CryoNet #33241 - #33243

CryoNet - Mon 17 Jan 2011

    #33241: Response to Metzger Part I [M2darwin]
    #33242: Response to Metzger & Base Part II [M2darwin]
    #33243: a dehydrating moss gathers no ice [oberon]

Rate This Digest: http://www.cryonet.org/cgi-bin/rate.cgi?msg=33241%2D33243

The Sun. Jan. 16, 2011 digest of messages #33233 - #33243 apparently
was too large (over 70 KB). I am resending it in two parts. - KQB


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Message #33241
From: [email protected]
Date: Sat, 15 Jan 2011 23:49:41 EST
Subject: Response to Metzger Part I

My attempted post to Cryonet early this AM bounced because it exceeded  the 
allowable file length. So, I've broken the file into two parts, pretty much 
 arbitrarily, and am now re-posting them.    


Mike Darwin
 
 
My responses are interleaved with the two posts below and start with  my 
initials (MD) and end with >>
 

Message  #33230
Date: Fri, 14 Jan 2011 16:51:49 -0500
From: "Perry E. Metzger"  <[email protected]>
Subject: Glass vs. Crystal  transitions
References:  <[email protected]>

It is rare that a topic  comes up here that is actually of direct
interest to improving  cryopreservation quality. However, as
Mike Darwin has had the temerity to  interrupt the usual chatter around
here by discussing actual experiment, I  thought I'd chime in:

> From: [email protected]
> Date: Thu, 13  Jan 2011 21:56:09 EST
> Subject: Intracellular Freezing &  Vitrification
[...]
> vitrify. A potential problem arises, especially  when ice growth
> inhibiting  molecules are also present, in that  such tissues will
> tend to supercool far below their true freezing  point,

One side note here. (I'm sure Mike is aware of this, but others  may
not be.) A uniform idealized substance (and sometimes a  uniform
idealized mixture) can have a single definable freezing point,  but
non-uniform substances do not. Biological tissues and individual  cells
are nearly the definition of a non-uniform substance -- if they  were
uniform, they would not function.

Furthermore, not every  substance has a well defined freezing point
even when it is perfectly  uniform. A good everyday example (just for
purposes of illustration) is  paraffin.

Why mention this? Because even in idealized conditions, getting  an
entire cell or block of tissue to undergo a phase transition at  the
same time may be a bit of a challenge, and because things one  learns
from uniform systems may warp when applied to non-uniform systems  like
biological tissues.
 
MD: First, before I get into specifics, I'd like to thank Perry for  asking 
some very good questions - so good in fact, that I'm sure I will not be  
able to answer all of them. My hope is that Brian Wowk will step up to the  
plate, especially where I don't know, or where I err.
 
Second, meaning no disrespect, Cryonet is not really the proper place  for 
these, or any discussions that involve complex scientific concepts that, by  
their very nature, cry out for visual aids and examples. I was hoping that  
a blog or interactive web 2.0 space would be available by now, but so far 
that  hasn't happened.
 
Now, to the matters at hand. Yes, as you point out, biological  systems are 
inhomogeneous and this is greatly amplified when a whole organ or  organism 
is considered. In fact, one of the primary obstacles to successful  renal 
vitrification at this point is that the renal medulla is comparatively  
poorly perfused compared to the cortex and may, in addition, have structural  
features that make cryoprotectant agent (CPA) equilibration more  problematic. 
Having said that, cryoprotective perfusion in a healthy animal  probably 
does a fair bit to decrease the inhomogeneity in various organs and  tissues 
because, if successful, it swamps the tissue with massive amounts of  
colligative agents AND it dehydrates the tissues to just about the maximum  degree 
possible. A cryoprotected brain is massively dehydrated at the end of CPA  
loading, and basically consists of CPA in a concentrated protein gel. All but  
the vascular extracellular space is abolished; and the tissue ground 
substance  is extremely dense - in fact it makes it very hard to see fine 
intracellular  structure with TEM. So, what you have is sort of a CPA loaded 
'brain-jerky.'  Still, there will be regional variations of some kinds, and if 
freezing is going  to take place, these may be material. But the point here is 
that the severe  dehydration, coupled with the massive freezing point 
depression, will probably  tend to collapse a lot of the normally present regional 
variation in things like  water, protein and lipid content.>>


> What Brian pointed out to me is that a
> 7.5 M solution  of glycerol has a melting point of ~ -50 deg C.Thus,
> even if tissues  containing 7.5 M glycerol solution nucleated
> perfectly upon passing  below their melting point during cooling,
> all the  ice growth would  take place at deep subzero temperatures
> where the mobility of water (and  cell membrane permeability to
> water) is putatively low. An added  complicating factor is that 7.5
> M glycerol solutions are virtually  certain  to supercool well below
> their thermodynamically ideal  freezing point - and indeed to do so
> by tens of  degrees.

Supercooling is common across most substances. Extremely pure  water
with no nucleation sites can be cooled to below -40C before it  will
undergo spontaneous "homogeneous nucleation". Crystal formation  itself
is a fairly complicated process, and without a nucleation center  the
transition can be disfavored even if the final crystal would  be
perfectly stable.
 
MD: Yes, water has two freezing points: 0 deg C, which is the heterogenous  
nucleating temperature, and -40 deg C, which is the homogenous nucleating  
temperature. In theory, if you could purge a solution of all exogenous  
nucleators, like bacterial proteins, gas bubbles, etc., then you could   cool 
water to -40 deg C and it would remain in a liquid state. In fact, this  works 
well for small volumes of water, and some living organisms survive by a  
combination of purging nucleators and using a modest amount of a colligative  
agent. But, for large volumes of water, thermodynamics and chance dictate 
that  you will get freezing, and sooner, rather than later.>>

The  specific issue, as I understand it, is this. (If someone
understands the  process better than I do, please chime up -- I know a
bunch of physical  chemistry but it is not my area of expertise.)

Forming an interface  between the two phases requires energy, which is
proportional to the size of  the surface area at the interface. Now,
there is a source of energy  available, to whit, the energy liberated
by the formation of the crystal  itself (the crystal necessarily has
lower free energy than the liquid), which  is proportional to the
volume of the forming crystal. However, the smaller an  object is, the
larger the ratio of its surface area to its volume. Thus, a  very small
spontaneously formed crystal will not liberate enough energy  to
account for the energy of the interface. Assuming a spherical  nucleus,
one can calculate a critical radius below which a spontaneously  formed
crystal is unstable. (A very similar process happens in  water
droplet formation in clouds, where below a critical radius  the
droplets are disfavored.)

However, as the temperature becomes  lower and lower, the critical
radius itself lowers (one can model this  mathematically though it has
been some years since I knew the details), until  finally you hit a
point where the critical radius is small enough that  spontaneously
formed crystals are larger than it and you get homogeneous  nucleation
followed by a spontaneous phase transition throughout the  liquid.
 
MD: Yes, I believe this is a succinct and accurate description, and it  
applies not just to freezing water, but to boiling water as well. With the  
advent of microwave ovens, many people are now familiar with superheating of  
water, as when a cup of water in the oven fails to boil at 100 deg C. As  
soon as it is nucleated, either by moving it or by adding a powder or a   tea 
bag, explosive boiling occurs as all that 'stored' heat that should have  
been disposed of by steam escaping during boiling, is rapidly dissipated. It is 
 the same phenomenon in reverse when freezing occurs after deep 
supercooling.  And, as you point out, crystal formation yields free energy - in this 
case in  the form of heat. If a LOT of ice forms quickly, the very rapid 
release of this  latent heat of fusion can, in theory, re-warm the system and 
cause damage  by inducing localized CPA toxicity,  melting and refreezing, and 
by driving  recrystallization in already frozen areas.>>

> This will be even  more true of glycerol
> solutions at these temperatures because of their  very high
> viscosity and  the resultant decreased mobility of water  in the
> solution.

I know little about the physical chemistry of  glycerol undergoing this
transition. Are the crystals formed pure water? If  so, the
concentration of glycerol of the surrounding liquid would rise  during
crystal formation, inhibiting further growth if diffusion was  slow
enough. That would tend to explain the phenomenon you describe  below:
 
MD: Yes, ice freezes out as pure water under these conditions, and with  
these molecules. Some putative cryoprotective agents such as the gases Xenon 
and  Argon can form non-ice structures called clathrates. However, to my 
knowledge,  the CPAs currently in use leave water either hydrogen bonded to the 
CPA and  present as a CPA-water liquid - or as ice, frozen out as pure 
water. In fact,  the definition of ice is a crystal of water, and a crystal of 
water is made up  only of H2O.>>

> As Brian points out, if you carefully examine a  vial of
> such a solution being slowly cooled to well below its  freezing
> point,  what you  notice first is the presence of a  few scattered
> large ice balls in the  solution. These are the  points in the
> solution where nucleation and  subsequent ice growth  first began,
> and they will typically have formed and begun  growing  at between
> -50 deg C and -60 deg C; at the warmest temperatures   that ice can
> form in a 7.5 M glycerol solution.

The "sentinel"  ice balls you describe may fail to grow faster
precisely because their  formation alters nearby concentration of
glycerol, a self limiting process  especially since the formation of
pure water ice requires energy since it is  entropically disfavored. It
would be interesting to learn if this is true.  However, with time, as
the liquids diffuse, they should grow until the  overall concentration
is sufficient to impede further change. The phenomenon,  as you
describe it, is self limiting even in a slowly cooled solution,  which
is slightly puzzling.
 
MD: Brian can better answer the second part of your question. But yes, as  
the ice front grows it is largely excluding not only glycerol (or other 
CPAs)  but also the salts and colloids present in the solution. The latter can 
amount  to as much as 6-10% of the volume, and more importantly, concentrated 
colloids  at subzero temperatures are VERY viscous, and probably comprise a 
huge barrier  to the already slowed kinetic diffusion of water. 
Additionally, the  concentration of CPAs and other dissolved solids will be highest 
right at the  crystal-solution interface. This will mean that the freezing 
point of the  microenvironment surrounding the ice crystals will be the lowest 
it is  anywhere, with the possible exception of the intracellular  spaces. 


And diffusion is greatly impeded at temperatures like -50 deg C not only  
because of cooling, per se, but because of the tremendous increase in 
viscosity  most CPAs undergo when they are cooled to these temperatures. So, you 
can't  simply do an Arrhenius-based calculation of the diffusion kinetics 
based on  water alone - you have to account for the high viscosity of the 
CPAs/carrier  solution.>>
 

> These sentinel ice balls almost certainly occur as a result of  the
> presence of bacterial ice nucleating proteins that are present  just
> about everywhere in the environment.

That may or may not be  the case. It should be straightforward to
determine which, however, if it  proved to be important.
 
MD: Well, what started Brian off on his criticisms of my previous post was  
that, in fact, the experiment HAS been done - and most embarrassingly, by 
me!  When we loaded our experimental dogs with 7.5 M glycerol we dropped the 
freezing  point of the tissues to ~50 deg C and we virtually guaranteed both 
supercooling  and inhomogenous freezing. And yet, we saw stunningly good 
histological and  ultrastructural preservation - with the exception of 
'random' areas of neuropil  that had a 'blasted' appearance - rather like what 
you'd see in an aerial  photograph after a hurricane of Tsunami hits an 
urbanized area. These were  probably areas of primary nucleating with large ice 
masses. Also, there large  holes or tears around many of the brain capillaries.
 
At the time we did that work we were very pressed for money - or more  
accurately, I was, since BioPreservation (my company) paid for that work. I cut  
corners in what seemed a perfectly reasonable way at the time, and that was 
to  do just one control that was glycerol perfused and fixed, but NOT 
frozen. I knew  what I was going to see, so I only submitted one sample for TEM 
and only paid  for a few high magnification 'survey shots' to confirm that 
everything looked as  expected. When we got the EMs back on the frozen 
dogs,they looked spectacular,  except for two things - the small 'blasted areas' of 
neuropil, and much more  disturbing, peri-capillary holes. There were these 
huge gaping holes that looked  like tears from ice around many (but not 
all) of the brain capillaries. 
 
Woebetide the scientist who knows what he will see BEFORE he sees it. As it 
 turns out, these peri-capillary holes are NOT from ice formation, but 
rather are  a result of the cerebral dehydration due to glycerolization. This 
was discovered  by Greg Fahy when they began vitrifying brains at 21CM, 
because they saw the  same exact holes in both vitrified and CPA perfused but not 
vitrified brains! I  mention this because, shockingly (to me) as I look back 
over the micrographs of  the 7.5M frozen brains, I find that if I exclude 
the peri-capillary ice holes,  which weren't really ice holes, high molarity 
glycerol yields structural  preservation that is arguably not that much 
worse than you get with  vitrification. Furthermore, Hugh Hixon told me some 
years ago that it is  possible to perfuse 8M glycerol in humans. If you can add 
ice inhibiting  molecules and cool at 0.5 deg C/min it may well be possible 
to vitrify. AND, if  you can perfuse the brain vasculature with ultra-cold 
gas, preferably helium (I  originally thought nitrogen would do, but there 
is now evidence that it is  undesirable) it should be easily possible to cool 
the brain homogeneously at  somewhere between 1-3 deg C min, depending upon 
the condition of the  vasculature. That should handily allow for 
'vitrification.' Indeed, just  extending blast cooling with cold gas to 
naso-oropharynx should double or triple  the currently achievable cooling rate of 0.3 deg 
C/min for human  heads. 
 
The is material because glycerol is far less membrane toxic than current  
vitrification solutions and in places where tight control of temperature is 
not  possible, it may be a much safer alternative. It is also vastly cheaper, 
easier  to prepare and handle, and perhaps most importantly, results in 
much less edema  in ischemic patients.>>

> Brian further notes that these initial ice balls (several
>  millimeters in diameter) grow as large as they do because they have
> lots  of time to do so during slow cooling.

That would seem on its surface to  be correct, but what limits them to
a few millimeters?
 
MD: I think this is definitely a Brian question, but my untutored answer  
would be viscosity and decreasing temperature. It's true that if you HOLD at 
a  favorable temperature, ice will grow to its maximum possible volume. In 
fact, it  was because I did these same experiments as a kid with dry ice that 
I failed to  understand what was happening. In order to prevent freezing of 
a glycerol water  solution at -77 deg C you need ~ 70% v/v glycerol! Since 
I didn't have sustained  access to LN2 as a teenager, all my 'work' with 
glycerol-water solutions  had to stop at dry ice temp. However, if you CONTINUE 
to cool, and do so with  some rapidity, you can stay ahead of the ability 
of ice to grow. Indeed, that is  what vitrification as it is currently 
practiced depends upon, because no  vitrification solution that is biologically 
innocuous has a critical cooling or  re-warming rate of infinity. You always 
have to cool or warm rapidly enough to  avoid devitrification. The ice 
inhibiting molecules greatly relax those cooling  rates and time constraints, but 
they don't abolish them.>>

>  However, what is easy to miss, or at least not to
> understand, is that as  the rest of the rest of the 7.5 M glycerol
> solution is further cooled,  it will grow cloudy. This cloudiness is
> due to the  formation of  millions of microscopic tiny ice balls
> that refract the light.   Those microscopic ice balls were areas in
> the solution that  nucleated  LONG after those few big ice balls
> formed, in fact, tens  of degrees C later,  when the solution was
> much more viscous. As a  result, those tiny ice balls  couldn't grow
> much before the glass  transition temperature (Tg) of the  solution
> was reached, which in  the case of 7.5M glycerol, is ~ -100 deg  C.

Again, that seems  reasonable, but are you sure of the details? One
would tend to believe that  an abrupt change in optical characteristics
has to be associated with a  partial phase change, but the exact nature
of the phase change is of  interest.

In particular, can you be sure these are not formed of some  sort of
organized glycerol-h2o crystals or small particles of glassy solid  and
are instead pure water? I would tend to agree that pure water  seems
most plausible, but mere plausibility isn't enough, and this  actually
may be important. Also, can you be sure the crystals grow no  larger
because they have no time to do so, or could it be because the  overall
concentration of glycerol has gone up enough to impede growth, or  for
some other reason? It seems to me that this would require  some
experimentation.
 
MD: I'll leave this question to Brian, because he can discourse at length  
on the magic that is differential scanning calorimetery (DSC). DSC can 
detect  the minutest of phase changes in a sample, and if your numbers all sum 
out  right, then you can be pretty sure "what's what" after cooling to any 
given  temperature. I was still at 21CM when Brian began this work, and it was 
horrible  - the kind of thing that would drive me barking mad. Basically you 
crimp a tiny  volume of solution 'just so' into sealed metal pans, and put 
them in the device  and cool them. Of course, the catch is, that you must do 
this thousands and  thousands of times to build a picture of how different 
solutions behave under  different regimens of cooling and re-warming. It is 
boring, repetitive  and truly dull  work.>>




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Message #33242
From: [email protected]
Date: Sat, 15 Jan 2011 23:50:27 EST
Subject: Response to Metzger & Base Part II

> In other words, the ice that makes the solution milky which   is
> most of the ice that will form in a 7.5 M glycerol solution -  is
> ice  that formed in a deeply supercooled state.This happens even  in
> the absence of  ice blockers because this is how  concentrated
> cryoprotectant solutions behave  when cooled slowly to  below their
> freezing point. Thus, ice formation in  supercooled  solutions in
> cryonics patients is not a new phenomenon that began   with
> vitrification.

Yes, that last sentence (which is one of the  punchlines in your note)
is almost certainly true.

> As Brian  points out: "It should also be clear from
> this that idea of nucleating  high molarity glycerol solutions
> externally would not have achieved  anything because such nucleated
> ice, like those ice balls in the flask  formed near Tm, could only
> have  grown a few millimeters into the  solution before cooling was
> complete. The  same goes for any ice  that nculeated at higher
> temepratures inside the patients  in poor  perfused areas.

Well, this sounds plausible, but I don't have a well  validated model
here of what's going on in a uniform solution, and biological  tissues
are very different from a uniform solution, so it is hard to know  how
well that applies in tissues. It is, I think, a mistake to believe  you
understand something simply because you have a plausible  but
unvalidated model. Experiments would probably be needed to  determine
the answers for real.  

MD: I too would be happier if there were more robust experiments and I  
suspect they are done or are being done at 21CM, but have no way of knowing.  
I've not been there in ~10 years, and maybe longer.>>

> In parts  of the brain that experienced good equilibration with the
> high  concentration solution, the penetration of ice from other areas
> would be  minimal, and most of the ice that formed in well-perfused
> areas would be  formed under conditions of deep supercooling.

Presuming that the  explanation of the opacity in the cooled solutions
is indeed homogenous  nucleation of some sort, this is again
potentially plausible. Of course,  tissues are very different from a
uniform solution -- some components of the  tissues might promote
nucleation at much higher temperatures, might impede  nucleation, etc.,
and in any case, the contents of cells and tissues are  very
non-uniform.
 
MD: Yes, and in any event, I am very unhappy with cryonics organizations  
calling what they are currently offering 'vitrification.' In most patients,  
vitrification will be inhomogeneous due to perfusion defects and I think 
that  some other, more accurate and more descriptive term should be employed, 
possibly  something like, Inhomogeneous Vitrification (IHV) or Very Low Ice  
Cryopreservation (VLIC). That would also be an incentive for the COs to  
develop a method of determining both WHERE and HOW MUCH ice is forming in  
patients and both correlate that with the patient's medical history  and 
Transport and CPA perfusion care.>>

> You
> can  bet whatever "blasted areas" or ice holes were seen  in your
> 7.5  Molar 1995 canine brains were areas hit by ice that started
>  growing  in a very supercooled state." What I take away from this
>  is that ice will still form and grow extracellularly under
> conditions  where the colligative CPA concentration is very  high
> and the  solution is very viscous. Where nucleation and ice growth
> occur close to  the melting point of the solution (Tm), the ice
> formed will be 'large  mass' ice, and likely mechanically
> disruptive. Where ice forms well  below Tm, it will likely be in
> microscopic domains that still begin  forming (nucleate)  outside of
> cells and consequently do little  damage.

Again, extremely plausible, but this is a question which  actual
experiments could answer, and in fact very quickly.

Now, going  beyond all that we've discussed up to here, this whole
topic brings to mind a  very basic question: has anyone in the cryonics
world or cryobiology world  done a deep exploration of the physical
chemistry literature on the formation  of glasses at low temperatures?
 
MD: You can probably bet next to your bottom dollar that 21CM has a  
veritable digital mountain of data on this. In fact, while Brian got his Ph.D.  in 
Medical Physics (imaging) he is now more properly a physical chemist  
specializing in the behavior of water at low temperatures in the presence of  
CPAs. His medical physics Ph.D. is probably nearly useless now, - judging by 
the  stunning advances in both imaging and treatment I'm seeing emerging in 
research  and medicine. For instance, direction diffusion tensor imagine now 
allows  sub-millimeter resolution of the brain in humans, and considerably 
better in  rats. Thus, it is now possible to map the general pathways of the 
white matter  circuitry - and a consequence we are getting our first picture 
of just how  devastating so called normal aging is to the human brain. I 
won't digress  further here beyond saying that by the time you are 40 you are 
already  f#%&@, big time.>>

I have to admit that until a few years ago I  wouldn't have even
thought to ask if such a literature existed, but I'm now  no longer so
ignorant. My suspicion is that the pchem guys know a whole lot  more
about this topic than most of us, and have a lot of  understanding
about the competition between crystal and glass formation and  what
shifts the equilibrium one way or the other.

I'm unsure as to  whether we would learn anything valuable by exploring
that topic in depth,  but there is a good chance we would. Knowing what
things are like in most of  science, I suspect that the pchem guys and
even conventional cryobiology  types have never really talked, though
of course I could be entirely wrong on  this.

Anyway, it doesn't hurt to ask...
 
MD: There is an exploding literature on these topics with entire books  
coming out just on the subject of Tg. In fairness, so much data is being  
generated that it would take a team of specialized cryobiological physical  
chemists to stay on top of it. But then, this is the case in so many areas of  
science - even cerebral ischemia and the related area of neuroprotection, the  
literature is simply impossible to track and  absorb.>> 


Message  #33232
From: "Eisab" <[email protected]>
References:  <[email protected]>
Subject:   latent  heat of fusion
Date: Fri, 14 Jan 2011 21:34:58 -0500

"Despite the  control applied to the cooling of cells, most of
the water present will  freeze at approximately -2 C to -5 C. The
change in state from liquid to  crystalline form results in the
release of energy in the form of heat; this  is known as the latent
heat of fusion. Warming of the sample occurs until the  equilibrium 
freezing 
point is reached, at which temperature ice
continues  to form. To minimize the detrimental effects of this
phenomenon, undercooling  must be minimized by artificially
inducing the formation of ice. This can be  accomplished by
seeding the suspension with ice or some other  nucleating
agent, or by rapidly dropping the temperature of the  external
environment to encourage ice crystal  formation."

http://www.nalgenelabware.com/techdata/Technical/cryo.pdf

I   wonder if the latent heat of fusion is a big problem in cryonics.

Basie  



MD: It used to be a proverbial pain the fanny, when we were freezing with  
low molarity concentrations of colligative agent. But with the advent of 
high  molarity cryoprotection, and much more aggressive cooling, the amount of 
ice  formed is so small that it is not much of a problem, if any. And of 
course, in  vitrification there is no latent heat of fusion and any energy 
released in  moving through Tg is both smeared out (rather than abrupt) and VERY 
small,  compared to that seen with ice formation.
 
Mike Darwin



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Message #33243
Date: Sat, 15 Jan 2011 22:42:18 -0800 (PST)
From: [email protected]
Subject: a dehydrating moss gathers no ice

  Could dehydration finally put vitrification over the top? Full
vitrification without any ice formation is not currently available due to
cryoprotectant toxicity. Faced with the Scylla of ice formation on the
one hand and the Charybdis of cryoprotectant toxicity on the other, even
the best cryobiologist navigators have failed to avoid numerous
shipwrecks. However according to myth, Aeneas was able to bypass the
deadly strait altogether. Was Aeneas onto something? Below a moss avoids
freezing by dehydrating first. Quote: "an elegantly simple solution to
the problem of freezing; remove that which freezes". It is not widely
recognized that human skin has the ability to survive complete
desiccation, and in this regard rivals anhydrobiotic organisms. Not all
human cells can be expected to survive complete desiccation, though
partial dehydration is not a problem. Similarly human cells can survive
non-vitrifiable concentrations of some cryoprotectants. I would like to
propose a novel solution to the problem of cryoprotectant
toxicity. Perfuse first with safe non-vitrifiable concentrations of
cryoprotectants, then partially dehydrate tissue to fully prevent the
formation of any ice crystals.
  One possible method for dehydration might be a second perfusion with a
high molar PEG solution. PEG solution does not penetrate tissue, due to
its high molecular weight, but it is highly hygroscopic and can remove
moisture. The goal would be to achieve vitrifiable concentrations of
cryoprotectants in tissue, by gradual dehydration, but leave the absolute
level of cryoprotectants in the tissue below toxic levels. An example of
an extreme case would be human skin, where no cryoprotectants are needed,
since there is no water left.

http://en.wikipedia.org/wiki/Scylla_and_Charybdis


Plant Cell Environ. 2010 Oct;33(10):1731-41. doi: 10.1111/j.1365-3040.2010.02178.x.
Freeze avoidance: a dehydrating moss gathers no ice.
LennAC T, Bryant G, Hocart CH, Huang CX, Ball MC. Plant Science Division, Research School of Biology, The Centre for Advanced Microscopy, The Australian National University, Canberra, Australia.
Abstract
  Using cryo-SEM with EDX fundamental structural and mechanical properties of the moss Ceratodon purpureus (Hedw.) Brid. were studied in relation to tolerance of freezing temperatures. In contrast to more complex plants, no ice accumulated within the moss during the freezing event. External ice induced desiccation with the response being a function of cell type; water-filled hydroid cells cavitated and were embolized at -4 A C while parenchyma cells of the inner cortex exhibited cytorrhysis, decreasing to a   20% of their original volume at a nadir temperature of -20 A C. Chlorophyll fluorescence showed that these winter acclimated mosses displayed no evidence of damage after thawing from -20 A C while GCMS showed that sugar concentrations were not sufficient to confer this level of freezi
 ng tolerance. In addition, differential scanning calorimetry showed internal ice nucleation occurred in hydrated moss at a  -12 A C while desiccated moss showed no evidence of freezing with 
 lowering of nadir temperature to -20 A C. Therefore the rapid dehydration of the moss provides an elegantly simple solution to the problem of freezing; remove that which freezes.
AC 2010 Blackwell Publishing Ltd.
PMID: 20525002

Transplantation. 2006 Jun 15;81(11):1583-8.
Human skin preserved long-term in anhydric pulverized sodium chloride retains cell molecular structure and resumes function after transplantation.
Olszewski WL, Moscicka M, Zolich D. Department of Surgical Research and Transplantology, Medical Research Center, Polish Academy of Sciences, Warsaw, Poland.
Abstract
  BACKGROUND: Human skin is needed to cover large areas of the body lost through burns, trauma, and extensive maxillofacial surgery. Contemporary methods of skin storage are limited by the period of preservation to a few days. Our previous findings showed that fixation and storage of human skin in anhydric sodium chloride at room temperature for weeks or months preserves its morphological and molecular structure. In this study, we examined whether skin grafts preserved in sodium chloride may be successfully transplanted.
METHODS: Skin was harvested from lower limbs of patients during elective surgery, placed in containers with anhydric salt powder, and kept at 22 degrees C for 3 to 12 weeks. Desalination and rehydration took place before transplantation. Desalinated fragments were transplanted onto the dorsum of scid mice.
RESULTS: All grafts were accepted by recipients. Three weeks after transplantation, keratinocytes synthesized keratins 10, 16, and 17 and expressed antigens specific for stem (p63) and transient (CD29) cells. Moreover, they proliferated vigorously, their basal layer cells incorporated bromdeoxyuridine and expressed proliferative cell nuclear antigen. Isolated from transplants and cultured in vitro, they remained viable and produced enzymes. Dermis retained its structure and expressed fibroblast-specific antigen. All graft cells remained human leukocyte antigen I.
CONCLUSION: Human skin preserved in anhydric sodium chloride at room temperature for months can be successfully transplanted to scid mice. We propose the concept of "spore-like" keratinocyte stem cells to explain the long-term ex vivo survival of keratinocytes. The mechanism of survival of fibroblasts remains to be determined.
PMID: 16770248

Ann Transplant. 2004;9(4):37-9.
Human skin preserved in anhydric sodium chloride for months can be successfully transplanted.
Olszewski WL, Moscicka M, Zolich D. Department of Surgical Research and Transplantology, Medical Research Center, Polish Academy of Sciences, Warsaw, Poland.
Abstract
  Human skin can be preserved in pulverized sodium chloride dehydrated at 240C for 2 hours at room temperature for periods of weeks or months and successfully transplanted to scid mouse, retaining its normal morphological structure. Fragment of skin of a size of 10 x 10 x 6 mm were harvested during elective vascular and orthopaedic surgery of lower limbs, dried of blood and placed in anhydric sodium chloride powder in tight sealed containers. Prior to transplantation to scid mice, the specimens were desalinated and rehydrated. Specimes preserved for 1 to 6 months and harvested 3-4 weeks after transplantation revealed intensive incorporation of bromdeoxyuridine (BrdU) into basal keratinocytes. They expressed p63 and CD29 (stem cells, and transient cells antigens), PCNA (proliferating cell n
 uclear antigen) and cytokeratin 16 specific for proliferating keratinocytes. Dermal fibroblasts and few large HLA II cells showed a normal structure. Bacterial flora of skin did not change a
 fter grafting. We conclude that human skin can survive in a dehydrated state in sodium chloride for months and after transplantation the epidermal basal layer cells give rise to keratinocyte progenies. Skin fibroblasts and some resident immune cells can also survive.
PMID: 15884435

Cryobiology. 2001 Sep;43(2):182-7.
Recovery of human mesenchymal stem cells following dehydration and rehydration.
Gordon SL, Oppenheimer SR, Mackay AM, Brunnabend J, Puhlev I, Levine F. Osiris Therapeutics, Inc., 2001 Aliceanna Street, Baltimore, MD 21231-3043, U.S.A.
Abstract
  As cell therapies advance from research laboratories to clinical application, there is the need to transport cells and tissues across long distances while maintaining cell viability and function. Currently cells are successfully stored and shipped under liquid nitrogen vapor. The ability to store these cells in the desiccated state at ambient temperature would provide tremendous economic and practical advantage. Human mesenchymal stem cells (hMSCs) have broad potential uses in tissue engineering and regeneration since they can differentiate along multiple lineages and support hematopoeisis. The current research applied recent technological advances in the dehydration and storage of human fibroblasts to hMSCs. Three conditions were tested: air-dried, air-dried and stored under vacuum (vac
 uum only), and incubated with 50 mM trehalose + 3% glycerol and then air-dried and stored under vacuum (vacuum + trehalose). Plates containing dehydrated hMSCs were shipped from San Diego to
  Baltimore overnight in separate FedEx cardboard boxes. The hMSCs were rehydrated with 3 ml of hMSC medium and were able to regain their spindle-shaped morphology and adhesive capability. In addition, they maintained high viability and proliferation capacity. Rehydrated and passaged cells continued to express the characteristic hMSC surface antigen panel. Additionally, cells showed constitutive levels of mRNA for a stromal factor and, when exposed to reagents known to induce differentiation, demonstrated upregulation of two tissue-specific messages indicative of differentiation potential for fat and bone. While our preliminary findings are encouraging, we still need to address consistency and duration of storage by considering factors such as cell water content, oxygen concentration, and 
 the presence of free radicals.
PMID: 11846472

As with anhydrobiotic organisms, humans cells require gradual desiccation in order to maintain viability.

J Physiol. 2004 Jul 1;558(Pt 1):181-91. Epub 2004 May 14.
Response of human cells to desiccation: comparison with hyperosmotic stress response.
Huang Z, Tunnacliffe A. Institute of Biotechnology University of Cambridge, Tennis Court Road, Cambridge CB2 1QT, UK.
Comment in:  J Physiol. 2004 Jul 1;558(Pt 1):3.
Abstract
  Increasing interest in anhydrobiosis ('life without water') has prompted the use of mammalian cells as a model in which candidate adaptations suspected of conferring desiccation tolerance can be tested. Despite this, there is no information on whether mammalian cells are able to sense and respond to desiccation. We have therefore examined the effect of desiccation on stress signalling pathways and on genes which are proposed to be expressed in response to water loss through osmotic stress. Depending on the severity of the drying regime, human cells survived for at least 24 h. Both SAPK/JNK and p38 mitogen-activated protein kinases (MAPKs) were activated within 30 min by desiccation as well as by all osmotica tested, and therefore MAPK pathways probably play an important role in both resp
 onses. Gene induction profiles differed under the two stress conditions, however: quantitative polymerase chain reaction (PCR) experiments showed that AR, BGT-1 and SMIT, which encode protei
 ns governing organic osmolyte accumulation, were induced by hypersalinity but not by desiccation. This was surprising, since these genes have been proposed to be regulated by ionic strength and cell volume, both of which should be significantly affected in drying cells. Further investigation demonstrated that AR, BGT-1 and SMIT expression was dependent on the nature of the osmolyte. This suggests that their regulation involves factors other than intracellular ionic strength and cell volume changes, consistent with the lack of induction by desiccation. Our results show for the first time that human cells react rapidly to desiccation by MAPK activation, and that the response partially overlaps with that to hyperosmotic stress.
PMID: 15146043
Free text>
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1664923/pdf/tjp0558-0181.pdf

Nat Rev Gastroenterol Hepatol. 2010 Oct;7(10):557-64. Epub 2010 Aug 24.
The safety of osmotically acting cathartics in colonic cleansing.
Nyberg C, Hendel J, Nielsen OH. Department of Gastroenterology, Medical Section, Herlev Hospital, University of Copenhagen, 75 Herlev Ringvej, DK-2730 Herlev, Denmark.
Abstract
  Efficient cleansing of the colon before a colonoscopy or a radiological examination is essential. The osmotically acting cathartics (those given the Anatomical Therapeutic Chemical code A06AD) currently used for this purpose comprise products based on three main substances: sodium phosphate, combinations of polyethylene glycol and electrolyte lavage solutions (PEG-ELS), and magnesium citrate. All these preparations give adequate cleansing results and have similar profiles in terms of the frequency and type of mild to moderate adverse effects. However, serious adverse events, such as severe hyperphosphatemia and irreversible kidney damage owing to acute phosphate nephropathy, have been reported after use of sodium-phosphate-based products. The aim of this Review is to provide an update on
  the potential safety issues related to the use of osmotically acting cathartics, especially disturbances of renal function and water and electrolyte balance. The available evidence indicate
 s that PEG-ELS-based products are the safest option. Magnesium-citrate-based, hypertonic products should be administered with caution to elderly individuals and patients who are prone to develop disturbances in water and electrolyte balance. Sodium-phosphate-based products can occasionally cause irreversible kidney damage and should not be routinely used in bowel-cleansing procedures.
PMID: 20736921

J Biochem. 2008 Jun;143(6):841-7.
CH2-units on (poly-)ethylene glycol radially dehydrate cytoplasm of resting skinned skeletal muscle.
Kimura M, Takemori S. Department of Molecular Physiology, Jikei University School of Medicine, Minato-ku, Tokyo 105-8461, Japan
Corrected and republished from:  J Biochem. 2008 Jan;143(1):123-9.
Abstract
  Observing the optical cross-section and electron micrographs of mechanically skinned fibres of frog skeletal muscle, we found that ethylene glycols (EGs) of small (mono-, di-, tri- and tetra-EGs; M(r) 62-194) and medium (poly-EGs; M(r) 900 and 3350) molecular weights efficiently dehydrate the fibres to shrink them radially without microscopic inhomogeneity. The medium-sized poly-EGs at 30% weight/weight concentration absorbed almost all the evaporable water from the fibre. Passive tension measurement at near slack sarcomere spacing indicated that this dehydration by EGs did not accompany longitudinal fibre shrinkage. Chemically relevant fully hydric alcohols (glycerol, threitol, ribitol and mannitol; M(r) 92-182) showed no appreciable dehydrating ability on fibres. An intimate correlatio
 n was found between fibre dehydration and CH(2)-concentration of the solutions. Viscosity measurements indicated that the hydrodynamic radii of the alcohols were comparable to those of the s
 mall EGs. Therefore, hydrodynamic radii are not a primary determinant of the dehydrating ability. Additionally, CH(2)-concentration of EGs but not alcohols was found to correlate intimately with the measured viscosity of the bulk solution of EGs. These results suggested that the interaction between water molecules and CH(2)-units in crowded cytoplasm of skeletal muscle affects cytoplasm as a whole to realize anisotropic fibre shrinkage.
PMID: 18583358

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