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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