPs, which prevent ice formation even better than fish AFPs, to successfully cool veins and arteries without the destructive formation of ice. “We’ve found that we can go below zero with these whole tissues for days on end without significant deterioration,” says the company’s president and chief science officer, Kelvin Brockbank.

In 2010, Brockbank began using insect AFPs in collaboration with Jack Duman, an expert in insect cryobiology and AFPs at the University of Notre Dame in Indiana who has studied freeze-tolerant and freeze-avoiding insects since the 1970s. With graduate student Kent Walters and biochemist Anthony Serianni, Duman recently discovered another antifreeze compound—this time from a small black beetle—that could be even more useful than previously identified insect AFPs. The Alaskan Upis ceramboides beetle is freeze-tolerant down to –60?°C, thanks in part to the production of the only known nonprotein antifreeze—a glycolipid consisting of a complex sugar called xylomannan and a fatty acid.2 The antifreeze appears to coat cell membranes. There, the researchers hypothesize, it not only inhibits ice-crystal formation in extracellular water but prevents ice from entering a cell, acting like armor against the cold.

“It’s phenomenal, perhaps better than any of these peptides to date,” says Brockbank. “This is absolutely going to have benefits—certainly for cells, probably for tissues, and possibly for organs.”



A delicate balance

Despite a few notable successes, scientists still lack the ability to preserve more complex tissues and organs. One of the biggest threats of freezing is not the physical damage from ice crystals—which look like tiny butcher knives under a microscope—but the effect of freezing on the flow of fluids into and out of cells.

When a tissue is exposed to below-freezing temperatures, the water between cells freezes first. As ice crystals form in the extracellular space, solutes such as metabolites, ions, and various proteins become concentrated due to the decreasing volumes of liquid water. Water therefore rushes out of the cell in an attempt to dilute the now-concentrated exterior environment, causing the cell to shrink and damaging the plasma membrane. At the same time, ion concentrations inside the cell increase, harming internal organelles. This outflux of water also puts extreme pressure on the shrinking cells: if a cell loses more than two-thirds of its water, the pressure becomes too great, and the cell collapses. “That is not survivable,” says Costanzo. “That’s damage that can’t be repaired.”

There is a way to avoid the damage from osmotic pressure, however—as the wood frog has clearly demonstrated. As summer turns into fall, and the days shorten and temperatures creep down, wood frogs begin to accumulate urea, and later glucose, in their skeletal muscle, liver, and blood. These natural cryoprotectants flow into the frog’s cells and serve to diffuse the concentration gradient between the interior and exterior of the cells as extracellular water freezes, preventing cell shrinkage. (See illustration.)

Another Ohio freeze-tolerant frog, the Cope’s gray treefrog (Hyla chrysoscelis), accumulates glycerol, instead of urea or glucose, as a cryoprotectant. Glycerol, it turns out, is a very effective cryoprotectant for many types of animal cells: in 1949, English biologist Christopher Polge first used glycerol to preserve fowl semen at −79 ?C, and within a year produced the first chicks from eggs fertilized with frozen sperm. Since then, glycerol has been used to safely freeze bacteria, mammalian embryos, and more. Other useful cryoprotectants have been identified in freeze-tolerant and freeze-avoiding animals, including trehalose, sucrose, and sorbitol.

However, testing these compounds for their ability to preserve larger human tissues has produced disappointing results. Alone, cryoprotectants have been unable to sufficiently protect a human organ from freezing damage: even if they protect individual cells from damage, they do not block the formation of ice between cells that interrupts vital cell-to-cell interactions in a tissue. In addition, cryoprotectants that are nontoxic for one species or tissue may be highly toxic for another. Glycerol, for example, damages human heart tissue. As yet, there is no known universally nontoxic cryoprotectant.

An additional risk of cryopreservation is the damage that can occur when a solution rapidly moves into and out of a cell, such as injuring the cell membrane or destabilizing the cytoskeleton. But Carissa Krane of the University of Dayton and David Goldstein of Wright State University in Ohio may have found a solution to that problem: a cell membrane protein that humans share with our distant freeze-loving frog relatives.

Goldstein takes a midnight stroll through the swamps of Ohio every year in late spring. Wading through water and mud, he listens for the shrill mating call of the Cope’s gray treefrog. During the night, Goldstein and his students capture 30 to 40 frogs and haul them back to his lab. The next morning, Goldstein calls Krane to tell her that her frogs are in.

About 5 years ago, Krane was busy using mice to study aquaporins—a class of membrane proteins discovered in the 1990s that regulate the flow of water into and out of cells—when Goldstein contacted her about a project on how glycerol and water move through cells in Cope’s gray treefrogs. She jumped onboard, and right away one of her graduate students identified an aquaporin in the frogs’ cells that allows the transfer of both water and glycerol through the cell membrane—an aquaglyceroporin. Last year, the team implicated the aquaglyceroporin, called HC-3, in freeze tolerance: blood cells from cold-acclimated frogs—those held in conditions simulating the approach of winter, and thus building up their glycerol stores—have a higher abundance of HC-3 than those of warm-acclimated frogs.3

Krane’s team sequenced the HC-3 gene and found a close ortholog called Aquaporin 3, or AQP3, in the human genome. The protein AQP3, surprisingly, is present in the same tissues in humans as HC-3 is in frogs—blood, liver, and skeletal muscle. “We actually have these proteins expressed in the same cells, but we don’t freeze,” says Krane. “If we can just understand how this natural process happens in a frog, maybe we can imagine a scenario down the road where we could prepare an organ harvested from a human donor by perfusing it with glycerol under cold conditions that upregulate these proteins,” she says.



Breaking records

The list of scientific challenges for cryopreserving organs is long, and that’s not even including the trouble of thawing tissues, which is “potentially more stressful than freezing,” says Goldstein. Ice crystals melting in the extracellular spaces of a tissue create pools of water, which can upset the osmotic balance and, this time around, cause cells to swell.

All told, the days of freezers full of human organs are still a ways off. “When I first started in the field in the ’80s, I thought there would be a couple different variants on a theme, and that we would solve preserving mammalian cells and tissues for medical applications forever,” says Brockbank. “But here I am, 20 years later, and we’re still far from it.”

But neither he nor other cryobiologists are giving up. One organism that might hold the answer is a small, flightless fly called the Antarctic midge (Belgica antarctica)—the world’s southernmost insect. The midge is the only known insect that spends its entire life in Antarctica. It survives for 2 years as a larva, frozen throughout the continent’s 7-month winters, then metamorphoses into a 5-millimeter-long adult that lives only 10 to 14 days in a rush of mating and laying eggs before it dies.

Over the last 8 years, Lee at Miami University has identified numerous ways that the Antarctic midge survives freezing, including elevated levels of glycerol, glucose, and trehalose—and aquaporins. But in this case, the aquaporins appear to help the flies, not to accumulate glycerol, but to move large amounts of water into and out of cells. To freeze without the dangers of ice, the midge simply gets rid of most of its body water.

Losing 15 percent or more of our body water is fatal to humans. Midge larvae, however, can survive a 70 percent water loss. “You can dry these little fly larvae out until they look like little raisins. They look terrible,” says Lee. “Then you add water, and they plump up and wriggle away. You can practically hear them laughing at you. They can handle this—it’s no big deal.”

Aquaporins may be at the heart of the midge’s unique dehydration ability. With David Denlinger at Ohio State University, Lee and colleagues recently sequenced the midge genome and have already identified a key aquaporin involved in the insect’s rapid dehydration.4 When they blocked aquaporin channels in midge tissue, the cells failed to survive freezing. “In hindsight, it’s a real clear thing,” Lee says. The ability to freeze without damage is “all about water moving around.”

COURTESY OF RICHARD LEETaking a cue from the midge, Lee’s team found that slightly dehydrating other insects increases their cold tolerance. The same might be true for mammalian organs: in the early 1990s, researchers at the University of Rochester in New York found that dehydration helped cryoprotectants reduce freezing damage in rat hearts.5 Still, dehydration stresses cells, especially the cytoskeleton, and there is little to no effort underway to apply this strategy to human organ cryopreservation.

Lee and Denlinger were surprised to find yet another midge adaptation to the cold: the larvae keep heat shock proteins (HSPs) turned on, in high production, all the time.6 “They didn’t read the textbook,” says Lee. In most organisms, HSPs, which help proteins fold and maintain their shapes, are turned on only when an organism is under severe stress. But continuous production of HSPs may be a common way to deal with continual cold stress: an Antarctic fish, Trematomus bernacchii, and an Antarctic ciliate, Euplotes focardii, also constitutively express such proteins.

“We think that when you’re living in a constantly cold environment . . . that it is necessary for you to maintain these chaperone proteins to help protect your proteins against abnormal aggregation and degradation,” says Lee. Activating HSPs in transplanted human organs, therefore, might be another strategy for cryopreservation, but it also remains untested.

From antifreeze armor to dramatic dryness to syrupy cryoprotectants, organisms use a catalog of molecular strategies to survive the cold. In fact, if there is one constant in the field, it is that there is no single way to freeze a frog—or any organism, for that matter. “Ever since people have been doing cryopreservation work, they’ve been looking for a magic bullet,” says Duman. “Maybe there is no real magic bullet. There certainly isn’t one for insects. They do lots of different things. Maybe that’s what we’ll need to do as well.”

Correction (February 6, 2013): This story has been updated to correctly reflect that Greg Fahy and colleagues vitrified a rabbit kidney at −130 °C, not −22 °C. The Scientist regrets the error.

1. G. Amir et al., “Improved viability and reduced apoptosis in sub-zero 21-hour preservation of transplanted rat hearts using anti-freeze proteins,” J Heart Lung Transplant, 24:1915-29, 2005.

2. K.R. Walters et al., “A nonprotein thermal hysteresis-producing xylomannan antifreeze in the freeze-tolerant Alaskan beetle Upis ceramboides,” PNAS 106:20210-15, 2009.

3. V. Mutyam et al., “Dynamic regulation of aquaglyceroporin expression in erythrocyte cultures from cold- and warm-acclimated Cope’s gray treefrog, Hyla chrysoscelis,” J Exp Zool A Ecol Genet Physiol, 315:424-37, 2011.

4. S.G. Goto et al., “Functional characterization of an aquaporin in the Antarctic midge Belgica antarctica,” J Insect Physiol, 57:1106-14, 2011.

5. M.C. Banker et al., “Freezing preservation of the mammalian heart explant. III. Tissue dehydration and cryoprotection by polyethylene glycol,” J Heart Lung Transplant. 11:619-23, 1992.

6. J.P. Rinehart et al., “Continuous up-regulation of heat shock proteins in larvae, but not adults, of a polar insect,” PNAS, 103:14223-27, 2006.