X-Message-Number: 3063
Date: 05 Sep 94 03:19:23 EDT
From: Mike Darwin <>
Subject: SCI.CRYONICS  Fahy's work


Bob Ettinger asks some specific questions about Greg Fahy's work with vitrifying

kidneys and suggests that I might be a good source of information.  I will do my

best.  I am in contact with Greg every few days about research matters unrelated
to cryonics or cryobiology (as some of you may know I am in the contract
research business and have customers who have nothing to do with cryonics and,
further, no interest in it!) and talk with him frequently about his progress.


I will try to answer Bob's questions as best I can, and I will also refer a copy
of Bob's post and this response to it to Greg in the event he wishes to respond
himself and amplify or correct any (mis)statements I make.


First, a little background.  Greg has been making steady progress, much of which
has been sheilded from public view by the need to get patents into place.  It
may come as a surprize to many Americans but in most countries he who patents
first has the patent.  Only in the US is it the case that he who documents that
he invented first,  get's the patent.  Even in the US as soon as you make a
public disclosure the clock is running and you have only a matter of months to
apply for your patent or the disclosure becomes public domain in the US.
Anywhere else it doesn't matter *who* discloses first, it is only who *patents*

first.  The upshot of all this is that much of Greg's work and progress has been

unavailable for publication.  This is rapidly changing and he is publishing with
great speed including full descriptions of the computer driven hardware for
introduction and removal of vitrification solutions.  Detailed information on
the methods and hardware is contained in the patent "A method for preserving
organs for transplantation by vitrification" US Patent No. 5,217,860.


I wish to correct what I believe was a misstatement on Bob's part (in another of
his posts) to the effect that there has been such dismal progress in

cryopreservation of the kidney.  This is not the case.  In fact, only Dr. Fahy's
lab has made a focused and *sustained* effort at achieving kidney

cryopreservation.  By this I mean that they did not just come up with an idea or
two, try a few things and walk away.  They has been a team of upwards of half a

dozen people working in a quite concerted way on the basis of *many* theoretical
as well as practical studies to solve this problem.  They have been miserably
funded and crammed into an area the size we use for administration here at BPI
and 21st; my operating room is about 3 times the size of Greg's lab at the Red
Cross.

What they have achieved, for all intents and purposes, is successful organ
cryopreservation, contrary to what Bob has said.  By this I mean that it is now
possible to render rabbit kidneys into the virtreous state in a fully
reversible, viable condition given the caveat of fast rewarming.  Slower rates
of rewarming allow for "devitrification" or freezing to occur during rewarming.


To briefly recap why we know this is so, I will point out the following:

1) Kidney slices which CAN be rewarmed at high enough rates to avoid freezing
have been successfully vitrified using the same solution in the same
concentrations used to perfuse (and vitrify) whole kidneys.  These slices have
been asssessed for function by very sensitive and rigorous criteria and do as
well as control slices.

2) Whole rabbit kidneys have been loaded and unloaded of 5M+ vitrification
solution and gone on on to function immediately after reimplantation and to
support the animal as the sole kidney.  I emphasize that this is a not a
one-time experimental result.  Greg spent several years perfecting this
technique, particularly the conditions under which reperfusion with blood is
carried out in order to make it uniformly and reproduceably successful.

3) A point closely related to point #2 above is that the concentration of
vitrification solution Greg has been able to introduce and remove is a)
sufficient to allow vitrification upon cooling to -135xC or lower, and b) to
allow such vitrification at ambient pressure (1 atmosphere), or in other words
without the use of ultra high pressures in the range of 2K to 5K atmospheres to
facilitate vitrification.  The use of such high pressures was necessary in the
past in order to minimize the concentration of vitrification agent(s) required
because they were toxic in a concentration sufficient to allow vitrification at
1 atmosphere of pressure.  

It took Greg several years of determined work to find ways to mitigate the

toxcicity of these agents and to learn the appropriate pharmacologic protocol to
apply upon reperfusion to prevent vascular injury.

Now, as to Bob's specific questions:

How long were they at this temperature? 

I believe that the kidneys were kept at -30xC only for 30 minutes to an hour or
two at most.

Is there a known time limit for storage at this temperature?  

Yes, there is a limit to storage at this temperature and it is a relatively
brief period of time if I recall correctly.  Why?  Several reasons: first very

high concentrations of agent are required to achieve vitrification and toxcicity

is still operational even at -130xC.  Toxcicity is NOT a problem upon cooling to
lower temperatures with solidification of the system.

Secondly, (and here I am less definite) there are physical (structural) chages
which go on in mammalian cell membranes at reduced temperatures and reduced

water concentrations.  The technical name for one such change is the lamellar to
Hex II transition.  This refers to a reordering of the membrane structure from
its normal lamellar configuration into a somewhat crystal -like (I am greatly
simplifying here) structure; the net effect is that the membrane is perforated
and rendered full of pores which prevent normal ion regulation.  These changes
are facilitated not only by cooling (which destabilizes membrane structure
independant of freezing) but also by the biophysical properties of the
cryoprotectant agents and the reduction of the water concentration in solution
(think about it, if you've replaced 50% or so of the volume of the tissue with
cryoprotectant(s),  then that means you've reduced the water concentration as
well!) Greg has found that the difference between survival and nonsurvival is
little at 1% vitification solution.  It has been a very long, tough battle to
get a 1-atmosphere vitrifiable system.

Were attempts made at lower temperatures?

My understanding is -30xC was chosen for a variety of reasons, not the least of
which is that prolonged holding of the vitrification solution at lower
temperatures (without actually cooling down low enough to vitrify) would result
in freezing.  Also, keep in mind that the toxcicity of this mixture vs.
temperature has been very well and very painfully establiashed by Greg.

However, with reference to my point above, I would note that slices of rabbit,
dog, and I believe human kidney have been vitrified and rewarmed  and
demonstrated normal function.  Further, such slices can be held in the vitreous
state "indefinitely."  I say "indefinitely" meaning that to my knowledge no
systematic studies have been conducted to determine storage limits at -135xC or
-196xC, however storage was for more than minutes, hours, or days.

Is enough non-proprietary information available to allow Europeans to do the
work the FDA will not allow here? 


The problem is not as simple as the FDA not approving the research.  The problem
is a bureaucratic one which apparently will have to resolved at the level of
Commissioner Kesseler himself.  The problem is also one of cost and rather

arcane technical expertise.  Simple microwave heating is not what is being used.
Radio Frquency (RF) rewarming is what is used and among others things it
required the purchase of an FM station transmitter at considerable cost (over
100K as I recall).  I do not know what the score is on the availability of this
technology but my guess is that it is still contyrolled by the FDA and not yet
available for public disclosure.  I will ask Greg when I talk to him next.

Finally, I wish to point out that Greg has also conducted extensive studies on
the ultrastructure of vitrified kidneys by fixing them at -30xC, by fixing them
at -130xC using solvent substitution and by examining them after rewarming and
unloading the agent.  These exhaustive studies have already revealed the
achievement of the cryonicists holy grail: essentially perfect ultrastructural
preservation.


The take hom message here is extremely simple.  If we were our kidneys we would,
right now, this very minute, have a technique which would allow for virtually
injury free, ultrastructurally nondisrupting cryopreservation.  The only caveat
would be that the technique would not yet be fully reversible in the sense that
apparatus to rewarm at sufficient rates to avoid freezing during warm-up is not
available.  

However, I would also point out here that the physics/mathmatics of rewarming
using the RF approach  seems to indicate no problems with masses at the least
the sze of the human liver.  I would further note that human livers are of a
comparable mass to human heads and mass larger than human brains.

Bob also asks if Greg uses glycerol.


The answer is no, he does not.  The last solution that I was aware of was called
VS4 (Vitrification Solution 4).  It contained DMSO, propylene glycol, formamide
and a variety of other compounds (salts, colloid, etc).  The exact composition
of the solution with respect to the ratio of the components to each other is
abosolutely critical to success both in terms of minimizing toxcicity and in
terms of glass forming (vitrifying) ability.  Glycerol is too poorly permeable
and too toxic at concentrations required for vitrification.

A *brief* bibliography:

I regret that one my repriints from Greg documenting his computer controlled
system (which is essential for success) for introduction and removal of VS4 is
out on loan.  I have the title, but not the cite.  Anyone with NLM Medline
access should be able to find it:

Fahy, G.M. Organ perfusion equipment for the introduction and removal of
cryoprotectants.

Other relevant papers:

Khirbadi, B, and Fahy, G. Cryopreservation of the mammalian kidney. I.
Transplantation of rabbit kidneys perfused with EC and RPS-2 at 2-4xC.
Cryobiology 1994;31:10-25

Fahy, G.M. Biological effects of vitrification and devitrification. In The
Biophysics of Organ Cryopreservation.  David Pegg and Arman Karow, Jr. Plenum
Press, New York, 1987, pp. 265-279.

This is just the briefest list.  The serious reader is urged to do a Medline
search using Dr. Fahy's name author.

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