[tt] Vitrification agents in cryonics: M22\

[Eugen Leitl]

http://www.depressedmetabolism.com/2008/07/08/vitrification-agents-in-cryonics-m22/

Vitrification agents in cryonics: M22

by Aschwin de Wolf ~ July 8th, 2008. Filed under: Alcor, Cryobiology,
Cryonics, Vitrification, Whole Body Cryopreservation.

M22 represents the culmination of decades of work in applied cryobiology by
researchers Gregory Fahy , Brian Wowk, and others to develop a vitrification
agent that can recover complex organs (such as the kidney) from cryogenic
temperatures without ice formation and minimal toxicity. In 2005, M22 was
licensed by the patent holder 21st Century Medicine (21CM) to the Alcor Life
Extension Foundation to replace their previous vitrification agent B2C. As a
result, the least toxic vitrification agent for complex organs that has been
documented in peer review journals is currently being used for cryonics
patients at Alcor.

M22 incorporates a number of important discoveries in cryobiology:

1. High concentrations of a cryoprotective agent (or a mixture of different
cryoprotective agents) can prevent ice formation during cooldown and warming.

2. The toxicity of some cryoprotectants can be neutralized by combining them
with other cryoprotective agents.

3. The general toxicity of a vitrification agent can be predicted by using a
measure called qv*, allowing for the rational formulation of less toxic
vitrification agents.

4. Within limits, non-penetrating agents can reduce the exposure of cells to
toxic amounts of cryoprotectants without reducing vitrification ability.

5. Synthetic “ice blockers” can be included in a vitrification mixture to
reduce the concentration of toxic cryoprotective agents necessary to achieve
vitrification.

6. Substituting methoxyl (-OCH3) for hydroxyl groups (-OH) in conventional
cryoprotective agents can decrease viscosity, increase permeability, and
reduce the critical cooling rate necessary to avoid ice formation.

7 Chilling injury can be eliminated by introducing the vitrification agent
with a hypertonic concentration of non-penetrating solutes.

8. In cryonics, with a minor proprietary modification, M22 can be used for
whole body perfusion without causing severe edema that has been a problem for
some other solutions.

Vitrification is the solidification of a liquid without crystallization. When
a solution is cooled down to the glass transition point (-123.3°C for M22)
the extreme elevation in viscosity will produce a glass in which all
translational molecular motions are arrested. Although water vitrifies at
cooling rates exceeding a million of degrees Celsius per second, such cooling
rates are relaxed when other solutes are substituted for water. In
cryobiology solutions with high concentrations of cryoprotective agents can
be used to vitrify complex organs such as the kidney or the brain.

Vitrification has a number of clear advantages over conventional
cryopreservation. The most important advantage is the elimination of ice
formation. Although the adverse effects of ice formation can be mitigated by
the use of cryoprotective agents (glycerol, DMSO) and optimization of cooling
rates, massive ice formation does not permit recovery of complex organs with
full viability. Another advantage is that vitrification eliminates the need
to strike a balance between the risk of intracellular freezing induced by
fast cooling on the one hand, and cell dehydration and solution concentration
induced by slow cooling on the other hand.

The challenge in formulating successful cryoprotective agents is to design
vitrification solutions that are non-toxic but allow for vitrification at
realistic cooling and warming rates. For more than a decade the least toxic
vitrification agent was Greg Fahy’s VS41A, which is an 55% weight/volume
equimolar mixture of DMSO and formamide plus propylene glycol. The “1A” in
VS41A reflects the solution’s ability to vitrify at normal atmosphere
pressure (as opposed to an older, more dilute solution, VS4, which requires
1000 atmospheres of pressures to vitrify). The equimolar concentrations of
DMSO and formamide reflect Baxter and Lathe’s research who concluded that
amides can neutralize the toxicity of DMSO, a finding that Greg Fahy later
revised in favor of the theory that it is actually DMSO that neutralizes the
toxicity of formamide. The ability of DMSO to neutralize the toxicity of
formamide (up to certain concentrations) allows for the formulation of
vitrification agents with reduced toxicity. This finding has been so
fundamental that an equimolar concentration of DMSO and formamide remains the
core of M22.

Another major step was made when the researchers at 21CM found that high
concentration of (penetrating) cryoprotectant agents do not necessarily
increase toxicity. Contrary to conventional cryobiology expectations, Fahy et
al. found that weaker glass formers favor higher viability. They proposed a
new compositional variable called qv* to predict the general toxicity of
vitrification solutions. Using qv* they made the “counter-intuitive” decision
to substitute a higher concentration of the weaker glass former ethylene
glycol for propylene glycol to create a solution called Veg, which produced a
substantial improvement in terms of viability as measured by K+/Na+ ratios.

Because cells contain higher concentrations of protein, the intracellular
space is more favorable to vitrification than the extracellular space. As a
consequence, the concentration of penetrating (toxic) cryoprotectants can be
reduced in favor of non-penetrating polymers like polyvinylpyrrolidone (PVP).
Variations of Veg in which the concentration of DMSO of formamide was reduced
in favor of PVP increased viability without decreasing its ability to
suppress ice formation. The concentration of penetrating cryoprotectants can
be further reduced by inclusion of non-penetrating “ice-blocking” polymers.
These ice-blockers also reduce the critical cooling and warming rates
necessary to avoid ice formation, which is an important requirement for
solutions that are used to vitrify complex organs such as the human brain.

Because concentrated vitrification solutions depress the homogeneous
nucleation temperature (Th) below the glass transition temperature (Tg), a
major obstacle to successful vitrification is the presence of heterogenous
nucleators. Some organisms have antifreeze proteins (AFPs) and anti-freeze
glycoproteins (AFGPs) that mitigate heterogenous nucleation by binding to
nucleators. Because adding such anti-nucleating proteins to vitrification
solutions would be prohibitively expensive and less effective, Greg Fahy
proposed the creation of synthetic ice-nucleation inhibiting polymers. In
2000 Wowk et al. published work that showed the effectiveness of a co-polymer
of polyvinyl alcohol (PVA) and vinyl acetate in inhibiting heterogenous
ice-nucleation. This co-polymer is now being sold by 21CM under the name
“X-1000″. X-1000 is particularly effective in glycerol solutions, presumably
because glycerol itself is a poor anti-nucleation agent. Increasing the
concentration of X-1000 in vitrification solutions decreases ice formation
and relaxes minimum cooling rates. Although X-1000 is presumed to be
non-toxic, the maximum concentration in vitrification solutions does not
exceed 1% w/v because no further benefits were observed beyond this
concentration. In 2002, 21CM announced the discovery of another synthetic
“ice-blocker” called Z-1000. Z-1000 is the polymer polyglycerol (PGL), which
specifically inhibits ice nucleating activity caused by the bacterium
Pseudomonas syringae. Mixtures of PVA and PGL are more effective in
inhibiting ice formation than either agent alone, suggesting the PVA and PGL
complement each other by inhibiting different sources (bacterial and
non-bacterial) of ice nucleation.

A variant of Veg that includes the low molecular weight polymer
polyvinylpyrrolidone K12, X-1000, and Z-1000 named VM3 improved viability in
renal cortical slices and decreased the critical cooling and warming rates
necessary to avoid ice formation and de-vitrification (ice formation during
rewarming) while maintaining the same molar concentration as VS41A. The
transition from Veg to VM3 reflects the two breakthroughs mentioned above:
reduction of cryoprotectant toxicity by inclusion of non-penetrating polymers
and ice blocking agents. VM3 also was the least toxic agent in vitrification
of rat hippocampal brain slices, which is of particular importance for
cryonics. The first vitrification agent ever to be introduced to cryonics was
a hyperstable variant of VM3 called B2C. B2C was used until late 2005, when
it was replaced by M22.

M22 takes advantage of two other discoveries: the ability to design better
glass formers by methoxylation of conventional polyols, and inhibition of
chilling injury by delivering the vitrification agent as a hypertonic
solution. Because hydroxyl groups can bind either to water or hydroxyl groups
on other cryoprotective agents, substituting methoxyl groups for hydroxyl
groups should decrease interaction between cryoprotectants and increase
interaction between the cryoprotectant and water. As a result, methoxylated
compounds have stronger ice inhibiting ability, thus reducing the critical
cooling rate for vitrification or reduce the concentration of (toxic)
cryoprotective agents in a solution. Methoxylated cryoprotectants also
decrease viscosity and increase cell permeability, allowing for shorter
perfusion times, and thus reduced cryoprotectant exposure at higher
temperatures. For example, the methoxylated glycerol derivative
3-methoxy-1,2-propanediol has a higher glass transition point and vitrifies
at ~ 5% lower concentration than the corresponding conventional
cryoprotective agent. Complete exploitation of these advantages is limited by
the fact that they are more toxic than their non-methoxylated compound, as
predicted by qv*. As can be seen in the table, the major difference between
VM3 and M22 is the reduction of PVP K12 in favor of the penetrating
cryoprotectants 3-methoxy-1,2-propanediol and n-methyl-formamide, and
increased concentration of the ice-blocker Z-1000. The final molar
concentration of 9.345 M demonstrates that more concentrated vitrification
agents do not necessarily have to be more toxic.

M22, so called because it was intended to introduced at -22 degrees Celsius,
constitutes a major landmark in vitrification of complex organs. In 2005
Fahy, Wowk et al. announced routine recovery of rabbit kidney slices from
temperatures around -45 degrees Celsius. Although consistent recovery of
vitrified organs is not yet feasible, continued progress in solution
composition and perfusion techniques inspire optimism that this may be
possible in the future. In 2007, Greg Fahy of 21CM reported recovery of
electrical activity in vitrified brain slices and induction of long-term
potentiation (LTP), which indicates that the structures for processing memory
are maintained after vitrification, storage and rewarming of brain tissue.
Visual evidence that M22 can preserve the ultrastructure of the brain better
than B2C was published on the Alcor website in 2005.

M22 also needs to be used in a suitable carrier solution to support cell
metabolism at low temperatures and decrease oxidative injury and edema. The
carrier solution for M22 is called LM5 to reflect the 50% reduction of
glucose (as compared to the older carrier solution RPS-2) in favor of
equimolar concentrations of mannitol and lactose, to address compatibility
problems with the ice blockers. The combination of the isotonic LM5 plus the
non-penetrating polymers in M22 creates a hypertonic solution, which has been
shown to eliminate chilling injury, which is the injury that is caused by
exposure to low temperatures as such. For cryonics, the composition of M22 is
further enhanced by including a proprietary components that allows perfusion
of whole body patients without edema.

The research breakthroughs discussed above allow for a global reconstruction
of the composition of M22 using the table. Maintained is the equimolar
combination of DMSO and formamide from Fahy’s older vitrification solutions
to reconcile strong glass formation ability and minimal toxicity. The
discovery of the  compositional variable qv* allows for substitution of
higher concentrations of the weaker glass former ethylene glycol for
propylene glycol. Substitution of a non-penetrating polymer, PVP K12, and the
ice-blockers X-1000 and Z-100 allow for further reduction of DMSO and
formamide, reduction of critical cooling rates, and increased stability
against ice formation. In M22, PVP K12 is reduced to optimize hypertonicity
of the non-penetrating agents for suppression of chilling injury. Added are
the methoxylated cryoprotectant 3-methoxy-1,2-propanediol and the highly
permeable amide n-methyl-formamide, producing the least toxic but most
concentrated vitrification solution to date.

The most striking differences between Alcor’s old perfusate and the newer
vitrification agents licensed from 21CM are complexity and cost. Until 2002,
Alcor patients were perfused with high molar glycerol in an MHP-2 based
carrier solution. M22 itself consists of 8 (!) different components, putting
the total number of components of M22 in carrier solution above 15. Such
perfusates makes great demands on preparation skills and quality controls.
Components such as the ice blockers and 3-methoxy-1,2-propanediol have put
the cost of Alcor’s whole body perfusate alone close to the cost of complete
cryopreservation arrangements at the Cryonics Institute (CI). This raises
obvious questions about costs and benefits. As evidenced by CI’s VM-1, potent
protection against ice formation can be achieved with a vitrification agent
that solely consists of DMSO and ethylene glycol. It is plausible to assume
that vitrification lessens demand on future repair technologies, but it
speculative to assume that minor differences in toxicity between different
vitrification agents will translate in earlier resuscitation and less
expensive repair protocols. However, more toxic vitrification solutions, such
as CI’s VM-1, may cause acute injury to endothelial cells. As Brian Wowk
notes, “good cryoprotection depends on good perfusion, which depends on
preservation of vascular integrity during perfusion. The ability to perfuse
M22 into whole bodies with tolerable edema is likely to be intimately related
to its low toxicity to vascular endothelium.” And of course, there are also
PR advantages to the fact that a cryonics organization uses a vitrification
agent that is also the state of the art in conventional cryopreservation of
organs.

M22 produces substantial brain shrinking during perfusion of (non-ischemic)
patients. As a matter of fact, cerebral dehydration may be a major
contributing factor to vitrification of the brain and even allow for reduced
concentrations of M22 for brain preservation. This does not mean that the
(expensive) non-penetrating polymers could be replaced for any high molecular
weight polymer because the ice blockers and non-penetrating cryoprotective
agents also protect the extracellular space against ice formation and are
effective in ischemic patients with a compromised blood brain barrier (BBB).
The limited ability of some components of M22 to cross the BBB and, and
differences in permeability of the various components of M22, does raise
questions about the exact composition of M22 beyond the BBB and within brain
cells after completion of cryoprotective perfusion.

Patients outside of the US may not fully benefit from cryopreservation with
M22 because of the of long cold ischemic times during transport. This raises
the question if cryonics patients can be perfused outside of the US and
shipped in dry ice. Experiments with VM-1 in bulk solution indicate that this
solution is very stable against ice formation, even during long storage
periods. M22 in bulk solution seems to form ice crystals overnight if stored
in dry ice. This does not necessarily mean that M22 cannot be used in
combination with dry ice for overseas patients because human tissue perfused
with M22 (or any cryoprotective agent) is not the same as M22 in pure
solution. But regardless of M22’s compatibility with dry ice shipping,
cryonics organizations may benefit from formulating a highly concentrated
inexpensive vitrification solution that is extremely robust against formation
of ice, which can be used for simple perfusion of non-US patients in
combination with dry ice shipping. The decreased cold ischemic times of such
a solution may outweigh the increased toxicity of such solutions.

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