X-Message-Number: 12624
Date: Sat, 23 Oct 1999 22:42:12 -0400
From: Mike Darwin <>
Subject: Can Organ Cryopreservation Be Achieved?

Alex Ber comments/ asks:

>I commend intellectual honesty of Mike Darwin recent posts, #12464. His 
>knowledge of cryobiological side of the problem is very impressive-#12595.
>However, I am puzzled. I'd like to ask you, Mike, do you think organ 
>cryopreservation is possible at all? (Without nanotech, just using
>means).  Is it true that most cryobioligists just gave up on organ 
>I thought that recent work by 21CM was close to success in vitrifying 
>organs-am I wrong?  What exactly is missing, in your opinion?
>I wish you good luck and hope your research will lead to finally achieving

>this elusive reversible cryopreservation.

1) do you think (sic reversibe, injury free) organ cryopreservation is
possible at all? (Without nanotech, just using biological means). 

The answer is yes. And definitely without nanotechnology. Some organs will
be easier to cryopreserve than others. The majority of people on this list
would argue that many organs can be cryopreserved well NOW such as skin,
bone, and so on. It is interesting to have been part of a business (21st
Century Medicine (21CM)) that actually entered the area of commercial
cryopreservation (and is continuing to do so, as I understand). One of the
amazing things discovered was the incredibly *low* and often downright
deceptive standards used by cryobiologists in making these claims. 

The reality is that *all* forms of commercially available cryopreservation
are little better than a disaster. Take corneas as an example, every
cryonicists know that corneas are frozen successfully. Oh yeah? Guess what,
there isn't a single cornea bank or cornea transplasnt center anywhere that
uses frozen corneas. The results are terrible: the occassional cornea may
tolerate cryopreservation with results good enough to allow vision to be
restored to some degree. But most are so damaged and scaed so intensely
following transplantation (due to freezing damage) that they are

Sperm is another classic example cryonicists love to quote to the feckless
and checkless media vultures. Reality: only ~1 in 10 *potential* donors
have sperm that can tolerate cryopreservation with sufficient robustness to
be accceptable. This means that every sperm bank has to screen circa 10
people to find ONE who is useable, and his "working life" is short. The
cost is astronomical in recruiting, processing evaluating and *rejecting*~9
out of 10 potential otherwise ideal donors.

Of those who are "acceptable" the best *real* numbers for post-thaw
viability are ~50% of the pre-freeze sperm count. Couples where the husband
has a low sperm count often have to have HUNDREDS of ejaculations stored to
get enough pooled motile sperm for one try! Thus, the development of
micro-injection techniques and other methods of augmenting male infertility
due to low sperm count.

Embryos do better, but only because a few of the cells have to survive to
reconstitute the blastocyst.

Skin suffers tremendous injury with the best available techniques.  This is
a major problem for the engineered tissue people such as Advanced
BioScienes since they need to be able to generate a stable INVENTORY. What
happens if 500 people are burned in a diaster? Right now, I've heard that
their cryopreserved product is grossly inferior to the fresh product and
they have a full-time consulting cryobiologist working on the problem, as I
understand it (rumor).  

In short, cryonicists got "snookered" by cryobologists who reported
(naturally enough) their *best* laboratory results, often using inferior or
inappropriate tests to validate viability after cryopreservation. One of my
favorites is dye exclusion where right after thawing ~90% of the cells are
"alive", and if you repeat the test an hour later the number is ~10%. Guess
which one is correct?

2) A question you did not ask but which is relevant to this list is "which
organs and by which standards?" I think that technology currently exists to
allow for reversible cryopreservation of the kidney, probably the pancreas,
gut and maybe the heart, with return of enough function to support life the
animal.  In the case of the kidney, pancreas and gut it may be that some
post-vitrifcation "recovery time" will be necessary. For instace, the
proximal tubule cells of the kidney are exquisitely sensitive to injury
from toxins and ischemia. 
Thus, many of the patients I dialyzed during my career as a dialysis tech
had "Acute Tubular Necrosis (ATN)" from causes ranging from too strenuous
exercise (muscle cell death resulting in release of nephrotoxic myoglobin
)rhabdomyolisis)), transfusion reaction, drug overdose, chemical ingestion,
ischemia (shock or aortic supra-renal aneurysm) and so on. In these cases
the tubules typically regenerate and, so in ~3-6 week,  the patient goes
back to a normal life with little or no increased risk of ever needing
dialysis again.

This is possible because we have a temporary substitute for the kidney.
Most dialysis patients would accept a period of ATN in exchange for good
kidney!  Unfortunately, heart failure people have a moe limited range of
options and the graft must, for many practical reasons, take full load
*immediately* post preservation.

I certainly think that the most recent generation of vitrification
solutions and ice blockers have a *good* chance of allowing recovery of
acute renal function in the rabbit without resort to dialysis, but with
some injury to the kidney. Many transplant surgeons would NOT consider this
acceptable and would only accept pristine organs that functioned as well
immediately upon implantation;  as well as conventionally preserved ones.
I'd put this number of surgeons at ~75% overall with ~90% of the "best"
demanding the best. So, much effort is being put into getting organs that
are PERFECT and as good as typically hypothermically preserved ones: 90% of
the effort into getting to the last 10% of the results!

The next issue is WHICH organ are you talking about. The brain is unique,
and here we have the problem of knowing the importance of which gross,
cell, and molecular level structues need to be preserved.

Virtually since the inception of the idea of using cryopreservation to
achieve human suspended animation (conservation of personal identity) the
question has loomed large as to what structures encode memory and personal
identity. Recently, Merkle and many others within the cryonics community
have argued strongly that memory, both declarative and procedural, are
stored in  make-or-break  connections between neurons with the
membrane-specific biochemical changes being simple, robust, and thus
presumably readily inferable to an advanced method of repair (such as
mature nanotechnology).

Recently, new laser membrane imaging methods have revealed that information
storage in the brain is occurring at a far higher density than most
scientists expert in the neurobiology of learning and memory have presumed
(with the possible exception of Dr. Daniel Alcon who's foresight is

Using a new method of infrared-guided laser stimulation, researchers at the
Max Planck Institute of Psychiatry in Munich, Germany have discovered that
information is stored in the brain with very high spatial density on the
surface of every single neuron (Science 1 October 1999).

The new method was developed by Hans-Ulrich Dodt from the Max Planck
Institute of Psychiatry. In the past, this researcher has developed a
method to visualize nerve cells in the depth of small pieces of rat brain.
To achieve this, Dodt used a microscope and infrared light instead of
normal light. In his new method, so-called "infrared-guided laser
stimulation", he coupled now a highly precise UV-laser with his infrared
microscope aiming the laser beam at neurons to be investigated. The method
allows the stimulation of selected target points on single neurons with
incredible  spatial precision.

A solution was added to the brain slices which contained neurotransmitter
in a special chemical form, which only becomes active if the so called,
 caged neurotransmitter  is illuminated by the UV-laser. Then, the
neurotransmitter is set free from its  cage  at the point at which the
laser aims. 

Using this technique it has become possible for scientists to do the same
in the laboratory what a synapse does in the brain, but now exactly at the
point and time when the scientist wants it. As this is something that
neuroscientists all over the world have always wanted, the method of
"infrared-guided laser stimulation" will probably be very quickly taken up
by many other laboratories.

Dot and his colleagues applied the new method to investigate the so-called
"long-term depression" (LTD), a very important molecular mechanism in the
brain. Actually, mechanisms like long-term depression and long-term
potentiation (LTP) are regarded by many researchers as the basis for memory
formation in the brain. It has been controversially debated how precise the
underlying modifications of the neuronal membrane can be and where these
modifications take place. The Max Planck researchers  have discovered that
these modifications are spatially highly restricted. Thus, information can
probably be stored with very high density on the surface of neurons. During
the experiments, it became apparent that a modification of the "receptor",
the postsynaptic neuron, is all that is needed to understand the mechanism
of long-term depression. Therefore, modifications of the amount of
neurotransmitter that is released during LTD can be neglected.

As the UV-laser stimulation allowed the release of the neurotransmitter
glutamate from an inactive form of caged glutamate in a very small region
on the neuron, the researchers could investigate how big the region on the
neuron was that experienced LTD. They found that this region was not bigger
than the resolution of their method, i.e. only 10 nanometers. Thus, even
single synapse may undergo long-term depression and each single synapse
could be used to store information separately from its neighbor. One could
compare this possibility for information storage in the brain with the
"high density information storage" on a CD-ROM.

The implications of these finding for brain cryopreservationists (and sadly
even more so for cryonicists) cannot be over-emphasized. It is now becoming
increasingly clear that it is necessary, but by no means sufficient to
preserve synaptic connectivity (which is overall NOT preserved by the
current best cryonics techniques), but that it is also necessary to
preserve the fine structure of the neuronal membrane at a level heretofore
unanticipated as being necessary. 

This is both challenging and worrisome for several reasons. First, major
alterations in membrane structure and chemistry are already documented in a
wide variety cryopreserved cells and tissues. Indeed, even the replacement
of ~50% of the water in cells (the minimum currently required to achieve
vitrification) likely results in major perturbation of membrane structure
with exacerbation of this upon cooling to subzero temperatures (chilling
injury?). So serious can this perturbation be that it results in cell lysis
under certain conditions. For example, Boutron and Darwin have both
observed that disaccharides which reduce the cell membrane toxicity of
glycol ethers and butanediols at near 0 C cause red cell membranes to
disappear upon cooling to approximately -40 C for reasons that are not even
remotely currently understood. 

Such vast changes in the structure of a membrane are of great concern if
the mechanism for declarative memory operates at the molecular-level in the
neuronal cell membrane. 

Conventional imaging techniques such as transmission electron microscopy
(TEM) applied to brains cryopreserved or even vitrified with today s
techniques show evidence suggestive of major alterations in membrane
morphology and topology. An added concern is that of the small subset of
humans who recover from prolonged global cerebral edema following closed
head trauma, a significant number experience apparently irreversible loss
of, or loss of access to, all or most of their declarative memories (global
retrograde amnesia) with relative conservation of procedural memories. It
has been argued that this  loss  of declarative memory is really a loss of
access to these memories, rather than a loss of the memory information
itself. This recent research into the mechanisms of memory in my opinon no
longer allows this arguably unacceptably risky proposition to continue to
be the default. 

Incidentally, I have long suspected this, and it is the reason for my
recent pessimism about currently cryopreserved patients' chances of
recovering with intact declaractive memories. Some may not care, so for
them suspended  animation arrived with Dolly the sheep. 

3)  Is it true that most cryobioligists just gave up on organ

Yes, and for many reasons. First, many were surgeons or others uniquely
unsuited to unraveling the pathophysiology of cryoinjury. They were
excessively optimistic and when "tinkering" approaches whose rasion de etre
was the mistaken impression of sucess with skin, sperm, corneas and the
like vanished, they left the field. Some were brilliant highly creative
minds like the cardiac surgeon Lillehei, who left lasting contributions
nonetheless. Other were just plain naieve bunglers.

Secondly, the kidney was the primary organ transplanted, and still is. The
development of Collins' solution and later UW solution extended hypothermic
storage times to the point where kidneys could be held for up to 3 days and
before that there was the Waters Company's perfusion system which allowed
48 hour preservation. This took the pressure off, and most kidneys were
transplanted by "turf:" he who got the gold (recovered the most organs) got
to USE them.  Thus, complicated immunomodulation strategies and other
techniques that were long-term preservation dependent and that might have
been developed were not, in part because people had a lot of incentive to
keep the organ on their turf for *their patient*. Clear-cut short term
benefit versus difficult to evaluate or quantify *possible* long term
benefit. UNDERSTANDABLE behavior as a result.

Third, the general calibre of cryobiologists has been low, and the best,
like David Pegg and to some extent Greg Fahy, have been academics who were
not "mission oriented." So, they dithered a lot. It wasn't THEIR money and
THEY weren't on dialysis. So, they took an incremental highly mechanistic
approach. Such approaches almost always suceed. The multibillion dollar war
on cancer launched by Nixon in *1972* will eventually suceed. But not very
fast. Jerry Lewis may well be dead before muscular dystrophy is cured even
though we know EXACTLY how it works and have lots of balls-out stategies
that might well work to cure it now. Trouble is, no one will try them for
many reasons, government regulation being not the least.

Finally, the financial incentive in MY opinion is not there for
cryopreserved organs at the moment and has not been there at all in the
past. There just aren't tht many transplants. Nor are there going to be
from cadaveric sources any time reliably soon. Now, it is in IMHO a much
better investment to work on transgenic and other gene-mediated solutions
to generating animal-source organs for the kidney, heart, and pancreas. The
liver will remain a problem due to its *massive* number of functions,
includig making vast numbers of human-specific proteins like albumin,
clotting factors, globulins and others too numerous to catalogue including
many human-specfic regulatory molecules.

There are other problems, but I've taken up too much space on this topic
already. I hasten to add that the folks at 21CM mostly do NOT agree with me
about much of what I've said and they may well be, and hopefully are right.

4) I thought that recent work by 21CM was close to success in vitrifying
organs-am I wrong?  What exactly is missing, in your opinion?

Yes, I think in principle they are. In practice they are spread thin with
many other more lucrative and remunerative short term uses for their
technology. This has in effect slowed kidney cryopreservation work.
However, I remain fairly optimistic that this work WILL start and will be

Having said this, I think 21CM is NOT suited to brain cryopreservation
research, nor is CCRI. What is missing a mission oriented approach which
uses the appropriate tools. *Brain slices NOT being one oif them!*

It is a general rule of thumb in any complex research project without
unlimited funding, that "things always take twice as long as initially
projected (and cost twice as much).   Even if a program of successful
reversible brain cryopreservation with demonstrated retention of memory and
identity were to be put a one-decade time-line, this would mean that people
60 years old now would be approaching their mean life expectancy. If
inevitable program delays are factored in, and such a timeline is doubled,
then a man of 60 will be 80 years old before the technology is even
demonstrated, let alone adequately deployed. Fifty years is a more
realistic timetable for the brain with no focused effort. Not because its
too expensive, far from it, it will likely be CHEAP.

As recent experience has made clear in dealing with the cryonics community,
even significant technological improvements in brain cryopreservation
techniques, or brain ischemic damage prevention, may not be implemented or
available until years after they are technologically and financially
possible. In fact, if this delay time is representative, and I believe it
is, then a minimum of 10 years should be factored in from the time a
discovery is validated in the laboratory until it is in its first clinical
iteration, even without governmental or other regulatory constraints, and I
think this really optimistic. CI isSTILL  using *pre-1960* techniques!

So, even proposals for mission-oriented research programs of this sort,
which by reason of complexity or cost would seem to imply far more than a
one-decade timeline, should  be assumed to be unacceptable.

The only path to succes for the brain near-term will be to use the most
rapid and appropriate methods known in cryobiology in particular, and
mission-oriented project design in general, to develop a unique program for
suspended animation of the brain.  To summarize, the specific goal has to
be to improve the state of brain cryopreservation and ultimately to achieve
suspended animation for the brain (and for whatever ancillary physiologic
structures are required to validate the brain s viability and working
status in an appropriate animal or human model).

This is not being done. Brains present unique problems, and only a handful
of people have any interest in solving them. Even they appear to be very
distracted and poorly motivated, perhaps because they think they already
have the prolem licked and "our friends of the future" using Nanotechnology
will make everything right. They should read a book called DATA SMOG or WHY
THINGS BITE BACK for some real perspective on life.

Mike Darwin

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