Biology on Ice

To learn how to better preserve human organs for transplantation, scientists may need to take a lesson from some remarkable frozen frogs.

By Wallace Ravven|Monday, August 01, 1994
Early spring, if you're willing to plod through snowbanks in northern woods, you just might get to watch a miracle unfold. As shallow ponds melt and then quickly freeze again, a tiny brown wood frog may get locked in the ice, just a few inches beneath the surface. Crouched as if contemplating a jump it will never get to make, the frog becomes frozen stiff. Its heart stops; its breathing ceases. But when the sun thaws the pond again, the frog can thaw, too. Ice melts in its body, its heartbeat returns, and its blood begins to circulate anew. The creature gulps for air and then, as if hours or days of suspended animation were just a small inconvenience, it hops away.

Turning into a block of ice might seem an odd way to cope with frigid temperatures, but for the wood frog and a number of other cold- blooded animals, it's an effective one. And it may one day prove effective for humans, too--or at least for human organs. By co-opting the physiological strategies frogs have evolved to survive freezing, a number of researchers think they can find ways to preserve human hearts, livers, and other organs for transplantation.

Right now a kidney can be kept alive for only three days, in a bath of proteins, enzymes, and ions at 4 degrees above freezing; a liver can be kept no more than 36 hours, and hearts and lungs rarely survive more than 6. Those numbers might be extended from hours and days to months and years if the bath's temperature could be dropped, but to attempt to freeze and then thaw a human organ is to drag it through a physiological minefield. As an organ freezes, ice crystals stab and rip into blood vessels, cells dehydrate, and their membranes collapse and leak, destroying the vital proteins inside. Thawing can be even more dangerous. As the temperature creeps up toward the melting point, the ice crystals fuse, squeezing the cells into tighter and tighter spaces and deforming them. That's why, despite researchers' success in freezing single cells like sperm and blood cells, and even simple tissues like corneas and immature embryos, freezing and restoring whole organs, with their complicated and vulnerable architecture, remains largely beyond reach.

But with an engineer's conviction, Boris Rubinsky, a Romanian- born scientist, insists that the minefield can be cleared if we can just learn enough about the terrain. To that end, ten years ago this bioengineer invented a microscope to study in exquisite detail how cells freeze and thaw. With his sensitive instrument he's discovered a new way to protect mammalian organs from the deadly damage that can occur at cold temperatures. And last year, with recipes picked up from a range of cold- tolerant animals, he and his colleagues at the University of California at Berkeley managed to freeze mammalian livers for six hours and revive them. That's not exactly stopping the hands of time, but it's certainly slowing them down.

Rubinsky scrutinizes everything from egg cells to rat livers in his Bio and Thermal Engineering Laboratory at Berkeley. An industrial- looking gray metal door opens onto a jumble of desks, computers, and liquid nitrogen tanks; amid the clutter, Rubinsky peers into his patented cryomicroscope. It looks much like a regular microscope, with an eyepiece and a stage on which a slide sits. The key to this particular scope is that one end of the stage is connected to a tank of liquid nitrogen, which can cool that end to -160 degrees Celsius; the other end is warmed to about body temperature. A small motor moves the slide between the two ends of the stage: the temperature can thus differ by 200 degrees from one end of the slide to the other.

Today the subject of Rubinsky's attention is a sample of blood freezing on the glass slide. A video camera aimed through the microscope records the action on a TV monitor a few feet away: on the cold side of the slide, opaque fingers of ice race through the clusters of red blood cells, rolling over them like a deadly mudflow and crushing the cells against the slide. Trapped in the narrowing space between ice blades, some of the blood cells are stretched; others are torn.

But when Rubinsky adds a solution of glycerol to lower the freezing point of the blood on the slide, the ice crystals change from sharp-edged blades to a series of feathery branches. The ice is less dangerous when it takes on this configuration--the unfrozen channels between the crystals are larger, and most of the red cells lodge, undamaged, in the gaps. "That's one of the secrets we've learned," says Rubinsky in his gentle Transylvanian accent. "The fate of the cells depends a lot on the structure of the ice crystals."

The knowledge that some sort of antifreeze can protect a living creature dates to as long ago as 1957, when physiologist Per Scholander reported on trips he'd made to Baffin Island and Labrador, where he caught fish "with the aid of Eskimos and dog teams." Scholander knew that the fish were living in water that was colder than the freezing point of their blood, and he realized that something must be preventing them from freezing but couldn't identify what that something was. A decade later Arthur DeVries, a young graduate student at Stanford, examined fish he had collected in the Antarctic and discovered that this antifreeze function resided in the protein portion of the blood. Within a few years he had identified and purified a number of these "antifreeze proteins."

DeVries also began to figure out how the antifreeze worked. The proteins, he learned, adhere to tiny ice crystals and physically interfere with their growth. These proteins give the fish just one extra degree of protection, but that one degree makes all the difference. "They would normally freeze at about -1 degree Celsius," explains DeVries, now a physiologist at the University of Illinois at Urbana-Champaign. "But the antifreeze protein in their blood protects them down to about -2 degrees-- the temperature of the water all around the continent of Antarctica. If they hadn't evolved the antifreeze protein, the whole Antarctic area would be devoid of fish."

Antifreeze proteins are one way to vanquish dangerous ice crystals, but they're not the only way. In 1989 Rubinsky took his cryomicroscope to the University of Bologna in Italy to help Israeli veterinarian Amir Arav learn how to preserve single-celled pig eggs, or oocytes, using a process called vitrification. To vitrify a liquid is to cool it so rapidly that its molecules don't have the chance to organize themselves into the delicate latticework that makes an ice crystal. Instead they become fixed in whatever position they happen to be in at the time, and the liquid turns into a rigid but unstructured glassy solid. With Rubinsky's instruments, and by immersing the eggs in a solution of protective chemicals such as glycerol and other alcohols, Rubinsky and Arav were able to control the rate of cooling precisely and chill the eggs to - 130 degrees Celsius without creating any detectable ice crystals; they were also able to watch what happened as they did so. Unfortunately, like others before them, what they saw was that the process of vitrification began to destroy the eggs' cell membranes, and thus the eggs.

The problem lies in the double layer of fatty lipids that is part of a cell membrane. These lipids are essentially liquid at body temperature. But at very cold temperatures they gel, just as fat congeals in a cooling skillet. During this change from liquid to gel (called the lipid phase transition), the membranes temporarily lose the ability to regulate the flow of ions and other compounds in and out of the cells. The consequences can be disastrous. Calcium, for instance, is found outside most cells in concentrations some 10,000 times greater than those found inside; but during the phase transition, calcium can flood through the altered membrane and destroy the cell.

As the eggs vitrified and the cell membranes became dangerously leaky, Rubinsky and Arav could only sit by and watch. Then, Arav remembers, "Rubinsky pulled some white powder out of his pocket and said, 'Look what I have. It's antifreeze protein.' "

DeVries had given Rubinsky samples of a number of these proteins, purified from the Antarctic fish. "I had already observed that the antifreeze proteins could modify the structure of ice crystals," recalls Rubinsky, "and I thought they might be beneficial when introduced into an organ. So I froze a liver with the antifreeze proteins and observed that the membrane looked intact. That kind of triggered the idea that perhaps they would preserve the membrane in oocytes as well."

"We put some of the powder in with the vitrification solution," Arav says, "and under the cryomicroscope we could see the cell membranes were staying intact." This was actually quite a surprise--after all, antifreeze proteins do their work by retarding ice crystal growth, but with vitrification there are no crystals to retard. Arav and Rubinsky worked well into the night to check their results, testing various cooling rates and studying the membranes' new resilience. Then, tiring of work, they headed out to a bar. But before they left, they put the remaining, unfrozen oocytes in an antifreeze-protein solution and placed them in the lab refrigerator, set at 4 degrees Celsius. They had no real reason to do such a thing; after all, Arav says, "nobody had ever preserved pig oocytes by refrigeration for even two hours." But 24 hours later, when they took the eggs out of the fridge and Arav looked at them under a microscope, he found that the oocytes' membranes were still intact.

It seemed that the same antifreeze proteins that adhere to ice crystals in cold-water fish can also somehow adhere to cell membranes at a range of temperatures both above and below freezing. They appear to kick in when the membrane is passing through its vulnerable phase transition. In addition to retarding freezing, the antifreeze proteins somehow shore up the membrane, plugging the holes. "These are remarkable proteins," says Rubinsky. "They appear to have more than one vital role to play in organisms that survive cold conditions."

As important as the proteins are, however, they are not the sole answer to the problem. Although they can impede the formation of ice crystals, they can only do so down to a certain temperature; below that, the proteins can't protect against the ravages of ice. Ice is made of pure water, so as it forms, dissolved salts and other ions are left behind in the fluid-filled spaces outside the cells. Water then begins to diffuse out of the cells until the total concentration of ions outside more closely equals that inside. But if too much water diffuses out, the cells shrink, their membranes shrivel, and the scaffolding within the cells breaks down. Ultimately, proteins lose the three-dimensional structure that allows them to work properly. In addition, there's always the danger that the ice crystals will puncture the cells, their organelles, or the blood vessels that nourish them.

Because the whole freeze-thaw process is so fraught with danger, for now organs for transplant simply can't be frozen. As a consequence, the time pressure involved in organ transplantation often forces doctors to accept poor tissue matches between donors and recipients. "With kidneys, a perfect match is achieved only about 5 percent of the time," says Greg Fahy, the head of organ preservation research at the Transplantation Laboratory of the American Red Cross. "Though the others are matched, they're just not matched well. With the liver and heart, preservation time is so short we don't even know the compatibility until after the transplant. Unfortunately, we're not likely to see big increases in the length of time organs can be preserved at above-freezing temperatures."

Fahy is convinced that, despite any problems with membrane leakage, the best possible future for transplantation will lie with vitrification. By racing past the temperatures at which water turns into ice, down to those at which it turns into glass, he says, vitrification offers the potential to elude ice damage. At temperatures of about -130 degrees Celsius, biological time is truly suspended.

The promise of vitrification, however, is still just a shimmer in the distance. The vitrification of organs, unlike the vitrification of single-celled eggs, requires lethally high concentrations of glycerol and other chemicals. These chemicals kill the organs they're trying to protect.

So when Rubinsky attempted to freeze and revive a mammalian liver, he turned not to high-tech vitrification but to the lower-tech wood frog. Along with Kenneth Storey, a wood-frog authority at Carleton University in Ottawa, he made the first-ever magnetic resonance images of a wood frog--or any animal, for that matter--in the process of freezing and thawing.

"For me, seeing is believing," says Rubinsky. "We had guesses as to what was occurring during freezing. The MRI showed us which guesses were correct."

Storey already knew that when a wood frog freezes, its liver produces enormous quantities of glucose--a simple sugar--which then acts as an antifreeze in the blood. Glucose levels throughout the body of a freezing frog can reach 100 times the normal glucose level in a human. And the sugar reaches its highest concentration in the brain and such other vital organs as the liver and the heart. So it wasn't a total surprise to Rubinsky and Storey when their MRI images showed that the liver, heart, and brain were the last parts of the body to freeze. The images also showed that the frog froze slowly, from the outside in toward the vital organs, and actually thawed from the inside out.

"It makes sense now that we know it," notes Storey. "If the animal's limbs thawed first, they might deteriorate before the heart and lungs could supply them with blood and oxygen."

Perhaps most important, Rubinsky's team clearly showed that the frog's survival depends on at least a partial dehydration of its cells. As expected, as ice formed outside the cell membranes, the cells began losing water until the concentration of solutes inside matched that in the fluid outside. But this had the effect of increasing the concentration of glucose inside the cells, which prevented the water remaining in the cells from freezing. The scientists found that the wood frog lost more than 60 percent of the water from its cells under freezing conditions, but the intracellular water that remained never froze.

Now that he thought he had enough details, Rubinsky was ready to proceed. He now knew that when freezing a mammalian organ, you should do so gradually, to give the organ's cells time to dehydrate. And he knew to use antifreeze--fish antifreeze proteins to protect cell membranes, and something like glucose to protect the cells' insides. Instead of glucose, however, he used glycerol because it passes more easily through mammalian cell membranes.

To give the glycerol time to seep into all the cells and protect the water within, Rubinsky knew he'd have to introduce it very gradually into the circulatory system. So with doctoral student Jen-Shin Hong he developed an engineer's dream: a temperature-controlled, computer-driven motorized perfusion system that is able to control precisely the pressure on four syringe pumps to assure that the right amounts of chemicals are injected into the organ at the right time. Using the new apparatus and some antifreeze proteins, the team succeeded last year in freezing three rat livers and reviving them six hours later.

Rubinsky wasn't the first to freeze a mammalian organ. A team at the University of Rochester froze a rat heart for three hours in 1991. And while six hours is impressive, it's still considerably shorter than the dozens of hours that a liver can be preserved with today's techniques. Of course, says Rubinsky, they could have tried to keep the livers frozen longer, but that wasn't the point. "The point of our experiment was not to develop the most efficient system," he says, "but to see if it is possible at all to bring the liver back from a frozen state."

Rubinsky thinks it's just a matter of more time and research--and close attention to details--until human organs will be routinely frozen for significant lengths of time. "Animals have evolved many ways to survive in extremely cold environments," he says. "I'm convinced that long-term organ preservation will come through mimicking the strategies that nature has already devised."
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