OFF the coast of Antarctica, where glaciers calve into the sea, schools of
fish play around the ice. The temperature of their blood is below the freezing
point of water, yet it still flows.
On the other side of the world, a wood frog sits motionless by an icy Ontario
pond. It is frozen solid. But as soon as the warmer temperatures of spring
arrive, the ice will melt from its veins, its heart will begin to beat, and it
will take a few clumsy hops. Within a day of thawing, the frog will be ready to
mate.
Unlike frogs and fish, human beings don’t take too kindly to freezing.
“Between two and five per cent ice in any mammal kills it dead as a doornail,”
says cryobiologist Kenneth Storey of Carleton University in Ottawa.
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But human beings, being human beings, want to get around that little problem.
Some, because freezing their bodies when they die offers the only hope of
immortality this side of heaven (see “When Hugh Hixon dies . . .” ). Others,
because freezing human tissues would be useful for medicine. Using lessons
Storey and others have gleaned from fish and frogs, scientists can now keep
cells alive after a bout of cooling or freezing that at one time would have made
them shrivel up and die.
And promising results with rat livers suggest that we may be on the brink of
successfully freezing whole human organs, too. That’s important because it means
transplant surgeons will be able to go straight to the freezer and pick out the
right pre-typed, pre-packaged body part for their patients, rather than waiting
on perpetual standby for suitable organs from dead donors. Meanwhile, if the
more fantastic goal of bringing someone back to life after a spell in the
freezer remains elusive, at least research is telling us what it would take to
perform such a miracle.
Today, only a few types of human tissue are routinely frozen. Sperm banks,
for example, store sperm in liquid nitrogen at –196 °C. Freezing kills
about a third of the cells, but that leaves plenty to play around with. Since
the late 1970s, frozen embryos have gone on to become healthy babies, cattle
and, most recently, pigs
(This Week, 28 March, p 17). And every year thousands
of cancer patients receive transplants of their own frozen bone marrow, to
replace the cells destroyed by radiation or chemotherapy. To most tissue,
however, freezing temperatures are far more toxic.
As blood starts to freeze, concentrations of ions and other solutes in the
plasma soar and the osmotic pressure increases, sucking water out of cells so
they become dangerously dehydrated. And once frozen, water turns into the
cellular equivalent of ground glass. Just above 0 °C, boomerang-shaped water
molecules begin to lock together, held in place by hydrogen bonds. At freezing
point, these clumps of molecules adopt the rigid honeycomb lattice of ice. As
the ice crystals grow, the sharp edges and corners slash and stab cell
membranes, turning tissue to mush.
Antarctic fish keep the crystals at bay with a simple sugar-coated protein,
or glycoprotein, that lowers the freezing temperature of the blood. The
antifreeze glycoprotein is made of many repeats of one sugar group attached to a
sequence of three amino acids. The triplets are slightly polar, and stick to the
slightly polar water molecules, coating the nascent ice crystals. With no place
for the next water molecule to latch onto, the crystals stop growing. Only if
the temperature drops several degrees below zero will enough new crystals form
to swamp the antifreeze and cause the fish’s blood to freeze after all.
Antifreeze proteins also seem to protect the fish from another huge problem
facing those who want to freeze human tissue—as cells cool, they spring
leaks. Cell membranes are a fluid mix of fats, embedded with proteins that act
as gatekeepers regulating which molecules can go in and out. But as a cell
cools, the fat in the membrane solidifies like pan drippings around leftover
roast. Molecules clump together, creating a patchwork of membrane slabs that
don’t quite fit, and ions and water can pour through the gaps.
Some cells leak less than others, depending on the mix of lipids in their
membranes. Pig eggs, for example, leak badly, shrivelling like raisins.
Reproductive biologists have tried to flash freeze eggs from a variety of
different species in liquid nitrogen to rush them through the leaky phase. But
that only postpones the problem. The cells spring leaks as they thaw, which is
difficult to do quickly without frying the egg.
Back in 1989, Boris Rubinsky, a biomechanical engineer at the University of
California at Berkeley, and Amir Arav, a visiting vet from the University of
Bologna, found a solution—at least to the problem of leakage. The
antifreeze glycoprotein, mixed with antifreeze proteins from Arctic fish,
protects pig eggs from membrane damage when they are frozen and bought back to
body temperature. “We’re still not sure how it works,” says Rubinsky. He
speculates that the proteins may simply coat the cell membrane, plugging any
leaks as they happen.
But it’s still not possible to freeze your prize sow’s eggs for later use.
Eggs are huge cells, and they rely heavily on a scaffolding of microtubules to
keep their shape and help them divide. In pigs, that scaffold crumbles in the
cold even if antifreeze protein is present.
Because of the problems with freezing animal eggs, for years no one attempted
to freeze human eggs, but recently that too became an option. Last October, the
births of babies born from eggs that had been frozen for up to 25 months were
announced by two research groups working independently at the University of
Bologna and at Reproductive Biology Associates, a private clinic in Atlanta.
“There are very big differences in the way [eggs and sperm of] different species
react to freezing,” says Michael Tucker, director of the Atlanta group.
The ability to store human blood platelets may also soon be transformed.
Platelets are essential for blood clotting, and transfusions of platelets are
routinely given to burns victims to reduce bleeding from their skin, and to
chemotherapy patients, whose own clotting systems are crippled by the drugs. But
platelets, which currently must be stored at room temperature, become
contaminated with bacteria and spoil in less than four days. In the US, roughly
one-fifth go off before they can be used for a transfusion. Cooling them would
increase their shelf life, but even refrigeration is out—if the
temperature drops below 15 °C their fragile membranes let in calcium,
triggering part of the clotting mechanism and rendering them useless.
Shelf life
At one-seventh the concentration found in the blood of the Antarctic fish,
the antifreeze glycoprotein lets the platelets survive temperatures as low as 4
°C without calcium leakage, according to a report in theJournal of
Cellular Physiology (vol 168, p 305) by John Crowe and Fern Tablin of the
University of California at Davis. Now Crowe and Tablin plan to transfuse mice
and pigs with the previously chilled human platelets to see if they can still
clot blood. If the tests are successful, clinical trials in monkeys and then
humans can begin.
Being able to store platelets for weeks rather than days would be
particularly valuable in the US, where a donor shortage means that blood is
imported from Europe, says Paul Holland, director of the Sacramento Blood Center
in California. If platelets could be stored, then people who now donate them
could donate blood instead, cutting down on imports. “We wouldn’t be wasting a
valuable resource,” he says.
But antifreeze proteins alone are not enough to get a whole organ through one
freeze-thaw cycle, at least that’s Rubinsky’s experience with rat livers. Organs
have a harder time surviving the deep freeze than cells, for several reasons.
They usually consist of dozens of different cell types, whose membranes are in
intimate physical contact. Not only does the sheer variety of cell types make it
more likely that at least one will react badly to freezing, but all those
different cells shrink, swell and freeze at different rates, tugging on one
another and creating complete havoc as the freeze sets in. Still, wood frogs
manage to do it. But how?
Wood frogs, it turns out, only look frozen solid. In reality, the water in
between their cells freezes, but not the water within them. To achieve this
semi-frozen state, the frogs adopt two main strategies. First their blood
contains ice nucleating proteins—molecules that actually encourage ice to
grow by mimicking its crystal lattice. “If you could fly over a nucleating
protein in a miniature airplane,” Storey says, “its surface would look like
ice.” With so many nucleators in the blood, no one crystal ever gets big enough
to damage tissue.
Glucose is the second trick. Just as the extremities begin to get icy, the
frog’s liver starts churning out glucose, which circulates round its body. “The
frogs start out with the same amount of glucose we have,” says Storey, “then go
right to being diabetic.” Glucose in the cells has the same effect as antifreeze
in a car radiator—it drives the freezing temperature down. Consequently,
the cells’ syrupy insides stay liquid even while the remaining 65 per cent of
the water in the frog has turned to ice.
Water freezing in the frog’s blood helps the process along by reducing the
amount of liquid in the blood and raising its osmotic pressure. That sucks water
from the cells, increasing the glucose concentration still further. Then, just
before the cell becomes dangerously dehydrated, the osmotic pressure inside the
cell, which has been increasing at the same time as the sugar concentration,
reaches an equilibrium with the outside. In this way, glucose protects the
cells’ interior from severe dehydration as well as from the ravages of ice
crystals. Without glucose, “the cells would go squish,” says Storey.
When the frog thaws, the organs that were the last to freeze and are the most
syrupy thaw first, so that the heart begins pumping blood before the extremities
thaw. That way no thawed part of the frog is deprived oxygen. “They’re clever,”
says Storey, “they thaw from the inside out.” Storey knows this because in the
early 1990s, he and Rubinsky put a frozen frog in a magnetic resonance imaging
chamber at the Lawrence Berkeley National Laboratory and watched it melt.
That experiment was enough to convince Rubinsky that sugar might be the key
to freezing rat livers. So a few years ago, he perfused fresh rat livers with
the fish antifreeze protein and glycerol, and then froze them. Glycerol has
similar antifreeze and anti-dehydration properties to glucose but it has smaller
molecules which cross cell membranes more easily. After six hours, the rat
livers, which were thawed and washed to remove the glycerol, could still produce
bile in a culture dish. Now, with money from A/F Protein in Waltham,
Massachusetts, a company Rubinsky co-founded to commercialise naturally
occurring antifreeze proteins, he’s getting ready to transplant freshly thawed
livers back into live rats. If he succeeds, it will be a first for
cryogenics.
Human organs from the deep freeze are still some way in the future, but we
may soon be able to freeze at least one type of cold-sensitive human cell for
transplant. Transplants of insulin-producing pancreatic cells from aborted
fetuses or adult organ donors can cure diabetes. But there’s a snag. Patients
have to take heavy-duty immunosuppressants, making the cure worse than the
disease. One potential way around the problem is to induce immune tolerance with
a transplant of bone marrow cells, before transplanting pancreatic cells from
the same donor. There’s also some evidence that giving a patient pancreatic
cells from a variety of different donors will reduce dependency on
immunosuppressants, although no one knows exactly how this works. For either
strategy, however, hospitals have to be able to store pancreatic cells for weeks
at a time. Now Gillian Beattie, a paediatrician at the University of California
in San Diego, thinks she may have found out how to do that.
Just as frogs use glucose, Arctic brine shrimp and many cold-tolerant insects
use a sugar called trehalose. Trehalose, which forms a syrup as thick as
stretchy toffee, is even better at lowering freezing points and stopping
dehydration than glycerol or glucose. But just like glucose, it crosses
membranes slowly. How then to get it into the cells so that it can work its
magic? The answer, Beattie says, is to take advantage of the cold-induced leaky
membranes. She and her UCSD colleague Alberto Hayek slowly cooled human
insulin-producing cells in a trehalose solution, and just as the lipid membrane
started to congeal at around 5 °C, the trehalose leaked in. Then Beattie
plunged the cells into liquid nitrogen to rapidly finish the freezing
process.
Time capsule
Every few months, Beattie revives some of the cells she froze two years ago.
So far the cells have survived, and still produce insulin both in the test tube
and when she implants them into mice, where the human insulin is easy to
distinguish from the mouse’s own. Unlike platelets, the insulin-producing cells
do not seem to be adversely affected by their temporarily leaky membranes. “When
they came back viable after two years, I was very excited,” Beattie says.
“There’s no reason to think we can’t preserve them for ever.”
In January, Beattie transplanted some of her thawed human pancreatic cells
into two diabetic rhesus monkeys. In a few weeks, she will know whether the
cells have taken and can still produce insulin.
Given the progress that’s been made, is there any hope of freezing and
reviving a whole human? Barring some unforeseen breakthrough, such cryogenic
time capsules will very likely remain impossible, according to most experts.
Scaling up techniques that work on bits of humans won’t work for the whole
thing. High levels of sugar trigger diabetic shock, for instance, and glycerol
would be toxic when you thawed out and started to metabolise it. And even if we
could handle such chemicals, getting them inside all the cells in the body would
be problematic.
That’s not to say that people like Storey don’t wish they could freeze
humans. “If by magic I could fill you with high levels of sugar and put
nucleating proteins in your blood,” says Storey, “then I could freeze you.”
Unfortunately, the operative word, he says, is “magic”.
The world record for survival in the deep freeze goes to bacteria discovered
in Siberia in the late 1980s. David Gilichinsky, a microbiologist at the Russian
Academy of Sciences in Moscow, extracted the frozen cells from dozens of metres
below the surface, in permafrost that has been at –10 °C or less for
at least three million years. Within hours, the cells were happily dividing
again.
No mammal has ever been frozen and thawed and lived to tell the tale, but in
1992 researchers at cryogenics company BioTime in Berkeley, California, came
close when they chilled Daniel the baboon down to about 2 °C for 55 minutes.
To achieve that feat, they replaced his blood with a patented cold-resistant
substitute they are developing in an attempt to lengthen the time surgeons have
to operate by reducing the patient’s metabolic rate.
When Hugh Hixon dies, he plans to have his body frozen in the hope that
medical technology will one day be able to revive him for another go. A
biochemist at the body freezing company Alcor in Scottsdale, Arizona, Hixon
readily admits that the chances are slim that science will ever coax another
life out of his frozen corpse. But he says a slim chance is better than none at
all.
The practice of cryonics, as the postmortem freezing is called, has won
hundreds of adherents who are prepared to pay as much as $120 000 up
front for a spot in a liquid nitrogen tank. Part of the money covers the cost of
topping up the nitrogen every so often, and some goes into a trust fund for the
customers to live off when they are brought back. Alcor now has 13 bodies in its
tanks, as well as the severed heads of another 23 people who opted for the
discount plan.
The money also covers the cost of the rather elaborate freezing procedure.
The moment a doctor pronounces someone dead—usually when the heart and
respiration stop, but before most cells have died—Alcor members receive a
large injection of heparin, which prevents the blood from clotting. Then an
Alcor team flushes out the blood with cool saline solution and chills the body
in an ice bath. They pump a glycerol-based antifreeze into the arteries to
retard crystal growth before lowering the body, or the skilfully removed head,
into the nitrogen pool.
Hixon says he is fully aware of the difficulties in bringing frozen bodies
back from the dead (see main story), including finding a way to reverse whatever
laid them low in the first place. Another problem is that below about −95
°C the organs begin to crack. “It’s nothing like a windshield popcorning,”
he says, “you just get a few big ones.” And if that doesn’t make you impossible
to revive, the fact that most of your cells will die from the cold should do the
job nicely.
Most cryobiologists say the best Hixon can hope for is to be cloned from one
of the cells that does survive. And that would merely be “giving birth” to an
identical twin, a child with none of the memories or experiences of the
cryonaut.
To Hixon that’s all beside the point. He says getting himself frozen is like
jumping out of a burning airplane with no parachute: it’s the only chance at a
longer life he has got. “It’s a lifeboat option,” he says. “And it sure beats
the heck out of the alternative.”
Old, cold bugs . . .
and cool baboons
When Hugh Hixon dies . . .
- Further reading:
Animal Life at Low Temperature
by John Davenport (Chapman & Hall, 1992) - To freeze or not to freeze—the dilemma for life below 0 degrees C
by K. B. Storey and J. M. Storey, The Biochemist, vol 19, p 8 (1997)