[tt] Aschwin de Wolf: vitrification agents in cryonics: VM-1

[Eugen Leitl]

http://depressedmetabolism.com/2008/03/31/vitrification-agents-in-cryonics-vm-1/

Vitrification agents in cryonics: VM-1

A major public misperception is that cryonics involves the freezing of dead
people. The objective of cryonics is not to preserve dead people with the
hope of future revival but to place critically ill patients in a state of
biostasis until a time when more advanced medical technologies might be
available to treat and cure them. Currently, all major cryonics organizations
induce metabolic arrest of the brain by attempting vitrification rather than
freezing.

Unless a patient has suffered a long period of circulatory arrest, after
which perfusion of the body or brain is no longer possible, metabolic arrest
is induced by cooling down the patient to cryogenic temperatures.
Vitrification can be defined as “the process of converting a material into a
glass-like amorphous solid that is free from any crystalline structure.”
Because vitrification of pure water would require extremely rapid cooling
rates, vitrification in cryonics is achieved by substituting the water of
patients with a highly concentrated cryoprotectant agent before cooling.

In 2001, Alcor introduced its first vitrification agent (B2C) for
neuropatients and extended this technology to whole body patients in 2005
with the introduction of M22. The Cryonics Institute introduced its own
vitrification agent, VM-1, to its membership in 2005. VM-1 was developed by
Dr. Yuri Pichugin and stands for Vitrification Mixture-1, which indicates
that it was the first vitrification agent to be introduced at CI. Before
VM-1, CI generally used the cryoprotective agent glycerol. VM-1 consists of
35% ethylene glycol and 35% dimethyl sulfoxide (w/w). It is introduced in a
carrier solution called m-RPS-2, consisting of potassium chloride, glucose,
and TRIS (alternatively called THAM). A more detailed review of the research
and components of the solution can be found on the Cryonics Institute
website.

VM-1 has been formulated and validated specifically for cryonics patients.
Although encouraging viability results have been obtained in brain slices,
the agent first and foremost reflects the search for a vitrification agent
that is an affordable, but also strong and stable, glass former. M22 is the
culmination of many years of research (mostly on kidney slices) by Greg Fahy
et al. to find vitrification agents that can successfully recover organs from
cryogenic temperatures for organ transplantation. M22 is being licensed to
Alcor by the cryobiology company 21st Century Medicine.

An interesting similarity between the two agents is that both contain the
same core components: ethylene glycol and DMSO. 21st Century Medicine
solutions additionally contain formamide, which has a low toxicity in the
presence of DMSO, allowing formulation of solutions of lower overall
toxicity. Solutions containing DMSO, an amide, and ethylene glycol are
protected by 21st Century Medicine’s M22 patent.

The strong glass forming ability and stability of VM-1 is further evidenced
by the following research findings. Pichugin did not observe ice formation or
devitrification when 20 ml glass vials of 60% and 65% VM-1 were cooled and
warmed with cooling and warming rates as low as 0.1 degrees Celsius per
minute. 65% VM-1 solutions with “homogenized rat brain tissues containing
natural nucleators” did not show visible ice crystals after 14 days at dry
ice temperature (-78.5 degrees Celsius). The stability of large volumes (2
liters — unfiltered) of VM-1 was investigated and no ice crystals were
observed after 21 days of storage at dry ice temperature. These results raise
the intriguing question of whether patients can be perfused with high
concentration VM-1 in the field and shipped to a cryonics facility on dry
ice. In cryonics we do not ship vials of cryoprotectant solution, or fully
equilibrated brain slices, but patients that have been exposed to variable
degrees of warm and cold ischemia. Questions about the nature and extent of
ice damage of poorly perfused areas during long holding and transport periods
at dry ice temperature still remain.

Unlike M22, VM-1 is not a suitable agent for perfusion of whole body
patients. In CI patients where whole body perfusion was attempted with one of
the components, ethylene glycol, serious edema resulted. Similar results have
been encountered in the past with DMSO as a mono-agent. Improved results may
be obtained by modifying the carrier solution of VM-1 to include an oncotic
agent and/or using glycerol for the rest of the body. Although high molar
glycerol can be perfused in (non-ischemic) patients without serious edema, CI
currently discourages members from choosing whole body perfusion in order to
ensure optimal perfusion of the brain.

The current carrier solution of VM-1, m-RPS-2, is basically a stripped down
and modified version of Fahy’s Renal Preservation Solution-2. As such, it
does not contain components that reduce free radical damage (such as
glutathione) or ATP precursors (such as adenine) to assist energy generation
during hypothermia. Perhaps a more controversial choice is the lack of an
oncotic agent to prevent and counter edema during perfusion. Pichugin
questions the value of such agents for perfusion of the brain. In practice,
CI has not encountered much edema during brain perfusion of its patients,
many of which have been exposed to considerable periods of warm and cold
ischemia. m-RPS-2’s lack of hypertonicity does not seem to make the carrier
solution suitable to inhibit chilling injury.

There are still some open questions about VM-1. Although VM-1 is designed as
a low cost agent to allow preservation of the brain without ice formation, no
published electron micrographs are available that show the quality of
ultrastructural preservation that can be obtained with VM-1. VM-1 has been
validated solely on the basis K+/Na viability assays. As electron micrographs
of brains perfused with vitrification agents B2C and M22 indicate, agents
that can inhibit ice formation can still produce strikingly visible
differences in terms of ultrastructural alterations. Although good
hippocampal slice viability results imply good ultrastructural preservation,
actual empirical evidence of this could make a stronger case.

During the final step of cryoprotective perfusion at CI, the current protocol
is to introduce 70% VM-1 at -7 degrees Celsius to reduce the time required to
achieve a minimum target concentration of 60% as measured by refractometry.
It is not clear what the biochemical effects of exposing the patient to such
concentrations of VM-1 are, although the “ideal” temperature for the final
step is lower than what is currently used by Alcor for M22. In practice, it
is doubtful that the patient’s brain is at such temperatures during a typical
perfusion. Such a protocol would require more rigorous control of the
perfusate and brain temperature using a subzero chiller.

Another advantage would be to introduce VM-1 in a more “linear” fashion using
a “closed circuit” in which the concentration is gradually increased. Because
such a protocol requires a more expensive, complicated, and challenging
perfusion circuit, the costs and risks of such a protocol need to be weighed
against the potential advantages. One straightforward compromise might be to
do “open circuit” perfusion but to close the circuit after target
concentration has been reached to allow for good equilibration of the cells
before terminating perfusion.

With VM-1, CI seems to have introduced an extremely cost effective and stable
vitrification solution. If CI will find the resources to do new experiments
to improve its composition and protocol, obtaining actual images of brain
slices perfused (and vitrified) with the solution seems to be an important
priority. It also needs to be stressed that results obtained in brain slice
experiments in different species are not necessarily a good indicator of what
can be expected in actual human cryonics patients who generally have been
exposed to long terminal periods, warm and cold ischemia, and longer
perfusion times at higher temperatures. It is clear that is there is an
urgent need for a research program that investigates the relationship between
such variables and outcome in terms of ice formation, viability and
ultrastructure. Investigations that have been done by Darwin and Pichugin
under more realistic conditions will be discussed in the future.