From: Eugene Leitl (Eugene.Leitl@lrz.uni-muenchen.de)
Date: Fri Feb 08 2002 - 07:51:58 MST
-- Eugen* Leitl leitl
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ICBMTO: N48 04'14.8'' E11 36'41.2'' http://www.leitl.org
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http://www.sciencemag.org/cgi/content/full/295/5557/1015
New Prospects for Putting Organs on Ice
Jocelyn Kaiser
After a lull, scientists are again exploring vitrification and other
techniques for deep-freezing tissues and organs
In the movie Vanilla Sky, Tom Cruise has his broken body frozen in the
hope that he'll someday be revived and healed. In reality, cryonics, as
this practice is known, remains the most speculative science fiction.
Researchers have failed for decades to deep-freeze and thaw most
tissues--let alone organs or animals--without damaging them, often
seriously. But recently, a few cryobiologists have celebrated successes
with ovaries and complex tissues like vascular grafts.
The work has sparked hope that donated organs can eventually be banked for
longer than the current few days, which would buy time for distributing
them and finding recipients who are good immunological matches. And as
progress is made engineering artificial livers, bladders, and other
tissues containing living cells, the need for better preservation
techniques is expected to grow (see Viewpoint on p. 1009). "Each company
at some point will need to store and transport their product," says Mike
Taylor of Organ Recovery Systems in Charleston, South Carolina.
Currently, transplant surgeons perfuse whole organs with a special
solution that enables them to be banked just above 0ºC for up to a few
days. Ideally, they would like to preserve organs at -196ºC, the boiling
point of liquid nitrogen--so cold that molecular motion virtually stops
and tissues cease to decay. Half a century ago, that seemed within easy
reach. Scientists found that blood and sperm could survive such deep
freezing if mixed with glycerol. The glycerol lowers the cells' freezing
point and keeps them from getting lethally salty when they do freeze and
water diffuses out. And since 1972, embryos have been frozen with liquid
nitrogen and later successfully implanted.
Organs, however, don't hold up so well below the freezing point. Water
leaked from cells during freezing forms ice crystals in the space between
cells, and this ice destroys fragile structures such as ducts and blood
vessels. Sometimes pieces of organs, such as pancreatic islet cells, work
adequately after freezing. But larger, more complex organs such as kidneys
don't function properly when sufficiently ravaged.
As a way around this problem, some researchers turned to vitrification, or
"ice-free" cryopreservation. The idea is to fill the organ with a viscous
fluid that turns into a glassy (not crystalline) solid at low
temperatures. This reduces the problems of ice, but toxicity of the
cryopreservant can still damage organs. And ice crystals tend to form as
vitrified tissue--especially large pieces--is warmed. Partial success came
in the 1980s, when Red Cross scientist Greg Fahy showed that rabbit
kidneys could withstand the high concentrations of cryoprotectants needed
to vitrify these organs. They worked when reimplanted, but Fahy had only
cooled them to -3ºC. Many experiments later, it's clear that
"vitrification is very, very complicated," says cryobiologist David Pegg
of the University of York, U.K., and in most labs vitrification of large
organs has been on hold.
Recently, cryobiologists have had better luck studying vitrification on a
smaller scale. For example, fine-tuning the solutions that are injected
into the organs, as well as heating and cooling rates, minimized injury to
2- to 3-centimeter-long pieces of rabbit veins, Taylor's team at Organ
Recovery Systems reported in the 18 March 2000 issue of Nature
Biotechnology. The vessels retained 80% of their function when dosed with
drugs that cause them to contract, compared to 20% following simple
freezing. When implanted in rabbits, the grafts appeared to work normally.
"We're the first to demonstrate [that vitrification works better than
freezing] in reasonably complex tissue," Taylor says. Two other groups
have recently shown that they can vitrify corneas, getting much less
damage than from freezing. But the scientists have yet to show whether
these corneas function in vivo.
Roger Gosden and colleagues at McGill University in Montreal have had
success even with conventional freezing with a small organ, they reported
last month in Nature. They froze rat ovaries, fallopian tubes, and
attached blood vessels in liquid nitrogen and transplanted them into
genetically identical rats whose ovaries had been removed. The animals
ovulated. Some ice damage occurred, so vitrification might be even more
successful, suggests Gosden, now at the Jones Institute for Reproductive
Medicine in Norfolk, Virginia.
Several groups are tackling problems that thwart both vitrification and
conventional freezing. Pegg's group at York, for instance, is working on
how to thaw tissues and avoid ice crystal formation. The team has
developed a technique that Pegg says can evenly and quickly heat pingpong
ball-sized clumps of cells embedded in gelatin to simulate large tissue.
Taylor's group, meanwhile, is collaborating with Carnegie Mellon
scientists on dosing tissues with iron compounds that are then excited
with magnets to generate heat.
Taking a cue from nature, researchers are also using natural antifreeze
proteins to help mop up crystals formed during freezing and thawing. Many
organisms, from carrots to fish to beetles, produce proteins that latch
onto and isolate growing ice crystals. The one drawback is that at
temperatures well below 0ºC, this system can backfire by causing ice
crystals to form spikes that disrupt cells. Several companies are
developing improved synthetic versions of these "ice blockers"; for
example, Taylor's company hopes to produce smaller molecules that attach
to ice crystals at the base as well as the face, which could prevent spike
formation.
Fahy, meanwhile, has never given up trying to vitrify large organs. "He
has soldiered the way on this for years and years," Pegg says. Now at a
company called 21st Century Medicine in Rancho Cucamonga, California, Fahy
has tested hundreds of vitrification solutions and patented the most
promising ones. "Greg has always been tantalizingly close to getting it to
work," says cryobiologist William Rall of the National Institutes of
Health. The company Fahy works for receives funding from the Life
Extension Foundation, which supports cryonics. Fahy says his work is
strictly limited to cryopreserving organs. But he adds, "If I'm
successful, perhaps it will remove some of the [cryonics] controversy."
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