Cryopreservation of the Brain by Vitrification

Perspectives and (Not So) Recent Advances

By Brian Wowk, Ph.D.

What’s old is new again; recently announced advances in brain tissue cryopreservation generate interest and excitement about old results for a new audience.

1960s counterculture personality Jerry Rubin once wrote that news media doesn’t report news, it creates news, because events don’t exist until they are broadcast.  With this in mind, and recent news media reports of frozen brain slices returning to metabolic function (Fudan University 2024), electrical activity in vitrified brain slices after rewarming (Cradle Healthcare 2024), and metabolic activity in whole brains hours after clinical death (Yale University 2019), it’s worth recounting the history of such findings.  While popular interest is new, raising the findings to mythical status (as Rubin would say), similar work has been in published scientific literature for decades.   It’s actually some of the foundational knowledge and technology that the practice of human cryopreservation (cryonics) is already based on.

Metabolism Recovery in Cryopreserved Brain Slices

In 2006, Pichugin, Fahy, and Morin published a paper showing excellent histological (cell and tissue structure) preservation and greater than 90% return of metabolic function in rat hippocampal brain slices after vitrification at a temperature of -130°C.  -130°C is below the glass transition temperature, which means that tissue can be stored for almost unlimited time at this or lower temperature.  

The Fudan University work covered by news media in 2024 preserved human brain tissue by cryoprotected freezing at -80°C instead of vitrification.  Unlike vitrification, freezing is an older cryopreservation method that involves ice crystal formation.  Freezing is most commonly used to preserve suspensions of isolated cells rather than tissue because crystals formed during freezing tend to disturb the organized structure of tissue.

The best results of the 2006 Pichugin, Fahy, Morin study were obtained using a vitrification solution called VM3.  VM3 is a close relative of M22 (used for rabbit kidney vitrification in 2009 and human cryopreservation since 2004), and of VMP (used for rat kidney vitrification in 2023), and also of VMPnoX (used by Cradle Healthcare in 2024).  In fact, VMP is VM3 minus one ingredient (polyvinylpyrrolidone), and VMPnoX is VMP minus one ingredient (X-1000 ice blocker).

There are no fewer than six distinct advances incorporated into this family of vitrification solutions developed 20 years ago by Dr. Gregory Fahy and his colleagues at 21st Century Medicine (21CM), including myself.  We therefore characterize VM3 and M22 as “6th generation” vitrification solutions.  The specific advances, including ice blockers, and especially the peculiarly low toxicity of the dimethyl sulfoxide/ethylene glycol/formamide (“DEF”) cryoprotectant combination, are discussed in more detail in my 2007 article, “How Cryoprotectants Work.”

That this family of solutions is still used by leading institutions studying large system cryopreservation in 2024 is a testament to both the height of the plateau in cryoprotectant mixture design space that the solutions rest upon, and perhaps also the small size of the field of cryobiology (comprising only a few hundred scientists, and a very small number of laboratories specifically studying organ cryopreservation).  While most of the identified principles built into these solutions will likely remain a foundation for future vitrification solutions, there is hope that high-throughput screening and better understanding of cryoprotectant toxicity might lead to future improvements in the majority “DEF” solution components that have been a mainstay since the turn of the century.  More powerful non-colligative cryoprotectants (“ice blockers”) to protect against extracellular ice formation and recrystallization, and even small molecule intracellular ice recrystallization inhibitors, are also possible.

As a historical note, support for the 2006 Pichugin, Fahy, Morin paper included essential funding from the Institute for Neural Cryobiology (INC), an organization started by a cryonicist, Dr. Thomas Donaldson, in 1993 to advance the science of brain cryopreservation.  INC continued operation under Paul Wakfer, whose grassroots fundraising made the study possible.  Dr. Yuri Pichugin subsequently became Director of Research at the Cryonics Institute (CI), where he invented CI’s VM-1 vitrification solution used for human cryopreservation.  (Coincidentally, VM-1 is a concentrated mixture of ethylene glycol and dimethyl sulfoxide, the two main ingredients of the MEDY freezing solution used in the Fudan University work.)  

Electrical Activity Recovery in Cryopreserved Brain Slices 

Post-cryopreservation electrical activity in neural tissue is very significant.  This is because nerve fibers aren’t passive electrical wires.  The propagation of electrical signals (action potentials) through nerve tissue requires ATP, the master energy molecule of the cell, to regenerate ion concentrations depleted by action potentials.  Stable capacity for electrical activity is therefore a step beyond restoration of metabolism because metabolism is necessary for it.  Structural integrity over macroscopic measurement distances is also required. The ability to perform electrophysiology experiments on brain tissue samples after cryopreservation also opens the possibility of studying more complex electrophysiological phenomena, such as LTP (long-term potentiation), which is related to mechanisms by which memories are encoded.    

In 2024, Cradle Health released a preprint (white paper) disclosing partial return of electrical activity in rat brain cerebellar slices after vitrification using VMPnoX, “the first report of recovery of electrical activity in acutely resected (and cryopreserved) mammalian brain tissue with accompanying protocols for validation and replication.”  This is an accurate claim subject to the parenthetical text I added, and subject to the text that I italicized.  However the later sentence, “This is, to our knowledge, the first demonstration of action potentials in cryopreserved and rewarmed acutely resected neural tissue,” did not reference important prior art.

In 2012, Fahy and collaborators published results showing return of full normal strength electrical activity (Fig. 2 of paper), and retention of ability to form an LTP response (Fig. 6 of paper), in hippocampal rabbit brain slices after vitrification using VM3.  These results were also presented to a cryonics audience at the 2011 Suspended Animation Conference, including high speed movies of field excitatory postsynaptic potentials (fEPSPs) propagating in two dimensions as revealed by voltage-sensitive dyes. This is very significant, not only because of the quality and depth of the results, including LTP findings, but because VM3 contains an ice blocker (X-1000) and colligative polymer cryoprotectant (polyvinylpyrrolidone) that were omitted from the VMPnoX solution used for the Cradle 2024 work.  VM3 is therefore much more stable against ice formation than VMPnoX.  VM3 has a critical warming rate (warming rate necessary to avoid ice formation) of only 3°C per minute.  It can therefore be used for larger tissue samples at slower cooling and warming rates than VMPnoX.

This may be why the Cradle 2024 experiments rewarmed cryopreserved tissue rapidly by induction heating of metal in contact with the tissue.  Special heating wasn’t necessary for the successful 2006 and 2012 brain tissue vitrification experiments using VM3.  However, since the 2012 electrical activity results by Fahy and his neuroscientist coworker, Dr. Yuansheng Tan, were disclosed as part of a larger paper about cryopreservation of precision-cut tissue slices, the full methodology of their 2012 experiments wasn’t published.  Therefore, to the best my knowledge, Cradle gets credit for the first disclosure of post-cryopreservation brain tissue electrophysiology in which detailed methodology was disclosed.  It’s not possible to credit individuals at this time because none were named in the Cradle white paper.  Future papers in scientific journals should remedy this, and also enable peer review.

As a further historical note, it was reported as far back as 1996  that while working under contract to CI at the Institute for Problems of Cryobiology and Cryomedicine (IPCC) in Ukraine, Dr. Pichugin and his colleagues obtained coordinated electrical activity in networks of neurons in neocortical rabbit brain tissue after rewarming from liquid nitrogen temperature using glycerol as a cryoprotectant.  This may qualify as the first public report in any forum of electrical activity in brain tissue after cooling to cryogenic temperatures.  Such experiments have understandably been of interest to the cryonics community for a long time.

Whole Brain Cryopreservation

Metabolic studies of tissue slices exposed to cryoprotectant solutions have been an invaluable screening mechanism for developing less toxic cryoprotectant mixtures for kidney vitrification since the early 1980s.  What kidney tissue slices “liked” has generally been liked by whole kidneys.

The brain is different story.  Unlike other organs, the brain doesn’t respond to cryoprotectants pumped through blood vessels (perfusion) the same way as tissue pieces soaked in cryoprotectants (diffusion).  What’s non-toxic to a brain tissue slice can still be toxic to a whole brain for reasons explained below.  (These reasons are unrelated to the heat transfer limitation of organs compared to tissue pieces, which is a separate problem.)  

For tissues and organs other than the brain, perfused cryoprotectants are able to leave blood vessels though capillary gap junctions (small holes in vessel walls) and then surround cells similar to the way that cryoprotectants can diffuse into tissue slices soaked in cryoprotectant solutions.  However blood vessels in the brain have no such holes.  Brain blood vessels are instead lined with a continuous layer of endothelial cells called the blood-brain barrier (BBB).  Therefore only the smallest molecules of a cryoprotectant solution can reach brain cells by first penetrating the cells of vessel walls and then slowly diffusing out the other side of the vessel walls.

Water molecules move through the BBB much faster than cryoprotectants.  This means that the initial response of a brain when perfused with cryoprotectants is to physically shrink as water leaves the brain and enters blood vessels faster than cryoprotectants can replace the water.  This happens because of something called osmosis, which can be described as water wanting to move from where there’s more water to less water.  This water movement will continue until “water activity” becomes equal on both sides of the BBB.

One of the measures of “water activity” is the freezing point of water in a solution.  This means that if a cryoprotectant mixture with a freezing point of, say, -55°C is perfused into a brain, water will leave the brain until the freezing point of brain tissue also becomes -55°C.  Interestingly, this means that brain tissue can be protected against ice formation even if no cryoprotectants penetrate the BBB at all.

The bad news is that if the freezing point of a brain is lowered to become equal to that of a vitrification solution only by water movement, the brain will be badly shrunken, and salts and proteins inside the brain will become highly concentrated.  While salts and proteins might seem like non-toxic cryoprotectants because they are natural, the opposite is actually true.  It was discovered early in the history of cryobiology that one of the mechanisms by which unnatural chemicals like dimethyl sulfoxide protect cells during freezing is by preventing elevation of salt concentration to toxic levels.

In practice, a brain with a normal intact BBB that’s perfused by a vitrification solution will end up protected against ice formation on the other side of the BBB by a combination of penetrating cryoprotectants that get through the BBB during tolerable perfusion time, plus abnormally high concentrations of salt and protein.  This stubborn elevation of salt and protein concentrations in whole brains doesn’t happen to the same extent in brain tissue pieces.  The translatability of brain tissue cryoprotectant toxicity studies to whole brains with an intact BBB is therefore uncertain.  Good brain tissue results, especially studies of penetrating cryoprotectant ingredients, are encouraging and suggestive of being on the right track because penetrating molecules will go through the BBB.  However they aren’t the end of the story for cryopreservation of whole brains with an intact BBB.

Why use a vitrification solution with any non-penetrating ingredients for cryopreserving a whole brain if none of those ingredients will leave brain blood vessels?  The answer is that preventing ice formation inside blood vessels, and especially in other tissue in contact with the brain, is important because ice that starts growing elsewhere can propagate into the brain.  Solution on the intravascular side of the BBB is also more likely to contain environmental proteins and other contaminants that trigger ice nucleation than the brain side of the BBB.  (This makes sense given that the BBB evolved for extra protection of the brain from blood-borne contaminants.)  Extra ice protection on the intravascular side of the BBB is therefore believed to be helpful.

What is the evidence that whole brains can be vitrified even though the cryoprotectant composition on the other side of the BBB won’t in general match the composition of the perfused solution, and whole brains will cool much slower than tissue pieces?  Whole brain vitrification using M22 was demonstrated by histological and ultrastructural study of rabbit brains cooled at simulated human brain cooling rates in 2004.  It was also shown to have been achieved in a human brain by post-cryopreservation CT scanning in 2012.  This CT scan also shows the remarkable extent to which physical shrinkage accompanies the cryoprotectant perfusion process when the BBB is intact.

Of course this is only morphological vitrification, which is the successful avoidance of physical ice crystal formation and damage during cooling.  Preservation of whole brain viability—the ability to resume whole brain function –after rewarming, unloading cryoprotectant, and reperfusion with warm oxygenated blood, is a much higher bar to cross. Dr. Isamu Suda was able to show electroencephalographic (EEG) recovery of a cat brain after freezing to -20°C using glycerol cryoprotectant in 1966, and even better results in 1974.  Such a demonstration after brain cryopreservation at cryogenic temperatures (arguably the neuroscience equivalent of a cryopreserved kidney producing urine) is still an unmet challenge, but foreseeable in light of progress in cryopreserving other organs so far this century.

Trivially, demonstrable successful reversible cryopreservation of the most complex and essential organ in the body is a necessary prerequisite for more ambitious cryopreservation goals, such as solid state suspended animation of animals or humans.  Even after demonstration for brains, real-time reversible cryopreservation of whole large animals is a vastly more difficult problem beyond the scope of this article.

Opening the Blood-brain Barrier (BBB) for Cryopreservation

The problem of cryopreserving a brain could be simplified by opening the BBB to create capillary gap junctions, thereby making the brain more like other organs in terms of being able to add and remove cryoprotectants.  As much as getting cryoprotectants into the brain is a problem, getting them out is an even bigger and more tedious problem because of the mathematics of osmosis and diffusion when the BBB remains intact.  Here again Yuri Pichugin’s name comes up in the history of brain cryopreservation research.  Dr. Pichugin recognized the importance of the BBB problem, and pioneered BBB-opening technology for cryopreservation during his time at the Cryonics Institute in the early 2000s.

 BBB opening was used by McIntyre and Fahy as part of their 2015 paper demonstrating aldehyde-stabilized cryopreservation (ASC), which is chemical fixation followed by vitrification.  That paper and associated data won the Small and Large Animal Brain Cryopreservation Prizes awarded by the Brain Preservation Foundation, a neuroscience prize for the first research group able to clearly demonstrate solid-state preservation of the connectome of a whole intact brain.  ASC achieved preservation of brain structure after vitrification that was indistinguishable from non-preserved brains when studied by electron microscopy at any magnification.

It should be noted that ASC is a structural preservation method that includes a chemical fixation step that ties up proteins with molecular crosslinks that leaves them inoperative.  This might be sufficient for the contemporary goal of cryonics, which is the preservation of sufficient identity-critical brain structure to permit future repair and restoration of the original cryopreserved person.  Such repair could conceivably include removal of the chemical crosslinks, and protein shape restoration.  (Eric Drexler’s 1986 book, Engines of Creation, contains a molecular nanotechnology “biostasis” revival narrative based on fixation and vitrification and the 2022 Robert Freitas book, Cryostasis Revival, contains a deeper description of such a process.)  But chemical fixation isn’t compatible with contemporary restoration of brain function by any simple means. 

BBB opening in the context of viable brain cryopreservation research is the next frontier of brain cryopreservation research because it puts the problem of brain cryopreservation on a more even footing with other organs, like the kidney, for which there has already been a great progress.  Brain tissue slice studies would also be more easily translatable to whole brains with an open BBB.  BBB opening has its own problems, which are beyond the scope of this article.  However it’s still an apparent “low hanging fruit” of brain cryopreservation development.

BBB opening has never been used clinically in cryonics.  There is a good reason for this.  Ischemia (stopped blood circulation) is empirically known to itself cause a time-dependent opening of the BBB.  Since clinical human cryopreservation always involves periods of warm and cold ischemia that are highly variable depending on case circumstances, it would seem difficult to correctly stack chemical BBB opening on top of BBB deterioration caused by ischemia.  This requires much more research by cryonics companies, or perhaps confining the technology to cases with minimum ischemic time.  Too much BBB disruption can lead to brain swelling and herniation during cryoprotectant perfusion.

Brain Function after Ischemia

Ischemia is somewhat off-topic from the subject of cryopreservation because ischemia is not intrinsic to cryopreservation.  However, ischemia is of great interest to cryonicists because of the idea that future technology might be able to repair ischemic brain injury.  This is believed to justify the practice of sometimes cryopreserving people long after cardiac arrest.  There was therefore excitement about the 2019 Yale University study resuscitating pig brains, even though the ischemic period wasn’t very long and much of it was cold ischemia.

Cryonicists should be aware that there is a substantial and long-standing body of literature showing recovery of various degrees of brain function, especially brain tissue function, after hours of warm ischemia (clinical death).  The oldest summary I’m aware of is in the “Death” section of A Brief Scientific Introduction to Cryonics by Thomas Donaldson in 1976, with additional references in his Prospects of a Cure for Death in 1990.  There’s also the “Post-Mortem Human and Animal Brains” section of The Cryobiological Case for Cryonics, and more recent function recovery references here. Structure persists for even longer, although there can be high variability.  The particular mixture of ingredients that Yale developed to resuscitate brains is a new advance, but the knowledge that there still is something to resuscitate hours after circulatory arrest is actually old.         

Toward a Distant Day

Incomplete knowledge of prior work notwithstanding, it’s exciting that new people and companies are taking an interest in the cryopreservation of brain tissue, large organs, and even whole animals (mammals?). By analogy to human space travel, those goals are similar in terms of community difficulty and timelines to the Moon, Mars, and Stars.  Demonstrable long-term recovery of a whole mammal from solid state cryopreservation at cryogenic temperature is not unlike star travel: It’s something conceivable in theoretical terms, but with problems so vast as to likely require solutions from a future century.  Yet, without people who dare to dream of things that are practically impossible, the impossible would never become practical.

In the meantime, I hope that newcomers to cryobiology motivated by medical suspended animation take time to study and leverage the 70 years of accumulated hard-won knowledge and publications of the cryobiology community.  I also hope that cryobiologists, who sometimes must labor mightily for many years to successfully cryopreserve even simple collections of cells, can be patient with those inspired by more audacious goals.  The challenges will manifest soon enough.