X-Message-Number: 33229
From:
Date: Thu, 13 Jan 2011 21:56:09 EST
Subject: Intracellular Freezing & Vitrification
A couple of weeks ago I made some comments in correspondence that were
subsequently translated into a posting on Cold Filter. I'm not a
cryobiologist, and I've never claimed to be one, but I do try to answer, and to
ask
questions that I feel confident have a good basis in fact. I don't always get
it right, and this is a case in point.
A long standing concern of mine has been that it is almost certainly case
that the 'average' cryonics patient subjected to vitrification protocols
(Alcor or CI) are not completely vitrifying. This is very likely to be the
case because warm and cold ischemia cause multiple infarcts, or defects in
circulation that prevent adequate distribution of the vitrifying agents - in
this case cryoprotective chemicals (CPAs) that depress the freezing point of
the solution ('colligative' CPAs). What this means in practice is that
there will be islands of tissues that will have a considerable amount of CPA
present, but not enough to 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, and then
undergo more or less instantaneous freezing. When that happens, the ice that
forms, forms inside the cells, and that is highly destructive - far more
destructive than if 'conventional' extracellular freezing had occurred
resulting
in cellular dehydration and subsequent vitrification of the intracellular
compartment.
However, theory does not always map reality, and it seems that in the case
of tissues loaded with very high concentrations of CPA that do undergo
freezing at low temperatures, other factors are in play. Brian Wowk was kind
enough to take a considerable amount of time to tutor me on this matter. And
while I confess that I still do not fully understand the mechanics of what
is going on, I do understand enough to realize that the situation is not as
cut and dried as I once thought, and that indeed, experiments I did myself
in collaboration with others at BioPreservation many years ago have a
direct bearing on this problem.
In the dog experiments we conducted in the mid and late 1990s the animals
were perfused with a 7.5 Molar (M) glycerol solution, which has a
concentration of ~69% w/v glycerol, and then frozen to ~ -90 deg C. They were
then
warmed up to -6 deg C and reperfused with fixative. 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. 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.
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. 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. Indeed, one
species of bacteria, Pseudomonas syringae, is so good at producing ice
nucleating proteins that it is actually grown, en masse, and used as an
additive
to water sprayed through cold air to make artificial snow for skiers. It
is marketed as a product called SnoMax.
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. 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. 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. 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. 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. 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.
Mike Darwin
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