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Research Findings of Advanced Neural Biosciences, Inc.

(Please note that the following paper may be found by some readers to be very "technical" in nature and some background reading and looking up of some scientific and/or technical terms may be necessary to aid in the reader's full understanding of the ANB research work).


Human Cryopreservation Research at Advanced Neural Biosciences 
By Aschwin de Wolf & Chana Phaedra 
In 2008 we obtained modest funding to establish a laboratory aimed at researching cryonics. Our 
first challenge was to establish a research program that (a) would distinguish itself from other 
research labs engaged in cryobiology research, and (b) would be feasible in terms of limited 
financial resources and time. We immediately recognized that our greatest contribution would be 
to investigate cryonics protocols under realistic conditions. In this article we introduce the reader 
to some of our most important and robust discoveries. 
Until cryonics becomes available as an elective medical procedure, all cryonics patients will 
experience varying degrees of cerebral ischemia. Even in “good” cases where stabilization 
procedures are initiated promptly after pronouncement of legal death, the agonal period prior to 
cardiopulmonary arrest can give rise to cerebral perfusion impairment. In the case of cryonics 
organizations that do not offer standby and stabilization services, we should expect at least 24 
hours of cold ischemia for a typical (remote) patient, often preceded by significant periods of 
warm ischemia due to no, or slow, cooling. 
The fact that no cryonics patient can completely escape some degree of cerebral ischemia forces 
cryonics organizations to deal with a fundamental question: how do our protocols and 
vitrification solutions perform under such conditions? In particular, in our lab we have been 
interested in the behavior of vitrification solutions in ischemic brains. It should not be a priori 
assumed that vitrification solutions preferred for non-ischemic tissues are preferred for ischemic 
tissues as well. A related line of research is whether the composition of carrier solutions can be 
altered to improve cryoprotectant perfusion in the ischemic brain. 
The investigation of cryonics procedures under realistic conditions is by no means exhausted by 
conducting experiments under ischemic conditions. Another major difference between 
cryobiology experiments conducted in the laboratory and the practice of cryonics is that the 
control over perfusion temperatures is limited in cryonics cases. Even the most sophisticated 
cryonics protocols expose the brain to toxic concentrations of the vitrification agent at high sub-
zero temperatures. Thus, our earliest investigations in 2009 were concerned with the effects of 
exposing red blood cells to high concentrations of VM-1 (the vitrification agent of the Cryonics 
Institute) in order to address the possibility that exposing a patient to high concentrations of this 
agent in the absence of rigorous temperature control could produce instantaneous red blood cell 
lysis (i.e., hemolysis). 
The Red Blood Cell as a Model for Cryoprotectant Toxicity 
Various approaches are available to investigate cryoprotectant toxicity, ranging from theoretical 
work in organic chemistry to cryopreservation of whole mammalian organisms. One simple 
model that allows for “high throughput” investigations of cryoprotectant toxicity uses red blood 
cells (erythrocytes). Although the toxic effects of various cryoprotective agents may differ 
among red blood cells, other cells, and organized tissues, positive results in a red blood cell 
model can be considered the first experimental hurdle that needs to be cleared before the agent is 
considered for testing in more advanced models. Because red blood cells are widely available 
for research, this model eliminates the need for animal experiments for initial screening studies. 
It also allows researchers to investigate human red blood cells. Other advantages include the 
reduced complexity of the model (packed red blood cells can be obtained as an off-the-shelf 
product) and lower costs. 
Red blood cells can be subjected to a number of different tests after exposing them to a 
cryoprotective agent. The most basic test is gross observation of the red blood cells in a 
cryoprotectant solution. When high concentrations of a cryoprotectant are introduced (such as in 
vitrification), a stepwise approach is necessary to avoid osmotic damage. If a cryoprotectant 
solution is extremely toxic rapid hemolysis will follow, which can be observed as a noticeable 
change of the color of the solution, hemolyzed cell debris sinking to the bottom of the test tube, 
or negligible difference between the pellet (if there is one at all) and the supernatant after 
centrifugation. It is important to keep in mind that these effects only indicate gross membrane 
damage and that absence of hemolysis is not equivalent to absence of cryoprotectant toxicity. 
In our investigations we did not observe instantaneous hemolysis of sheep red blood cells when 
70% of VM-1 (in carrier solution) was introduced in a stepwise fashion either at room 
temperature or close to the freezing point of water. Morphological studies with light microscopy 
showed slight alterations for VM-1 (dehydration, decreased uniformity) but we have not seen the 
extreme alterations and destruction that have been observed in solutions that were formulated to 
produce hemolysis. Eliminating the step-wise approach and exposing the red blood cells to 70% 
of VM-1 at once, however, did produce hemolysis. This effect was more pronounced at lower 
temperatures, presumably because at low temperatures the rate of diffusion of cryoprotectants is 
further depressed than the rate of diffusion of water, causing more pronounced osmotic damage. 
VM-1 consists of 35% dimethyl sulfoxide (DMSO) and 35% ethylene glycol (EG). 
Cryobiologist Yuri Pichugin identified this binary cryoprotectant as one of the least toxic (non-
patented) binary vitrification solutions for the vitrification of rat hippocampal brain slices. 
DMSO is a stronger glass former than EG, but in the case of DMSO as a mono-agent, stepwise 
exposure of red blood cells to a 70% solution produced complete instantaneous hemolysis. This 
observation corroborates the contribution of specific toxicity to hemolysis of red blood cells and 
the need for toxicity neutralization in vitrification solutions. 
Since red blood cell hemolysis assays are not optimal for quantifying minor differences in 
cryoprotectant toxicity, or for investigating the effects of cryoprotectants on organized nervous 
tissue, we limit our use of this method to preliminary investigations of new variants of VM-1 
and/or alternative carrier solution composition. 
Perfusion of the Ischemic Brain 
The brain distinguishes itself from most other organs by its high energy utilization. When the 
brain is deprived of oxygen and other energy substrates, a complex biochemical cascade ensues 
that ultimately results in decomposition. Since we do not know the degree of degradation that 
still permits meaningful reconstruction of the original state of the brain, the most conservative 
approach is to limit ischemia as much as is practically possible. 
The human brain is too large to use immersion as a method to replace water with a 
cryoprotectant. This fact necessitates the use of vascular perfusion to prepare the brain for 
exposure to cryogenic temperatures. As a consequence, the ability to protect the brain against ice 
formation is not an independent challenge but depends on the state of the brain at the time of 
cryoprotective perfusion. It is at this juncture of ischemia and cryoprotective perfusion where we 
have conducted most of our experiments. 
In a non-ischemic brain, sub-optimal equilibration of the vitrification solution may be 
compensated by dehydration. This phenomenon is of limited relevance to patients with extensive 
cerebral ischemia because, as ischemia progresses, the blood-brain barrier through which such 
dehydration is mediated will become progressively disrupted. For example, cryoprotective 
perfusion of the non-ischemic rat produces severe dehydration of the brain. After 24 hours of 
cold ischemia, this dehydration is sharply reduced, and after 48 hours there is no evidence of 
cerebral dehydration after cryoprotectant perfusion. This phenomenon allowed us to investigate 
cryoprotective perfusion under ischemic conditions without modifications to the carrier solution 
to limit cryoprotectant-induced shrinking of the brain. 
Our first approach to study the effect of ischemia on perfusion impairment in the brain was to 
add India ink to the perfusate. Areas with no, or poor, perfusion are distinguished by residual 
blood and absence of ink. In those studies we limited ourselves to investigating the perfusability 
of the brain without subsequent freezing to obtain a basic understanding of this phenomenon 
without additional variables. 
Inspection of the brain after ink perfusion showed that 60 minutes of ischemia at room 
temperature is sufficient to produce noticeable perfusion impairment with the degree and 
distribution of the impairment worsening progressively as the duration of warm ischemia 
Two interventions that are presumed to mitigate perfusion impairment are antithrombotic therapy 
and induction of hypothermia. Administration of the anti-coagulant heparin prior to ischemia and 
the thrombolytic streptokinase following ischemia failed to improve perfusion. This outcome 
corroborates that ischemia-induced “no-reflow” is not confined to blood clotting and suggests a 
role for the involvement of blood in a non-coagulating fashion. Scientific and clinical reviews of 
the no-reflow phenomenon have identified several other factors that contribute to perfusion 
impairment including red cell aggregation, vasogenic and cellular edema, free radical damage, 
and inflammatory mediators. Some studies, including the cerebral resuscitation studies of Peter 
Safar and colleagues, have found benefits from a combination of high perfusion pressures and 
hemodilution. Our studies into such protocols for short periods of ischemia are inconclusive and 
for longer (>24 hours) periods of cold ischemia we have found that higher perfusion pressures 
during cryoprotective perfusion increase ice formation after cooling to cryogenic temperatures. 
One of our most robust findings is that rapid induction of hypothermia after circulatory arrest 
mitigates the no-reflow phenomenon. Perfusion impairment was greatly reduced when the brain 
was cooled in situ using a miniature portable ice bath. The whole-body cooling rate in these 
experiments exceeded 1°C per minute. Since such cooling rates are not practically feasible 
during external cooling in human cryopreservation stabilization without an aggressive 
combination of different cooling modalities, including cyclic lung lavage, we repeated these 
experiments at a cooling rate (~ 0.18 °C per minute) that is practical for human cryopreservation 
and observed the same benefits. These findings strongly corroborate the current practice of rapid 
induction of hypothermia in cryonics and suggest that even modest decreases of brain 
temperature can significantly mitigate perfusion impairment, even if the reduction in metabolic 
demand cannot prevent exhaustion of energy in the brain. 
Another consistent finding in our research is that blood substitution prior to circulatory arrest 
strongly reduces perfusion impairment. In the India ink model we did not observe evidence of 
perfusion impairment after up to 72 hours of cold ischemia following blood substitution with m-
RPS-2 (the carrier solution of VM-1). One limitation of this model is that complete washout of 
the blood prior to ischemia excludes observation of residual blood after perfusion as an indicator 
of perfusion impairment. Filling of vessels with India ink correlates strongly with the degree of 
perfusion impairment but it does not rule out the presence of small pockets of poorly perfused 
areas in the brain. Because India ink perfusion may not completely predict the degree of 
cryoprotectant equilibration that is possible after warm and cold ischemia, we further refined our 
model and introduced observation of the degree of ice formation after cryoprotectant perfusion 
and cooling as an endpoint. 
Cryoprotective Perfusion of the Ischemic Brain 
As a general rule, cryonics interventions aimed at preventing and mitigating ischemic injury are 
not evaluated with cryoprotective perfusion and ice formation as an endpoint. As a consequence, 
there is a serious lack of knowledge about the efficacy of cryonics stabilization protocols on 
reducing ice formation. One of the most valuable research models in our lab has been to conduct 
cryoprotective perfusion under various conditions of (cold) ischemia. Space limitations prevent us from disclosing all our findings, but our most important discoveries are discussed below. Most 
of our investigations into cryopreservation of the ischemic brain have been conducted with VM-
1, the vitrification solution of the Cryonics Institute. 
The most fundamental and robust finding in these experiments is that the duration of warm and 
cold ischemia is positively associated with perfusion impairment and ice formation after cooling 
to liquid nitrogen temperatures. Our studies corroborate the pioneering feline work that cryonics 
researcher Michael Darwin did in this area with electron micrographs in the 1980s. In the rat 
brain we have identified a consistent hierarchy of vulnerability to cold ischemia-induced 
perfusion impairment as revealed by inspection of the perfused brain and signs of ice formation 
after cryogenic cooling. The following four major areas are ranked by increasing vulnerability: 
Cerebral cortex; cerebral subcortex; cerebellar cortex; cerebellar subcortex. 
We do not have a full understanding of the reason behind this ranking but these findings may be 
somewhat comforting in light of our current understanding that the most identity-critical 
information is stored in the cortex of the brain and that the cerebellum may be the least important 
area in this regard. Notwithstanding this, our research has been aimed at overcoming perfusion 
impairment and ice formation in patients with extensive ischemic exposure. 
We have studied a number of different interventions to improve outcome in cold ischemic brains 
and the majority of our experiments involved alteration of the cryoprotectant carrier solution. 
We started by adding various non-permeating salts and sugars and high molecular weight 
polymers to the carrier solution in order to mitigate edema under the expectation that this would 
improve outcome. This approach did not produce the desired outcome and meaningful reduction 
of interstitial edema was not observed either. 
We did observe improved outcome in terms of reduction of perfusion impairment in the presence 
of suitable concentrations of the high molecular weight polymers PVP K360, dextran 500, and 
dextran sulfate 500. We initially attributed these encouraging outcomes to the ability of these 
polymers to “seal” leaky membranes, although this interpretation seemed to be at odds with the 
lack of edema reduction observed. 
A real breakthrough occurred when we designed a number of solutions that were made 
equiviscous with a dextran sulfate 500 based carrier solution – our most successful carrier 
solution to date. All these solutions produced comparable results in terms of overcoming 
perfusion impairment, indicating that the advantageous properties of these higher molecular 
weight solutions was not specific to their chemical composition but may be mediated through 
higher viscosity. This interpretation was further corroborated by our observation that we could 
also produce improved outcome when we conducted cryoprotective perfusion at lower subzero 
temperatures, which also increases viscosity of the solutions. Protocols that gradually decreased 
viscosity during cryoprotective perfusion with the aim of taking advantage of the vessel-clearing 
properties of higher viscosity solutions at the start of perfusion and improved equilibration of the vitrification solution towards the end of perfusion failed to improve upon protocols in which the 
viscosity was kept constant (for a given pressure) across all steps. 
Contrary to what one would expect from the vast literature on the no-reflow phenomenon, 
conducting cryoprotectant perfusion at high pressures (> 100 mmHg) in brains with 24 and 48 
hours of cold ischemia worsened the outcome. We speculate that these high pressures “push” 
more perfusate with low glass-forming properties into the interstitial space, limiting the 
equilibration of the higher concentrations of the vitrification solution during later stages of 
perfusion. As a matter of fact, many of our best results were obtained when we lowered the 
perfusion pressure below our standard arterial line pressure of 100 mmHg. We also observed 
improved perfusion and reduced ice formation when we eliminated one or two steps in our three-
step perfusion protocol. This finding may offer some important clues to the mechanisms that 
contribute to improved cryoprotectant perfusion in the ischemic brain. Since starting with such 
high initial concentrations of the cryoprotectant at the start of cryoprotective perfusion clearly 
contradicts basic cryobiology practice to minimize osmotic injury and consequent cell rupture, 
we have not explored this approach in much detail. 
So far, we have employed three distinct cooling methods. In our earliest cooling experiments we 
used liquid nitrogen plunging to cool samples to liquid nitrogen temperatures. To avoid 
fracturing, we later modified a small lab dewar to allow a more gradual descent of the 
temperature to -130°C (slightly below the glass transition temperature of VM-1). Currently we 
employ an ultra-low temperature electrical freezer that can cool samples to -130 degrees Celsius, 
which also permits us to store our samples for longer periods our time. Our findings concerning 
ice formation after cryoprotective perfusion of the ischemic brain have been identical for all 
three cooling methods. The distribution of ice formation generally follows the areas of perfusion 
impairment observed prior to cooling, which validated the investigations we conducted with 
India ink. We have not found any benefits for the addition of pharmacological agents to the 
carrier solution. Our best understanding about cold ischemia-induced cryoprotective perfusion 
impairment is that two major contributing factors are red cell aggregation (i.e., hyperviscosity) 
and edema. 
Organ Preservation Solutions 
Remote blood substitution in cryonics has a number of important (theoretical) arguments in favor 
of the practice. Replacing the blood with an organ preservation solution extends the period that 
organs can be received from static storage in clinical organ preservation. The procedure also 
permits a faster cooling rate in the field than is possible with external cooling alone. The 
mannitol-based perfusate MHP-2 that is currently used by the Alcor Life Extension Foundation 
has been developed in a series of experiments where dogs were recovered after 5 hours of 
asanguineous ultraprofound hypothermia. 

Cryobiology researcher Yuri Pichugin has questioned the value of remote blood substitution in 
cryonics because none of the organ preservation solutions that he tested (including MHP-2 and 
UW Solution) could maintain viability of hippocampal brain slices for periods that are typical of 
transport times in cryonics practice. Our own research, however, has been informed by the 
possibility that remote blood substitution may fall short in terms of preserving viability but could 
still confer benefits in terms of improving cryoprotective perfusion. 
We have compared controls (i.e., no blood substitution) against the following washout solutions: 
m-RPS-2, RPS-2 and MHP-2; and observed that blood substitution does confer significant 
benefits in terms of improving cryoprotective perfusion and reducing ice formation. In particular, 
MHP-2 outperformed the other solutions and has allowed us to conduct cryoprotective perfusion 
after 48 hours of cold bloodless ischemia with no ice formation in the brain after cooling below 
the glass transition temperature. Even at 72 hours, ice formation is relatively minor compared to 
72 hours of cold ischemia in which the blood is left in the brain, which produces severe perfusion 
impairment and ice formation. These experiments vindicate the practice of remote blood 
substitution in cryonics, but also emphasize that the composition of the organ preservation 
solution matters a great deal. 
None of the organ preservation solutions we have tested (including more advanced recent 
formulations from colleagues) mitigate the severe vasogenic edema that is observed during 
cryopreservation after prolonged periods of cold ischemia. We have designed a number of 
experiments to improve upon the formulation of MHP-2 but none of these variants has been 
successful so far in decreasing edema and frequently produced worse results than MHP-2 in 
reducing ice formation after bloodless cold ischemia. 
Cryopreservation after Chemical Fixation 
The idea to chemically fix the brain prior to cryopreservation has remained a topic of interest 
among cryonics advocates. As a matter of fact, this procedure was discussed in Eric Drexler’s 
classic treatment of molecular nanotechnology, Engines of Creation. One argument that could be 
offered in favor of this procedure is that it halts the development of ischemia in patients with 
long expected delays between pronouncement of legal death and cryopreservation. For a long 
time this idea has been met with skepticism because of (unpublished) experimental observations 
that such protocols risk producing intracellular freezing during cooling. Because the current 
generation of cryoprotectants is designed to eliminate ice formation altogether we revisited this 
topic and designed experiments to study the effects of cryopreservation after chemical fixation. 
When there is no ischemic delay prior to chemical fixation, chemical fixation still permits 
cryoprotective perfusion, and no ice formation in the brain was observed after cooling to liquid 
nitrogen temperatures after up to two weeks of hypothermic storage of the fixed brain in vivo. 
These experiments have been unique in that no whole body edema was observed during 
cryoprotective perfusion. We did, however, observe severe dehydration of the brain following cryoprotective perfusion of the fixed brain, a phenomenon we were not able to eliminate when 
we added an agent to open the blood brain barrier to our carrier solution. 
A practical limitation of cryopreservation after fixation is that delays between pronouncement of 
legal death and fixation could compromise the efficacy of this procedure and produce the kind of 
freezing damage that has traditionally been associated with this procedure. When we delayed 
chemical fixation by an hour, washout of the blood and fixation were incomplete and extensive 
ice formation followed cryoprotective perfusion. This phenomenon may be overcome by 
alteration of the fixative carrier solution and different perfusion protocols, but it is doubtful that 
such sophisticated protocols can be realized in most of the cases where the combination of 
chemical fixation and cryoprotection may be attractive. 
Electron Microscopy of the Ischemic Brain 
In collaboration with Dr. Michael Perry of the Alcor Life Extension Foundation we have 
prepared brain tissue samples for electron microscopy for time points up to 81 hours of 
normothermic ischemia. Since the rat brain cools at a much faster rate than the human brain after 
circulatory arrest, we decided that using an incubator to keep the in vivo brain at body 
temperature would be a better and more conservative approximation of what would be expected 
to occur in human brains. The electron micrographs have given us insight into the ultrastructural 
properties of the brain after various periods of warm ischemia. Dr. Perry is using these images to 
develop an algorithm that models the state of ischemic tissue after various periods of warm 
Dr. Perry has also supported investigations to examine the degree of fixation and long-term 
effects of delayed fixation of the brain. Preliminary results of these experiments indicate that 
even short delays between circulatory arrest and chemical fixation of the brain produce 
incomplete fixation and risk of progressive decomposition of poorly fixed areas over time. 
Whether such findings discredit chemical fixation as a low cost alternative to cryonics cannot be 
conclusively resolved by experimental research due to our incomplete understanding of the 
neuroanatomical basis of identity and the capabilities of future cell repair technologies. One 
might also argue that a straight freeze is preferable to chemical fixation but that chemical 
fixation is still preferable to complete decomposition. 
Implications for Cryonics Protocols 
To date, our investigations into cryopreservation of the ischemic brain strongly support the 
practice of standby and stabilization in cryonics. In particular, rapid induction of hypothermia 
after pronouncement of death and remote blood substitution with an organ preservation solution 
can limit the degree of perfusion impairment and ice formation after cryoprotective perfusion 
and cooling. We have identified some emerging principles for alteration of carrier solutions and 
cryoprotective perfusion protocols that can overcome no-reflow in the brain after cold ischemia 
and reduce ice formation. In patients with varying levels of ischemia, such protocols are still confined to the experimental stage until ultrastructural and viability assays have validated the use 
of these solutions and protocols. 
Our research suggests that chemical fixation of the brain prior to cryoprotective perfusion could 
be beneficial in case of prolonged (transport) delays, but adverse effects of ischemia limit the use 
of such protocols to a very narrow set of circumstances in which there is negligible delay 
between circulatory arrest and chemical fixation. 
Future Developments 
Future developments in our lab concern further refinements of perfusion and cooling protocols. 
In our more recent experiments we have been conducting cryoprotective perfusion using an open 
ramp system that gradually introduces the vitrification agent to the brain (as opposed to distinct 
steps of increasing concentration) combined with cooling to just below the glass transition 
temperature of the vitrification solution. We will keep upgrading our cryoprotective setup to 
make it conform to conventional perfusion equipment; ultimately, we hope to introduce 
computer controlled features. We also aim to alter our circuit to conduct cryoprotective perfusion 
at controlled high subzero temperatures. 
A major portion of our time and resources in the coming years will be devoted to developing a 
set of viability assays that can be used to screen the toxicity of improved vitrification solutions. 
Such assays will not be confined to in vitro brain slice work but will include whole brain in situ 
electrophysiology as well. 
We have also received financial support to develop a whole body resuscitation model, which will 
allow us to validate organ preservation solutions and vitrification solutions at hypothermic and 
high subzero temperatures. 
Our effort to simulate realistic cryonics conditions in our lab remains a work in progress. So far 
we have mostly limited ourselves to cryoprotective perfusion after either warm or cold ischemia, 
with a strong emphasis on cold ischemia. Recent observations in our lab indicate that there is a 
distinct pathophysiology associated with warm ischemia (and hyperthermia) that limits simplistic 
extrapolations between cold and warm ischemia using the Arrhenius Equation. 
In a more realistic cryonics model, variable periods of warm ischemia precede cold ischemia. In 
particular, we aim to investigate the efficacy of blood substitution when blood substitution is 
delayed; a scenario that is common in cryonics practice and that basically constitutes the rule for 
organizations that do not offer standby and stabilization services. 
Advanced Neural Biosciences, Inc., was incorporated in 2008 and conducts neural cryobiology 
research. We have received funding and equipment from the Immortalist Society, the Life 
Extension Foundation, the Cryonics Institute, and the Alcor Life Extension Foundation. We are 
extremely grateful to Alan Mole, Mark Plus, York Porter, Ben Best, Jordan Sparks, Luke 
Parrish, James Clement, and Dr. Peter Gouras for additional financial, logistical, and general 

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