X-Message-Number: 6776
From:
Date: Sat, 17 Aug 1996 21:58:43 -0400
Subject: SCI. CRYONICS Fahy article
FAHY ARTICLE
In connection with the Prometheus Project, Dr. Thomas Donaldson has suggested
that everyone interested, if not already acquainted with the rationale behind
the vitrification approach to cryopreservation, should read the article by
Dr. G.M. Fahy, "Organ Preservation," in ADVANCES IN ANTI-AGING MEDICINE, ed.
R.M. Klatz, published by Mary Ann Liebert Inc., Larchmont NY 10538.
Following is my own attempt to summarize the article (or that part of it that
relates most directly to the rationale for vitrification):
Very low temperatures ordinarily cause ice formation in biological specimens,
which generally causes some damage at least in relatively large organs of
adult mammals. [I omit discussion of mechanisms of damage.] However, some
solutions do not freeze (form noticeable ice crystals) at any temperature;
instead, they just get stiffer, like glass or tar. This process is called
"vitrification" or "glassification" (from the Latin word for "glass").
Vitrification potentially has much the same long-term storage advantages as
freezing, but without the mechanical and chemical changes caused by formation
of ice crystals.
Problems with vitrification:
(1) The necessary high concentrations of vitrification-inducing
cryoprotective agents (CPAs) can be toxic, either generally or in process of
introduction or removal.
(2) When a specimen is cooled without freezing, damage may occur which is not
well understood, although it may be partly a result of additional time of
exposure to the CPA.
(3) When a vitrified specimen is warmed, crystallization ("devitrification")
tends to occur. To minimize this kind of damage, warming must be uniform and
rapid--on the order of 300 deg C per minute; total rewarming time (to 0 C)
must not be more than about 20 seconds. (This seems to imply a banking
temperature of about - 100 C.)
Dealing with the problems:
TOXICITY: To minimize toxicity, Dr. Fahy's group have used a
computer-operated organ perfusion machine to coordinate rate of cooling with
rate of perfusion, using mostly rabbit kidneys but also rat livers.
Around 1989 they obtained 100% survival of transplanted kidneys, using a low
concentration of CPA that would only permit vitrification when used with high
pressures, 500 to 1,000 atmospheres. (I believe this means they obtained the
survival in the absence of pressure or cooling, testing only for toxicity of
CPA.) Vitrification using the combination of high pressure and CPA perfusate
allowed no survival.
COOLING INJURY
Cooling injury was found to be more severe with higher concentrations of CPA.
Therefore a relatively low concentration of CPA was used initially, and the
rest needed to vitrify added when the temperature was near - 30 C.
"At the lowest concentrations needed for vitrification, 100% survival
resulted, and at the next higher concentration, 100% survival was attained
after cooling to - 32 C. These results emboldened us enough to try
concentrations that will vitrify with no applied pressure."
As at other points in the article, there seems to be a certain lack of
clarity here. We are not given any clearly stated data on vitrification
temperatures (or glass transition temperatures) as related to CPA
concentrations and to pressure, or explicitly and in full what was done when.
(Of course, space was a consideration, and possibly proprietary information.)
I think the quotation above means using CPA and cooling without pressure.
Next, apparently they used a similar approach with higher concentrations,
enough to vitrify without pressure. In their [then] most recent series, both
of two kidneys survived, with good life support function after
transplantation. Another of them also survived after further cooling to - 46
C.
Tissue slice experiments suggest that further cooling (beyond - 46 C) will
induce little or no further injury.
DEVITRIFICATION:
Ruggera and Fahy have had "success" in rewarming test CPA solutions at rates
of over 400 C/min. Plans were under way to finish perfecting the heating
technique.
QUESTIONS:
The following quotation is somewhat puzzling to me:
"Vitrification has been successfully applied to human islets of Langerhans,
human monocytes, human red blood cells, human liver cells in culture, certain
kinds of plants and plant tissues, and to animal embryos and egg cells. It
has been applied with partial success to human corneas. It is also clear
that, if earlier researchers had understood the scientific issues as well as
we do today, they could have successfully vitrified whole organs
(particularly guinea pig uteri and adult frog hearts) in 1965. Our laboratory
has obtained encouragement that it may be possible to vitrify both rabbit
kidneys and rat livers. These observations illustrate the universal nature of
cryopreservation by vitrification and imply that success with one type of
major organ can be followed by success with virtually any other type of organ
or human tissue desired for banking."
The above quotation has several strange features. He says that it "may" be
possible to vitrify rabbit kidneys and rat livers, but it is "clear" that
researchers in 1965 "could have successfully vitrified whole organs."
Nor do I see why the named successes imply further successes with "virtually
any other type..." WHY do the named successes show the "universal" nature of
crypreservation by vitrification? It seems to me one would have been just as
much justified, in 1948, when Rostand cryopreserved frog sperm with
glycerol--or at most a few years later, when many cell and tissue types had
been cryopreserved with glycerol and DMSO--to say that the way was now clear
to cryopreserve anything by similar methods. In fact, the researchers of that
time knew and stated clearly that methods required for other types of
specimen, and especially for whole adult mammalian organs, might not be
simple extensions of previous ones. As far as I can see, that is still true.
FOR ALL WE KNOW, THERE MAY BE MORE DIFFERENCES IN CRYOPRESERVATION
REQUIREMENTS BETWEEN DIFFERENT REGIONS OF THE BRAIN THAN BETWEEN (SAY) HEARTS
AND LIVERS.
Olga Visser has also said that her novel CPA, which has had apparently
complete success with rat hearts (THE BIGGEST BREAK-THROUGH SINCE 1948) and
at least partial success with pig hearts and other organs, has in her opinion
the potential to work with all organs--although with many individual
modifications required. With all due respect--and I am extremely hopeful
about her technology, and full of admiration for her achievements against
odds, as well as for Greg Fahy's achievements--I am uneasy with short term
optimism about all these things, even though I yield to no one on long term
optimism.
Thomas Donaldson seemed to feel that Fahy's article would do much to convince
readers of the high probability of success of the Prometheus Project as
currently outlined. It doesn't seem terribly persuasive to me in that
context.
Admittedly, there are many relevant things I don't know. For example, the
article says it took Fahy's laboratory at least 5 years (from 1981 to 1986,
and maybe longer, if I read him correctly) to solve the problem of correct
rate of introduction and removal of cryoprotectant. I don't know how many
person-hours were involved or how much money, but many such questions would
need clear answers to permit any kind of reasonable guesstimate about future
projects--their expected duration, cost, and probability of success. In many
cases we don't even know the QUESTIONS yet, let alone the answers.
Work! work! work! by all means--but let's not make dubious promises to
ourselves or to each other. It just isn't that easy.
Robert Ettinger
Cryonics Institute
Immortalist Society
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