© JOELLE BOLT
Icing
Organs
Why scientists are so near and yet so far from being able
to cryopreserve organs
BY MEGAN SCUDELLARI
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.
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.
“I’ve been doing this for 25 years now,
and still, whenever we freeze the frogs and
watch them recover, it seems magical,”
says Costanzo, a cryobiologist at Miami
University in Ohio. But though it may look
like hocus pocus, the frog’s dramatic transformation is cold, hard fact.
For most animals on the planet, prolonged exposure to temperatures below
freezing means death. But for the wood
Organ cryopreservation
would transform medicine the
way refrigeration transformed
the food industry.
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
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then thawing them with ease and watching them hatch and live normal lives. (See
“Glass Menagerie” sidebar on page 38.)
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.
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
At temperatures below 0 °C the cells of human organs shrivel
and collapse, extracellular matrices rip apart, and blood
vessels disintegrate.
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 on opposite page.) 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
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.
But other teams working to use antifreeze proteins in cryopreservation forge
ahead. South Carolina-based Cell & Tissue
Systems, a cryopreservation and tissue-
© JOELLE BOLT
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.
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 longneglected 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,
MECHANISMS OF CRYOPROTECTION
A handful of species have learned how to survive in freezing climates. To do so, the animals must counteract the damaging effects of ice crystal
formation, or keep from freezing altogether. Here are a few ways they do it.
ANTIFREEZE PROTEINS
Antifreeze proteins (AFPs) Q
1 , first
identified in the blood of Antarctic fishes,
have repeating structures that bind to the
surface of ice crystals and prevent them
from growing into larger crystals Q
2 . AFPs
isolated from the blood of these fishes have
been used successfully to preserve rat and
pig hearts at below-freezing temperatures
for up to 24 hours.
CRYOPROTECTANTS
As the temperature drops, extracellular
water begins to freeze, leaving behind a
slush of concentrated solutes. In an attempt
to dilute those solutes, water rushes out of
the cell Q
3 , causing significant cell shrinkage
and death. But cryoprotective compounds
such as glycerol, glucose, urea, and trehalose
accumulate inside cells to help equalize the
imbalance of solutes, preventing water loss
and cell damage Q
4 . Scientists have found that
during the fall, wood frogs accumulate urea,
and later glucose, to preserve their organs
when the frogs freeze solid during the winter.
AQUAPORINS
Water can make its way through a cell
membrane unaided through the process of
osmosis, but a quicker way into or out of a
cell is through an aquaporin—a membrane
protein that regulates the flow of water into
and out of cells Q
5 . Scientists have found that
aquaporins help some freeze-tolerant frogs
move not only water but glycerol into cells
in preparation for freezing. Aquaporins also
help freeze-avoiding insects move water out
of cells during cryoprotective dehydration.
1
Q
Solutes
Antifreeze
proteins
Water
molecules
4
Q
Cryoprotectants
such as glycerol and
urea increase the
concentration of
solutes inside the
cell to help prevent
a harmful outflux of
water.
As ice forms in the
extracellular space,
water from inside
the cell rushes out
through aquaporins
to dilute the higher
concentrations of
solutes.
3
Q
AFPs bind to ice
crystals, preventing
them from growing Q
2
into larger crystals
Aquaporin
5
Q
CREDIT
C
R E D IT
T LINE
Ice
crystal
KIDNEY ICE CUBE: A rabbit kidney that has
not been perfused with vitrification solution is
frozen solid at a temperature of –140° C (left).
A perfused kidney is preserved, but not frozen,
at the same temperature.
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 −22 °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. 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.
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.
COURTESY OF GREG FAHY
GLASS MENAGERIE
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 freezetolerant 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.”
When a tissue is exposed to belowfreezing 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.”
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.
Ever since people have been doing cryopreservation work,
they’ve been looking for a magic bullet. Maybe there is
no real magic bullet.
—Jack Duman, University of Notre Dame
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 on page 37.)
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,
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 freezeloving 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 con-
tacted 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
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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,
THAWED ALIVE: Larvae of Antarctic midge
survive for 2 years, though they are frozen
throughout the continent’s 7-month winters.
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
J
that’s what we’ll need to do as well.”
References
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.
COURTESY OF RICHARD LEE
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.”
Taking 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.