X-Message-Number: 33162
Date: Thu, 30 Dec 2010 05:02:30 EST
Subject: Cryopreservation since 1990 

Content-Language: en

Date: Wed, 29 Dec 2010 11:21:21  -0800 
Subject: Cryopreservation since  1990 
From: Brian Wowk  <> 
Mike Darwin  wrote: 
>>Finally, the quality of  cryopreservation most patients are now receiving 
is dismal and has, on average,  deteriorated since 1990.>> 
I think Brian's response fails  to account of two short, but very important 
words in my post:"on average." I'm  not going to spend the time required to 
tot up the statistics, but you can go  here: 

http://www.cryonics.org/patients.html, and you can go here:  
http://alcor.org/cases.html, and then do the 
math  yourself. 
I am in complete agreement with  Brian, that increasing the degree of 
cryoprotection (ice suppression), by  increasing CPA concentration, trumps 
everything else that could be done to  improve the quality of care for 

cryopatients. But that assumes that this is  actually being implemented. And 
yes, I 
know it is being implemented  occasionally, and I hope it will be implemented 
more consistently in the future.   
And no, I do not believe that  patients who have experienced days of cold 
ischemia, absent any stabilization  beyond being packed in ice hours or 
minutes post-cardiac arrest, will achieve  even, let alone good equilibration 
with CPA, or that CPA perfusion will be as  "benign' as is the case in 

patients with vastly less warm and cold  ischemia.  I don't think these are  
controversial or unsupported statements.  
>>Nor are the effects of  incomplete perfusion with vitrification solution 
as bad as Mike fears.  Attempting to vitrify small tissue  pieces with 
sub-vitrifiable concentrations of cryoprotectant is very bad because  rapid 
cooling can cause them to freeze  intracellularly. 
However human heads are so much  larger than tissue pieces that even when 
cooling a head at the maximum possible  rate, that rate in the brain is not 
much faster than the canonical 1 degC per  minute that allows an average cell 
to dehydrate in response to growing  extracellular ice and thereby avoid 
intracellular freezing.  In other words, it appears plausible if  not probable 
that the effect of poor perfusion or incomplete equilibration with  

vitrification solution in human cryopatients is freezing of those tissues in a
manner similar to that seen with low concentration glycerol in the  1980s.>>  
This statement surprises me, and  maybe Brian can add some clarifying 
detail that will resolve my puzzlement.   
I don't know anything about  trying to rapidly cool small samples of tissue 
with sub-vitrifiable  concentration(s) of CPAs in them. It's not an 

experiment I would do to try to  resolve this issue, for exactly the reason that
Brian subsequently points out:  the maximum core cooling rate of a human brain 
(in situ in the head) is quite  low. From the ata I've seen, the maximum 
achievable rate for an average sized  human head is ~0.3 - 0.4 deg C/min, 

which is even slower (and therefore likely  more favorable in the case of intact
organs) than the value of 1 deg C/min,  which Brian gives. So, as near as I 
can tell, this is not a particularly  relevant experiment (though I admit 
it is an interesting and worthwhile  one). 
What we are really interested in  is what happens when we cool tissue 

loaded with a high concentration of CPAs,  *and* ice inhibiting molecules, 
to the glass transition temperature (Tg).  This is a very different 
situation than usually pertains when tissue is frozen  in the presence of 
cryoprotectants for several reasons. First, tissue loaded  with very high 

concentrations of CPA (but still not sufficiently high enough to  permit 
will have a strong tendency to undergo supercooling.  Supercooling occurs 
when ice does not form at the "true' (thermodynamically  prescribed) 

freezing point of the solution or tissue, and the system remains in  a liquid 
well below this point. When freezing does occur under such  conditions, it 
happens with extreme rapidity, and there is usually insufficient  time for 
the extracellular ice mass to osmotically extract water from the cells.  The 
result is intracellular freezing.  
This is a posited, if not proved  problem, in cryopreserving large masses 
of tissues that have been treated with  cryoprotectant, where it is thought 
that regions of supercooling in may occur  resulting in patch damage from 
intracellular freezing. In particular, it is  posited (quite reasonably) that 
this will occur most often under conditions  where there is minimal 
extracellular space and/or where the small volume of  extracellular space is 

comprised of high viscosity fluid. This is a perfect  description of a brain 
regions of sub-vitrifiable CPA present, or worse  still, where most or all of 
the brain has failed to reach the concentration  needed to vitrify at the 
cooling rate the patient actually experiences.   
The brain has virtually no  extracellular space with even the briefest 

exposure to ischemia. In fact, there  was a great deal of debate for many years
as to whether the brain had ANY  non-vascular extracellular space, because 
of the difficulty of fixing brains  under conditions that do not "abolish' 
the extracellular space. In addition, CPA  perfusion can result in severe 
cerebral dehydration which will further increase  tissue and intravascular 
fluid viscosity, and reduce/eliminate non-vascular  extracellular space.  
There is also the issue of the  ice inhibiting molecules X-1000 and Z-1000, 
that are present in 21st Century  Medicine vitrification solutions (such as 
B2C and M-22 which have been used on  human patients). Ice blocking 

molecules are wonderful things in the context of  vitrification. They slow or 
ice formation during cooling and re-warming at  rates slow enough that 
extensive freezing would normally occur. However, a  consequence of this 

suppression of ice growth is that they, in effect, act as  agents for the 
of supercooling under conditions of  freezing. 
Finally, Arav, et al., the  inventors of "directional freezing,' state 
that: "when crystallization starts at  low temperatures, water will not leave 
the cells because dehydration occurs only  in a certain range of high subzero 
temperatures, and at lower temperatures the  membrane becomes impermeable. 
Therefore, supercooling must be avoided and  crystallization should start at 
atemperature as close as possible to the  freezing point of the solution." 
(Arav A, Natan Y. Directional freezing: a  solution to the methodological 
challenges to preserve large organs. Semin Reprod  Med. 2009 Nov;27(6):438-42. 
Epub 2009 Oct 5. Review. PubMed PMID: 19806511.) The  freezing point of 

tissues loaded with a sub-vitrifiable concentration of CPAs  could be quite low
(i.e., -10 deg C, or even considerably lower) and if Arav, et  al., are 
correct, then this would be yet another reason for concern over the  
possibility of intracellular freezing under these conditions.   
Despite the long road to reach  it, my point is fairly simple: absent 

experimental evidence demonstrating that  the occurrence of freezing in 
brains under conditions likely to attend  sub-vitrifiable cooling and 

freezing of human brains (e.g., brains in heads),  there should be no 
that the attendant freezing damage is less than,  or equivalent to, that 
which would occur under conditions of "conventional' slow  freezing in the 
presence of cryoprotectant with adequate nucleation of the  tissue. Indeed, it 
would seem (at least to me) that the presumption would favor  intracellular 
freezing. The point is, the experiment must be done, and the  results 
On a different, but perhaps  related topic, all these many years I have 
been deeply troubled by Audrey  Smith's hamsters, and by my own red-eared 

slider turtles. Both can tolerate  truly incredible amounts of ice formation in
their kidneys and brains. Since the  kidneys are located in the 

retroperitoneal space, it is reasonable to presume  that in Smith's hamsters 
experienced at least 40-50% ice formation. In the  case of the brain, it is 
probably reasonable to assume that approximately 60% of  its water content is 

converted into ice. And yet, if as little as 8% ice is  formed in the medulla of
the inadequately vitrified rabbit kidney, it is  lethally injured. How can 
these two seemingly contradictory facts be reconciled?   
An experiment I've long wanted  to do, but could never figure out how to 
carry out, is to find out EXACTLY how  and where ice is forming in frozen 
hamsters and frozen turtles. I can't help but  wonder if the answer to this 
question may prove critical to achieving workable  cryopreservation for some 
organs and tissues. Currently, rabbit kidneys are  failing to survive mostly 
(leaving viscoelastic injury aside) because of the  formation of a "trivial' 
amount of ice, compared to what is acutely tolerable in  WHOLE RABBITS. 
Smith not only froze hamsters, she froze rabbits as well, and  while none 

survived long term, some did recover acutely, and were ambulatory. I  continue 
wonder if a great deal of "load' could be taken off vitrification if  it 
were only possible to control the locus and extent of ice formation.   
This is perhaps not realistic,  but it is certainly an idea that deserves 
some additional thought. I'm not a  cryobiologist, and I cannot evaluate the 
scientific rigor of directional  freezing. But, that may be beside the point 
if other investigators demonstrate  that it is much less injurious than 
conventional cryopreservation of complex (as  well as simple) tissues, and/or 
explain the mechanics. I'd be very interested in  Brian's take on this? 
Selected  References: 
Elami A, Gavish Z, Korach A,  Houminer E, Schneider A, Schwalb H, Arav A. 
Successful restoration of function  of frozen and thawed isolated rat hearts. 
J Thorac Cardiovasc Surg. 2008  Mar;135(3):666-72, 672.e1. PubMed PMID: 
Gavish Z, Ben-Haim M, Arav A.  Cryopreservation of whole murine and porcine 
livers. Rejuvenation Res. 2008  Aug;11(4):765-72. PubMed PMID: 18729808. 
Hubel A, Darr TB, Chang A,  Dantzig J. Cell partitioning during the 
directional solidification of trehalose  solutions. Cryobiology. 2007 
Dec;55(3):182-8. Epub 2007 Aug 10. PubMed PMID:  17884036. 
Robeck TR, Steinman KJ, Montano  GA, Katsumata E, Osborn S, Dalton L, Dunn 
JL, Schmitt T, Reidarson T, O'Brien  JK. Deep intra-uterine artificial 

inseminations using cryopreserved spermatozoa  in beluga (Delphinapterus leucas.
Theriogenology. 2010 Oct 1;74(6):989-1001.  Epub 2010 Jun 8. PubMed PMID: 
Mike  Darwin

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