X-Message-Number: 11231
Date: Sun, 7 Feb 1999 13:34:21 -0500 (EST)
From: Charles Platt <>
Subject: 21st Century Medicine Seminar Summary: Part 2
The following text is the second of three parts of a
summary of the seminar sponsored by 21st Century Medicine in
Ontario, California, on November 8th last year. This seminar
presented preliminary results of research that promises to
eliminate damage in cryopreserved organs caused by ice,
toxicity, and rewarming.
Originally I had hoped to circulate my summary of this
exciting research last December. I was delayed by problems
transcribing the tape, and by conflicting obligations. I
regret the delay but am now able to offer my summary,
accompanied by excellent reproductions of brain electron
micrographs, in a 16-page leaflet. Members of CryoCare
Foundation will receive this publication automatically;
anyone else can receive a copy free by sending a self-
addressed 9" x 12" envelope to Charles Platt, P.O.Box M,
Jerome, AZ 86331.
Alcor members will find a version of this text, with
lower-resolution photos, in the current issue of Cryonics
magazine. An abridged version, with fewer photographs, is
scheduled to appear in The Immortalist.
The complete seminar is available on a set of five video
tapes for $50. Individual tapes are also available for $15
each. Call toll-free 877-277-0322 or send a check or money
order payable to Life Extension Foundation, Box 229120 Dept.
21MED, Hollywood, FL 33022.
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Part Two
Real-World Applications
After the descriptions of these discoveries, Fahy listed
some immediate, potentially lucrative applications. First,
there's the transplant field. "Most kidneys are not matched
to the recipient," Fahy said. "This causes rejection. . . .
95 percent of the time we have a bad match between the
recipient and the donor in terms of tissue type. Livers and
hearts are an even worse problem."
Obviously if organs can be kept "on ice," this would
allow time for better matching. About 20,000 organs are
transplanted each year in the United States; if a banking
system enabled more efficient use of these organs by solving
the problem of rejection, this would justify the expense of
setting up the system and paying a royalty to 21st Century
Medicine for its preservation techniques.
But according to Fahy, "The really big market is in
artificial tissues and organs, because there's no limitation
on supply." He estimated that the total market is for at
least 100,000 implants a year.
Initially he hopes to cryopreserve kidneys, since this
is the organ that has been used most extensively in
experiments and is best understood. "I've had clinicians tell
me that as soon as I think I'm ready, they'll go ahead and
transplant a human kidney," Fahy said.
At the same time, he noted that 21st Century Medicine is
"negotiating a contract with a major university which is
skilled in cardiac preservation, and we will test out our new
vitrification solutions in that laboratory." 21st Century
Medicine will retain the commercial rights to results of the
research.
"We also have some possibilities for going into liver
cryopreservation," Fahy went on. "We're now negotiating a
contract with a liver transplant laboratory that is
interested in developing short term liver cryopreservation at
relatively high subzero temperatures, and we will move
forward at their expense on that, but 21st Century Medicine
again will own the commercial rights."
Summing up, Fahy said, "We are now building a perfusion
machine to actually do the experiments in house. The surgical
facilities are ready to go. . . . And we're already engaged
in friendly negotiations with a number of organ preservation
labs, to go further."
Other Markets
Fahy said he had never paid much attention to freezing
small systems such as sperm, because the problem seemed "too
trivial" compared with preserving a large, complexly
structured organ such as a kidney. Still, 21st Century
Medicine can acquire revenue and credibility if its research
improves existing procedures such as cryopreservation of
sperm or corneas.
Fahy valued the market for human sperm at $20 million,
while bovine sperm is a $200 million market, and human
corneas are a $400 million market. He said, however, that
when he investigated these areas, he found that between 90
and 95 percent of human sperm donors are rejected because
their sperm cannot survive the primitive preservation
procedures, while vitrifying human corneas has been
considered so difficult, no one is even trying to do it
anymore. "Currently there are about 50,000 cornea transplants
a year," Fahy said, "but I'm told by the top people in the
field that if you could bank corneas without limit, this
market would expand by a minimum of 5-fold, so the corneas
alone would be worth $2 billion a year, and we'd get whatever
royalty we could charge on that."
He showed a video tape in which sperm died after being
frozen and thawed in a 1 molar glycerol solution, but
survived in his new VX cryoprotectant. "We're new at this,"
he admitted. But, "Our result, preliminary though it may be,
is better than what's out there, so it is possible we can
help the sperm bankers with their problem of expanding the
donor pool and saving money. That means a market for 21st
Century Medicine."
He predicted similar success in vitrifying corneas,
though he has not tried this yet using VX. "We're currently
collaborating with a major-name medical clinic to have a
venture to demonstrate that we can cryopreserve corneas by
vitrification, using the new technology."
Brain Cryopreservation
For cryonicists, the most exciting aspect of the
conference came at the end, where Brian Wowk and Gregory Fahy
revealed results of their first two experiments applying new
cryoprotectant formulas to rabbit brains. "In general," Fahy
commented, "we think we have achieved the equivalent of
complete vitrification of the brain."
A year ago, no one had any idea that this might be
achieved so quickly. Moreover, the procedure does not require
extremely rapid cooling, high atmospheric pressures, or other
exotic techniques, and a sample brain has been not only
cryopreserved but rewarmed with virtually no structural
damage. There is presumably some chemical damage from
toxicity, which would prevent restoration of function.
Additional research will be needed to address this.
Currently, under ideal circumstances (which are often
unavailable), a cryonics patient is perfused with a solution
of glycerol reaching a final concentration of 7 to 7.5 molar,
after which the patient is cooled at approximately one-tenth
of a degree per minute. This is the best we can hope for. But
as Brian Wowk demonstrated at the conference, the results are
extremely unsatisfactory. He showed a slide (reproduced in
Photo 5) of a two-liter solution of 7.3 molar glycerol that
was cooled at 0.1 to 0.3 degrees per minute, to a temperature
of -100 degrees. The chalky white appearance is caused by
millions of tiny ice crystals in the solution. In a human
brain, each crystal is likely to cause significant damage.
Photo 8 illustrates this damage. The picture is a
reproduction of an electron micrograph of a canine brain that
was perfused with 7.5 molar glycerol, cooled using optimal
cryonics protocol, and then rewarmed. The white, "empty"
areas almost certainly were caused by ice forming and
displacing or destroying tissue. After rewarming, the ice
melts and debris remains. Remember, this is the best we can
hope for, using current procedures.
Photo 6 shows an obvious improvement. This flask
contains a 7.2 molar glycerol solution to which 1 percent of
Wowk's "X1" ice blocker was added before freezing. The
solution is now partially vitrified, meaning that it has
turned into a uniform glasslike substance interspersed with
hundreds of ice balls a few millimeters across, as opposed to
millions of tiny ice crystals. The large pale object at the
bottom of the flask is not ice; it is a stir bar. Wowk
estimates that ice now constitutes only 10 percent of the
mixture, by volume.
This is still less than ideal, but it can be achieved
right now just by adding the X1 ice blocker that Wowk has
discovered. No special cooling technology is required.
What if we use a 7.5 molar glycerol solution with 2
percent X1? Photo 7 shows the result. There is now virtually
no ice, and almost 100-percent vitrification has occurred.
The 7.5 molar solution is so viscous, it can be used on
human patients only with difficulty. Also, there's no
guarantee that the insides of cells will be completely
protected, because the X1 ice blocker does not penetrate cell
membranes.
If cooling can be done more rapidly, however, internal
cell damage should be minimized, because (in very simple
terms) ice has less time to form.
Until relatively recently, no one knew how to cool a
human patient faster than 0.1 degree Celsius per minute. The
new technique of perfluorocarbon perfusion, however, offers a
radical improvement. First, the patient would be perfused
normally with cryoprotectant. Then the vascular system would
be flushed with a perfluorocarbon, which is nontoxic and
remains free-flowing at temperatures as low as -130 degrees.
Potentially this can produce a cooling rate of almost 10
degrees per minute--100 times the best rate for a cryonics
patient using conventional methods. Because the temperature
differential diminishes as cooling takes place, the cooling
rate will diminish also; but 1 degree per minute is still
possible even at -110 degrees. This has actually been
verified in dog experiments.
The procedure will require a specially insulated room
where perfluorocarbon can be sprayed onto the patient and
perfused through the patient under remote control. A
prototype cold room has been built at 21st Century Medicine.
Perfluorocarbon cooling is such a powerful technique, it
enables vitrification with lower concentrations of
cryoprotectant. A 7 molar solution of glycerol, with X1 ice
blocker added, should be sufficient. Unfortunately, even a 7
molar glycerol solution is biochemically toxic to cells.
Perhaps chemical damage will be much easier to undo in the
future than structural damage, but still we would prefer,
obviously, to do no damage at all.
Wowk and Fahy have taken a step in that direction.
Shortly before the conference, assisted by biologist
Christopher Rasch, surgeon Yasumitsu Okouchi, and Mike
Darwin, Fahy perfused two rabbits (the first consisting of
the upper body, the second consisting of the head only) using
two different perfusates. The composition of the perfusates
is not public information at this time, but one of them
relied more on concepts developed by Brian Wowk in his
research into methoxylated compounds, while the other
incorporated ideas relating to the VX series of
cryoprotectants formulated by Fahy.
Photo 9 shows a perfusion in progress, using a closed
circuit in which concentration was gradually ramped up, as
shown in Graph 1. This procedure is roughly similar to
published protocols involved in rabbit-kidney
cryopreservation.
After perfusion, Photo 10 shows one of the specimens
being cooled in the plastic bucket just in front of the
stainless-steel dewar. Note the electric drill clamped over
the bucket, which provided rapid stirring, promoting heat
exchange.
Graph 2 shows the rate of cooling. Note the small bump
between 125 and 130 minutes, and -30 and -40 degrees C. This
bump suggests that a small amount of water froze, briefly
liberating latent heat as it turned to ice.
Photo 11 is a very high-magnification electron
micrograph showing the condition of the brain after
rewarming. An intact synapse and presynaptic neurotransmitter
vesicles are visible, with good postsynaptic density. Some
shrinkage has occurred because of the high concentration of
cryoprotectant, creating the small white spaces around the
fine axons, dendrites, and synapses shown. Fahy feels that
this shrinkage is not very significant because the structure
seems intact.
Photo 12 is at a slightly lower magnification (the
original electron micrographs range from 10,000 to 40,000
magnification) showing an intact axon with clearly defined
cell membrane. "We do see some cavities on the local level,"
Fahy commented when he showed this picture at the conference.
But these cavities are minimal compared with the damage in
brain tissue perfused conventionally with glycerol.
Photo 13 provides a broader overview showing no apparent
ice holes. There are some slightly shrunken neurons, but
again the membranes are intact and structure is clearly
visible.
Not all areas of the brain were preserved so
successfully. Photo 14 shows cells that have been damaged by
ice, toxicity, or inadequate oncotic pressure allowing tissue
edema. "Nevertheless this seems a substantial advance over
glycerol," Fahy commented.
In their second rabbit experiment, Fahy's team attempted
the complete avoidance of ice within the brain. "We did
various things to optimize the perfusion," he told his
audience at the conference, though he would not reveal
specific details. Graph 3 shows the increase in concentration
of perfusate over time, while Graph 4 is a remarkably smooth
cooling/warming curve, showing no kinks or bumps that would
indicate ice formation as the temperature fell, and no ice
forming either during the rewarming phase.
Electron micrographs of this brain revealed truly
exceptional results. Photo 15 shows an axon containing
neurofilaments--conglomerations of individual molecules.
These are clearly discernible in the original picture but may
be harder to see here because of the limits of halftone
printing. "We have never seen those [filaments] in any
cryopreserved brain, ever," Mike Darwin commented, as the
slide was shown at the conference.
"This is a level of preservation that's really
unprecedented," Fahy agreed.
A lower-magnification overview of the second brain, in
photo 16, shows no pockets of cell damage of the kind seen in
the first brain. There are moderately dehydrated but
basically intact cells amid shrinkage spaces that are
moderate and probably not a source for concern. Intact myelin
sheaths are visible around axonal processes.
All the electron micrographs mentioned so far were of
the cerebral cortex. Photo 17 is of the hippocampus, which is
the area most sensitive to ischemic insults. Although the
cells seem dehydrated and shrunken, they remain well
connected to the neuropil surrounding them.
Fahy was quick to warn his audience that these two
experiments are just the first that have been done using the
new processes developed this year at 21st Century Medicine.
"We expect that we can go farther than this fairly rapidly,"
he said, "now that we have a better feel for the kind of
cooling and warming rates that we're dealing with."
Sitting next to Fahy, Mike Darwin added that "I've spent
twenty years doing cryoprotective perfusions and subsequent
evaluations of brains. . . . my opinion is a very dismal one
about the utility of current procedures, particularly in
preserving the fine connections of the cells to the neuropil,
which is probably where you're at, where your identity is
really encoded." But he went on: "I just cannot overemphasize
the difference between this [new work] and the previous work
that has been done. We've eliminated virtually all of these
terrible tears, massive tears that occur at 10 and 30 micron
intervals, and the ultrastructure is remarkably better. I
think that within very short order we're going to have
significant viability, 50 percent viability, in brains that
are treated with techniques that yield the same kind of
ultrastructural results."
Of course, we don't know how easily the work will
translate and scale to human brains. Also, the researchers at
21st Century Medicine have been exploring several different
approaches, in parallel, to the same basic problems of
reducing damage. "We have not completely and fully combined
[these ideas] to get the most powerful possible approach to
cryopreservation," Fahy said, adding that the next step will
involve "fine tuning all the parameters in order to get the
best possible result. But it's just a straightforward
process, there's nothing magical about it."
"The magic is the money and the time," Darwin commented.
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