Landmark Brain Preservation Research - Dr. Greg Fahy on Compelling New Evidence

In this episode, Daniel Walters sits down with Dr. Greg Fahy, past president of the Society for Cryobiology and one of the most respected figures in the field, to discuss landmark new research on brain preservation.

The paper, Ultrastructural and Histological Cryopreservation of Mammalian Brains by Vitrification, provides some of the first direct evidence that mammalian brains can be vitrified with preservation of their ultrastructure. It's the result of decades of work, an extraordinary collaboration with Alcor, and the final wish of a scientist who chose to make his own brain part of the study.

In this conversation:

• How Dr. Fahy came to cryobiology and his path through organ preservation research
• The science behind vitrification and what makes M22 a sixth-generation cryoprotectant
• Why Stephen Coles, a leading biogerontologist, wanted his brain to be part of the study
• A daring experiment that could have produced some very unfavorable results
• What the research revealed about ultrastructure, shrinkage, and cracking during cryopreservation
• What this means for the cryopreservation vs chemo-fixation debate
• A look at what's next, including additional human cases, blood-brain barrier research, and an open question Dr. Fahy and his team are exploring. 

Join us for this in-depth discussion of this landmark research. 

Click here to view the full paper.

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Transcript

Daniel Walters: Okay, welcome to another episode of the Alcor Podcast. I'm your host, Daniel Walters, and today we have a very special guest and a conversation I've been looking forward to for quite a while.

If you've been following the cryobiology and brain preservation community at all recently, you've probably heard some of the buzz around this new research that came out earlier this year. It's titled Ultrastructural and Histological Cryopreservation of Mammalian Brains by Vitrification — a bit of a mouthful — and it comes from a team at 21st Century Medicine in participation with Alcor. It has generated a lot of attention.

The basic question at the heart of this research is: can you preserve mammalian brain tissue at cryogenic temperatures using vitrification, rewarm it, and find that the fundamental architecture is still intact? That question has been hanging over the field for a long time, and this new research takes it on directly.

There's also a pretty remarkable human story behind this work, involving a scientist listed as one of the co-authors who knew that his own brain tissue would become part of the study. And Alcor played a real role here too, helping fund an experiment that could have gone either way, with no guarantee of good results. We'll get to all of that.

Our guest today is Dr. Greg Fahy, lead author on this new research. He is the past president of the Society for Cryobiology — the field's leading professional organization — and one of the most respected figures in cryobiology. He's also the chief scientific officer at 21st Century Medicine, where he and his team have spent years doing work that very few other labs in the world are even equipped to do.

So without further ado, I'd like to welcome Dr. Greg Fahy to the Alcor Podcast. Dr. Fahy, thank you for joining us.

Dr. Greg Fahy: Thank you, Daniel. It's a pleasure to be with you today.

Daniel: You've been in this field for a long time. How did you first get drawn into cryobiology, and what made you stick with it for so long?

Greg: Well, it's a long story, but one of the reasons I got into cryobiology is I thought it was really important to make a lot of advances in the field, especially in organ cryobiology. At the time I had to make a decision about my career path, there was virtually no activity in that area. My other love in science is gerontology research, and I really wanted to become a biogerontologist, but I figured I would make more of an impact in cryobiology because it was an understudied, neglected field.

I became aware of the shortage of organs for transplantation when I was in high school. I read an article — probably in Time magazine or one of the other news magazines — that talked about the fact that if you needed an organ to save your life, whether you lived or died would be decided by a committee that would sit around and decide whether you got that organ or somebody else. I thought, what a horrible situation it would be to be in. So we need to do something to improve that. Organ banking is one of the things that can help alleviate the organ shortage and also make organ transplantation much more effective — there's all kinds of reasons for that. But essentially that was my orientation going into my career choice, which was graduate school at the Medical College of Georgia.

There was one organ banker in the United States at the time, and that was Armand Karow. He was actually a pharmacologist. One problem of cryobiology is that there are no institutions of cryobiology to speak of. That's beginning to change now, but for most of the history of the field, in order to be a cryobiologist, you had to be something else primarily. So Armand Karow was a pharmacologist, and I got my PhD in pharmacology, but I really was not that interested in pharmacology — I was really more interested in cryobiology.

I got into it with the idea that if we could preserve an organ at cryogenic temperatures, we could help a lot of people and we could also open up a lot of minds to broader possibilities. So I thought it would be a strategic thing to do.

Daniel: Could you tell us a little bit about 21st Century Medicine? How did you get involved there, what's the mission, and what makes you excited to go to work every day?

Greg: Yeah, so 21st Century Medicine is really a very unique institution. It's one of the very few places where you can be a cryobiologist full-time. We have a pretty limited capacity to accept new investigators into our company because we're a small company, but we do have a number of really top-notch people working in our group, and we focus on the problems that are too hard for other people to pay attention to — at least until recently. That turns out to be organ banking.

We really specialize in developing technologies that can cryopreserve organs. Most cryobiologists may be dealing with simple cells or simple tissues, but we want entire vascularized organs because we think that's one of the grand challenges of cryobiology. The reason we are able to do this is that we have support by visionaries who understand our goal and the importance of our goal, and the fact that the goal is very, very, very hard to achieve. We've been at this for a long, long time, as you pointed out in the introduction. That's what it takes in order to make this work, because there's so many different problems you have to unravel in order to get to the final solution.

We're very excited because we're making great progress these days, moving toward what we think will be reversible organ cryopreservation in the fairly near future. Unfortunately, I've had that feeling many times in the past, and those expectations have been dashed, but we're really, I think, narrowing it down now. We can talk more about that in detail, but we've got a new cryoprotective agent, we have new perfusion techniques, and we have a lot of results — including vitrified kidneys that do support the life of the recipient when they go into the original donor. In one case we had a rabbit maintain excellent clinical health for almost two years before we had to take the kidney out to have a look at it.

This is great progress. It's reality, it's not just speculation. We hold the world record for the largest organs that have been banked successfully so far with life support afterwards. We've inspired a lot of research elsewhere, particularly nowadays at the University of Minnesota and satellite labs associated with that. But we were the pioneers, and in my opinion, we're still in the lead. So it is a very exciting place and time for us, and we're working very, very hard to try to deliver the final product — demonstrations that what seemed impossible not too many years ago is actually possible and achievable in the near future.

Daniel: You mentioned the rabbit kidneys. One of the landmark moments in the field was when you showed that a whole rabbit kidney could be vitrified, rewarmed, and retransplanted and support life. I'm sure there have been many interim steps, but that really felt like the last big splash before this new research. I know you could do two whole other podcasts just on that, but could you briefly tell us that story and what it meant to the field at the time?

Greg: Yeah, so I will admit that the first organ we banked successfully was done in 2002. At the time, we had a surgeon who was pretty adventurous. Near the end of the year, it's hard to get anything done because you have Christmas and the holiday season coming along, people go on vacations and so forth. So we decided to do a moonshot just to see what would happen. We didn't really expect it to work, but by George, we put this kidney down, we brought it back, put it in the rabbit, and it actually survived.

My surgeon had to come in over the Christmas holidays, but it was well worth it. It took us a long time to publish that — I actually delayed publishing for seven years because we knew we had problems with ice formation in that kidney, and I figured after a fairly short time we would be able to overcome that and then publish a really good study showing a complete success without the ice. But unfortunately, certain irritating things happened in the background. A couple of the chemicals we needed for those experiments were taken off the market, and we went through a lot of experiments trying to find substitutes in vain, so we had to re-engineer the entire system from the ground up. Then our surgeon left and had to be replaced with another surgeon, and that took years for him to learn it. Eventually we got an opportunity to publish that study as kind of a review article, and so we did that, and the world found out about it in 2009.

You are right — that was, at the time, the greatest achievement in the field of cryobiology ever. It's been rivaled since. The University of Minnesota published a study not long ago showing that they could vitrify rat kidneys successfully — five out of five rat kidneys successfully. That was the next major advance. But then we published in 2025 being able to vitrify a rabbit kidney once again, but this time without clinical signs of ice damage. That was yet another leapfrog forward, and I still think we hold the lead at this moment. The progress continues — we're moving on to new directions now, and I think the prospects are just getting better all the time.

Daniel: Before we jump directly into this research, just because we're going to use some words that may not be familiar to everybody — for the listeners who are new to this, quickly: what is vitrification exactly, and why was it such a breakthrough compared to traditional freezing?

Greg: Right. So it turns out that when I was in graduate school, I wanted to understand the cryobiology of organs, and so I did a lot of work on the fundamental aspects of organ banking. At the time, there was only one way to preserve anything at cryogenic temperatures, and that was by freezing it. But I did some studies with the electron microscope, taking kidney tissue and freezing it in the normal way, but having a look at it in the frozen state to see what it actually looked like.

At first I thought I must have done the experiment wrong or something, because I couldn't make any sense out of what I was seeing. Nothing looked familiar. After a while I learned to understand what I was looking at. The fields were largely replaced with ice — very large cavities — and the tissue was very distorted. You could see cells being ripped apart from other cells, you could see the vascular system of the kidney being damaged.

Sure enough, a few years later, when we modified freezing techniques to make them more gentle according to cryobiological theory, and we found that we had cellular viability in frozen rabbit kidneys and then in frozen dog kidneys, we were pretty excited that maybe we'd overcome the problems of freezing. But it turned out that when you transplanted, say, a frozen dog kidney into a dog, after a few minutes of a pink kidney, the kidney would then start turning dark and blue and start urinating what looked like whole blood. So we knew that this distortion I'd seen in graduate school — of cells being ripped apart — was not an easy thing to overcome.

So I turned to supercooling. It's possible to take a solution and cool it below the theoretical freezing point without having it freeze — that's called supercooling. I thought, well, maybe I can supercool organs at dry ice temperature. It turned out that could be done for maybe a day or so, but not for much longer than that. Then it dawned on me that if I continued to cool this in the supercooled state below the freezing point, but without any ice, maybe I could cool all the way down to a low enough temperature that the system would lock up.

As you approach absolute zero, of course, you're subtracting energy from all of the molecules in the solution, and molecules need energy to move around. If they don't move, there's no change, and so there's no deterioration of a biological system over time. Eventually you reach a point called the glass transition, in which you don't have enough thermal energy to drive molecular motions, but you still don't have a frozen state. The water molecules are not reorganized into an ice crystal, and so when the system locks up in this non-frozen state, you say that it becomes a glass or a vitreous state. That's called vitrification.

I got the idea of trying to vitrify organs in 1980. I published an abstract on it in 1981, and the rest is history, as they say. I introduced the idea in a significant way in '83, and then we actually tried applying this to embryos. We were actually able to vitrify mouse embryos successfully, and that captured a lot of attention at the time, and has since really helped reproductive biology and reproductive medicine. But it was a stepping stone along the way to showing that you could vitrify a living system and have it survive afterwards — and that was the theoretical way of banking an organ at cryogenic temperatures without ice damage. So that's what vitrification was all about.

Daniel: Your team at 21st Century Medicine developed a vitrification solution called M22, which is part of this new research. What's the story behind M22? What makes it different from simpler alternatives?

Greg: So we tend to call M22 a sixth-generation vitrification solution. A vitrification solution is a cryoprotectant water solution that you can expose a cell to and allow it to escape ice formation on cooling — in other words, it allows the solution and the organ or cell to vitrify.

A long, long time ago, Basile J. Luyet — who was a priest and one of the early figureheads in the history of cryobiology — not having any other ideas about how to preserve things, decided that maybe if you could cool something, a living system, sufficiently quickly, you could outrun ice formation. You could cool it so quickly that water would not have time to reorganize into ice, and then you could rewarm it so fast that you'd still remain in the amorphous, non-crystalline state, and that therefore you could survive. He tried that for a long time, and along the way he dabbled with using certain chemicals we now know of as cryoprotective agents to dehydrate the tissue. If you expose a piece of chick brain or a piece of chick heart to ethylene glycol, it will suck the water out of that tissue osmotically. That means that it's easier to vitrify because you don't have as much water to freeze, so you can outrun ice formation more easily. But he didn't really use that as a cryoprotective agent at the time.

That might be considered, in a sense, a first-generation vitrification solution — a single cryoprotective agent that allows a system to vitrify, at least in theory. When I published on vitrification, I was already a couple of generations beyond that. Molecules, of course, vary in size — some molecules are small and can penetrate cells, they go right through the cell membrane, and others are too big for that and they stay outside of the cell. I reasoned that the protein inside of a cell would assist the cytoplasm — the contents of the cell — in vitrifying, because that replaces some water. So, what if you put something outside of the cell that sort of had the same function as the protein inside the cell? That means you don't have to use as much of the agent that goes inside the cell to vitrify the inside of the cell.

So we had polymer, we had toxicity neutralization, and we had cryoprotection. Toxicity neutralization is an interesting phenomenon. There are certain chemicals that if you add them to cells, they are toxic — but if you add them to cells in the same concentrations in the presence of other chemicals, the other chemicals reverse or prevent the toxicity of the first chemical. The classic example is a chemical called formamide. Formamide is really interesting because it's a really, really small molecule. It goes into cells very quickly, but it's pretty toxic. But if I put a toxic level of formamide in contact with kidney tissue, and then expose the kidney tissue to the same concentration of formamide but in the presence of DMSO, I don't see the same toxicity. Very severe toxicity disappears when the same level is exposed in the presence of dimethyl sulfoxide. The beauty of that is it allows the total solution concentration to get very high without toxicity. That is what you want to do if you want to prevent ice from forming — you have to use a lot of cryoprotective agent and replace a lot of water. And how do you do that without killing the cell? Well, you use toxicity neutralization.

So in the early days, we had this principle of toxicity neutralization and extracellular agents, and also another principle, which I call mutual dilution. That means if you have cryoprotectant A and it's toxic, and cryoprotectant B and it's toxic, but you combine them in such a way that you have half the concentration of A and half the concentration of B, then A is not toxic anymore because it's not at a toxic concentration, and B is not toxic for the same reason. But now the total solution concentration is high, and so you can still survive and maybe vitrify.

So that was the early days. Generously, I might call that kind of concoction, relying on those three principles, a first-generation vitrification solution. M22 is a sixth-generation vitrification solution — we've added a lot of technology on top of that.

One of them is methoxylated cryoprotective agents, which was discovered by 21st Century Medicine before I joined the company. Without getting into too many technical details, methoxylation kind of beats some of the rules of toxicity that allow you to escape from some of the drawbacks of agents that tie up water molecules. Cryoprotective agent toxicity has to do with the availability of water to hydrate biological molecules. Methoxylated glycerol is pretty good at vitrifying, and yet it spares living cells from toxic effects that would otherwise be caused by disturbing the relationship of water with the molecules in the cell. Without getting too technical, that would be one of the next generations.

Another generation is ice blockers. Normal cryoprotective agents don't interact with water specifically — they just dilute it basically, and they hydrogen bond with it, so they limit its ability to move around and reorganize into ice. But I figured in the 1990s that we needed something more specific than that, something that would recognize the surface of ice, adhere to it, and stop it from growing, just like antifreeze proteins do in polar fish. So we came up with the first ice blocker, which we called X-1000, and we also came up with a second ice blocker called polyglycerol, or Z-1000. We have two ice blockers in the solution, and that's yet another principle, because these ice blockers inhibit ice formation in two different ways, and they're synergistic when they're present in the solution together. That allows you to lower the total concentration of permeating cryoprotective agents and therefore control the toxicity of the solution. You don't need as much brute-force cryoprotection if you can have finesse cryoprotection by attacking the ice directly with these ice blockers. That would be the fourth generation.

The fifth generation is enhanced mutual dilution. The more cryoprotectants in the mixture, the less you can have of each one, and so the less opportunity there is for specific chemical effects of that cryoprotectant. We have N-methylformamide in our solution as a way of diluting all of the other cryoprotectants in the solution. And miraculously, that works very well. It permeates pretty well as well. That was a great choice for making M22.

The last feature I would call your attention to is chilling injury protection. It's not necessarily enough to prevent ice formation if you want to vitrify a complex system. Sometimes, if you cool a cell from a high temperature to a lower temperature, it's injured just by the act of cooling. Even if ice does not form, there's something about being colder that's a problem. We discovered this phenomenon in which, by adjusting the non-penetrating components of the cryoprotectant mix and the carrier solution that all the cryoprotectants are dissolved in, we could actually control chilling injury. That's built into M22 as well.

It's a very sophisticated solution. It was developed in the early 2000s, around 2002 or so, and I've been working to improve upon it ever since. We're now 24 years later, and I have found solutions that are less toxic, but they're less perfusible — and that doesn't work. I've found solutions that are more perfusible but more toxic — and that doesn't work. You have to have perfusibility, lack of toxicity, and high vitrification tendency. To get all of those things together in one solution at one time is very hard. I think M22 is still the world leader in that department, although we're now working on finally maybe an improvement this year.

Daniel: Nice. And I believe Alcor adopted M22 for its preservation procedures shortly after in 2005, and still uses it today. It's more expensive than the other solutions, but for Alcor's purposes, that's a trade-off they are absolutely willing to make for the best level of preservation possible.

There was previous work on preserving brain structure in regards to fixation, and I want to mention it because that might be more familiar in terms of traditional practices. But the issue was — and tell me if I'm oversimplifying it — essentially fixation eliminated viability, while vitrification offered viability through the potential of reversal, but hadn't yet had the kind of detailed imaging to fully prove that structural preservation was there. Is that essentially the right way to think about it?

Greg: Yeah, pretty much. There's really two issues: structure and viability. I think they're both quite important, for somewhat different reasons. Structure is sort of a prerequisite for everything else. If you don't preserve the structure, there's no hope of preserving viability. But structural preservation alone may not be sufficient, because you may induce other changes that are hard to deal with later on.

The paper you're referring to was published by myself and Robert McIntyre — who's now Aurelia Song — at 21st Century Medicine. I'd proposed this idea before I even knew of his existence, at a previous meeting in which we were competing for the Brain Preservation Prize. I realized that I could cheat to win the prize by perfuse-fixing the brain before we introduced the cryoprotective agent. Robert came to our lab and we perfected that and we published it with very nice results in Cryobiology. But it was a cheat because, of course, if you perfuse-fix something, then of course you can vitrify it — it's obvious. That's the easy way out. There's no challenge associated with that, really. There are still some osmotic issues in a fixed brain, but you can deal with those sorts of things.

One issue with fixation and then preservation is that the end point of that can be very useful if you are a neurobiologist and you want to understand the anatomy of the brain, because there are obstacles in the way of getting good quality brain material for study in certain cases. If you want to study brain aging, the National Institute of Aging and Jackson Labs can sell you an old animal, but what if the animal dies of old age before they sell it to you? It may be hard to guarantee that you have the material you need when you want it. If you could preserve the brains of old animals rather than letting them die, you could get something out of that. Also, old humans or humans with rare diseases — human brain banks now freeze the brain, and that destroys the neuroanatomy. It will preserve proteins and nucleic acids, but destroys the anatomy.

So the ASC method — Aldehyde-Stabilized Cryopreservation — is a way of preserving the neuroanatomy, but it completely, totally destroys the function. You can detect certain proteins and you can detect certain nucleic acids, but you can't actually get any functional data. It would be much better to have a viable system that was cryopreserved, so you could study that.

One of the issues with the neurobiology of aldehyde-fixed brains is memory. What is memory and where is memory stored? We all understand that memory is related to synapses and the weight of synapses in the brain. If you look at a brain in the electron microscope, you can kind of see those synapses, unless the plane of the section misses them, which is going to happen quite frequently. If you're doing serial sections of a brain, you'll see some of the anatomy. You will not see all of the anatomy, and maybe some of what you miss is important. Maybe you can see how much weight was given to a given synapse by its density, and maybe you can't.

But one of the things you're not going to see is things like Protein Kinase C populations in the axonal membrane. When I was studying neurobiology, I learned some neurobiology from Daniel Alkon, who in my view is one of the deans of memory research. He discovered that memory is encoded — both in sea snails and in rabbits — by the movement of a protein from the cytosol onto the cell membrane in the axons that convey messages from one cell to the other. That movement of Protein Kinase C to the cell membrane permanently changes the electrical properties of the membrane, so that when you stimulate it, you get a different response than you had before. It's not just synaptic changes, it's also presynaptic changes — and you will never see that in serial sections with the electron microscope. You would have to ablate the tissue molecule by molecule to build up a database showing the locations of all of the molecules in all of the axons in the entire brain, and that starts getting to be undoable just from a data processing point of view at some point.

So, wouldn't it be a lot better if you could preserve the Protein Kinase C distribution in the brain in the vitrified state? Then maybe you could actually learn something about memory by actually interrogating the system and seeing if it remembers things or not. That sort of thing is becoming possible. You saw probably the news reports that came out from Alexander German and his group showing that you could take a mouse brain and vitrify it and warm it back up again and recover some function of even the hippocampus in some cases in those mouse brains. We know that this sort of thing is possible — it's a matter of refinement. Why would you settle for fixation if you can have viability and not just fixation? It doesn't make a lot of sense. I'm firmly going after the reversibility side of things.

One other point I want to make, which I didn't make before when we were talking about rabbit kidneys versus mouse kidneys or rat kidneys, is that I deliberately chose big organs — rabbit organs — because they're hard. I could have done rat kidneys 20 years ago. We could have done mouse brains 10, 20 years ago. We didn't want to do that because we didn't want to get a misleading result. There's a lot of publicity that's come out of these small organ studies, like the rat kidney at the University of Minnesota and the mouse brain in Europe, but that's only possible because they can take a lot of shortcuts that don't apply to large brains. We insist on doing it the hard way, looking at large organs. And — as one of the things we will discuss later about the paper — we discovered that these more difficult, more rigorous methods of preserving larger organs can be translated all the way up to a human organ scale.

Daniel: Now that we have a good amount of background and setup, I want to segue into the research itself. In your own words, what was the key question you were trying to answer, and why had it gone unanswered for so long?

Greg: It's an interesting question. We were kind of moved into this area of brain preservation research from two points of view. For the first one, we had done the studies on Aldehyde-Stabilized Cryopreservation, as you know, as we had discussed before, and we wanted to know if you could go to the next level — be able to get structural preservation without the aldehyde step. That was one dimension, but there was actually another one.

I was actually invited by John Baust, a senior cryobiologist who was the editor of a cryobiology journal, to write a review about the science behind cryonics. I agreed to write it, but then I realized I couldn't do it because the data — we didn't have any information, right? What happens to the brain, nobody knows. What you know is the cryotransport phase. In other words, in a cryonics scenario — it's not a laboratory situation, but you have pristine brains to work with. You have a downtime between preservation and the availability of the brain. There's no information about whether that gap completely prevented the brain from being preserved or not. So it seemed like it would be a good question to answer from a scientific point of view, because there's just a vacuum in the literature about the whole fundamental basis of this practice of preserving people.

And then the third dimension, actually, is that I had a colleague and friend, Stephen Coles, who developed pancreatic cancer and had a desire to have his brain cryopreserved. That provided an opportunity to learn something about brain cryopreservation through that pathway too. There's really three strands that sort of converged on this research. The good news is that those research strands were in agreement with each other and gave each other mutual validation.

Daniel: Now, you mentioned Dr. Coles — I believe he passed away in 2014, if I remember correctly. He was one of the leading biogerontologists at UCLA, very, very well known in the field, and I believe one of the world's leading experts on supercentenarians.

Greg: Yeah, Dr. Coles ran a discussion group called the LA-GRG — the Los Angeles Gerontology Research Group. It was a forum for discussion about aging and aging intervention. He had branches elsewhere — if he traveled to Washington, DC, he interviewed me for one of the episodes of that discussion in DC. Basically, he was interested in supercentenarians, and he arranged for autopsies of supercentenarians. He was instrumental in discovering that supercentenarians tend to die of conditions that don't affect most of the rest of us, and they don't tend to die of the things that kill us. So he made some pretty good contributions to our understanding of aging. But unfortunately, he was late to the game about brain preservation, because he was afraid that brain cracking, which he had heard about, would make that whole exercise pointless. He didn't really pay too much attention to the whole issue until he developed pancreatic cancer and decided that it might be an opportunity to answer some of those questions.

Daniel: He was ultimately preserved with Alcor. But from what I understand, he wanted to be listed as a co-author on this research, and from a previous talk, you said you promised him that would happen. So here we are, over a decade later, and his name is listed as a co-author. I imagine that has to carry some emotional weight for you.

Greg: Well, it's really gratifying in a way, because I was able to grant one of his last wishes in that regard. He also wished for Steven Harris to be a co-author on the paper, because Steve was his personal physician and was helpful in arranging for what happened to Dr. Coles. So Steven is on there as well.

I had great sympathy for Dr. Coles. He did not have arrangements to have his brain preserved, and it was necessary to work out an agreement with Alcor in which he would be Alcor's basically first research case. Alcor had a research budget that it was using for other purposes, and Dr. Coles had no budget to have himself cryopreserved. So it occurred to me that maybe if you put those two things together, he becomes a very, very, very valuable research project of Alcor. He can get cryopreserved and have the benefits of that, and also answer a lot of basic scientific questions that Alcor would want to have answered, and that everyone interested in brain preservation would like to have answered.

By some minor miracle, it was all agreed to and it happened smoothly — and it happened in no small part because Dr. Coles was very cognizant of medicine and biology and was able to arrange things so that the experiment could take place under good conditions.

Daniel: I'd like to toot Alcor's horn a little bit here, because I think they deserve it. You had said publicly in the past — and I quote — that Alcor was "very, very brave" to enable this experiment. As you mentioned a little bit earlier, it could have led to terrible results and it could have been embarrassing, but it had to be done. And you thought it was the best research money Alcor had ever spent. Do you stand by that assertion — that it's the best research money they've spent so far?

Greg: Yeah, I would stand by that. I do want to salute Alcor, because this is the ultimate proof of sincerity and lack of subterfuge or fraud or whatever like that — to be willing to put your life on the line, essentially, to put your product under the microscope, literally, and allow the world to see whether your procedure, quote unquote, works or doesn't work. It's absolutely necessary if you are committed to coming up with a procedure that has value.

If you are a complete, fraudulent outfit that didn't even believe in what you're doing, you wouldn't want such scrutiny, because what if it was embarrassing? It could have easily turned out to be a disaster. Nobody had any idea what we were going to see. I was actually pretty surprised that the preservation was as good as it was. I was surprised by other things that came out too — such as the fact that Dr. Coles' brain was so saturated with the M22 solution that we were not able to freeze it in our laboratory when we got the biopsies back from it. There were many good features that came out of this, and it was very brave and honest of Alcor to enable this to happen. I think it speaks volumes about the integrity of the organization.

Daniel: And I believe, just to give kudos to the other people from Alcor who are listed on this research as well — Hugh Hixon, Steve Graber, and as you mentioned, the late Steven Harris before he passed in 2023.

Greg: Yes. I will just say along those lines that this was really a heroic effort on Alcor's part. Nobody had ever done anything like this before. I came up with the protocol for this, and it was very unfamiliar. At the time that this was done, the surgeon that normally assists Alcor in cases like this was not available. They had a new surgeon who was not familiar with this at all, and he had to learn on the fly. Nobody quite knew what to do with the brain samples, and so I was in communication with people up to the last minute. Of course, time was short in everybody's direction, including mine, but we were just barely able to pull it off.

I have to salute the Alcor team. They were flexible, they were cooperative, they wanted this to work. They did everything necessary, and yeah, it was not easy for anybody — but everybody stretched and went the extra mile, and we got this accomplished. The results speak for themselves. It was very much worth it. It was a little bit traumatic at the time.

Daniel: There's one more aspect before we jump into the results. In terms of brain shrinkage — is that one of the core questions this research actually looks at? To frame it for a layperson: when you preserve with M22, from what I understand, the brain can shrink quite a bit, and it was unclear if that shrinkage actually created irreversible damage, or whether you could rehydrate it and the structure would still be intact. Could you explain a little bit of that? The shrinkage aspect is something I hear quite often when people talk about this.

Greg: Yeah, you've put your finger right on it. There had been some previous studies of brain structure. There was a study that was published by Lemler et al. in Annals of the New York Academy of Sciences showing some evidence for brain structural preservation in the presence of M22. But that was at a pretty low level of magnification, and neuroanatomists were not satisfied that the finer structures on a very high magnification level were still preserved.

Unfortunately, since neurobiologists are not cryobiologists, they're not used to looking at dehydrated tissue. If you take a cable that's this diameter and you shrink it down to something that looks like this, what do you make of that? The neurobiologists can't tell what that means. Now, a cryobiologist is not bothered by that, because we see cells shrinking in frozen states all the time, and we know that those cells can come back and they're perfectly fine. But a neurobiologist is not so sanguine about it. Plus, if you have a neuron over here and a neuron over there and they're connected with a cable, and now you stretch that cable by shrinking them, are you going to rip that cable apart? There's all kinds of long-range interactions that you might be concerned about. So it was sort of unknown.

We actually did some studies on brain anatomy a long time ago, and it looked intact to me as a cryobiologist — but A, that's rabbit brains under good conditions, it's not human brains under less-than-perfect conditions, and it was a limited study. The question is: what can you do to look at this question in more detail? What can you do to generate data that would be more satisfying to a neurobiologist?

There's two ways of looking at that. As you pointed out, one is just to rehydrate the system and see if the structure becomes more recognizable. We did a lot of experiments along those lines with rabbit brains, and we did a little bit of that even in Dr. Coles' brain. But the other way to look at it is just to blow up the scale even more. Okay, everything is shrunken — well, just use higher magnification and look at it. Are the cell membranes there? Are the organelles there? Do you see any ripped axons or anything like that? That was the central question.

There was another question too, and that was Dr. Coles' concern about cracking. If you cool an organ into liquid nitrogen, it has a great tendency to crack. We have found in our lab — and I found back in 1980 — that you can cool an organ into liquid nitrogen without it cracking, but you have to cool it very, very, very slowly. And once it's in that low temperature, it's never going to relax into a safe state. If you tap it, it can crack, because the tension cannot be relieved in the vitrified state. In any case, cracking had been observed in patients before, and Dr. Coles was concerned about that.

At our lab, we're aware of this as an issue for organ banking. So we developed this technology called intermediate temperature storage, or CIVS — Controlled Isothermal Vapor Storage. We had the ability of limiting cooling to a temperature that's cold enough to allow preservation for unlimited periods of time, but not cold enough to cause cracking. Alcor, to its credit, actually has one of those units in its inventory. Dr. Coles was given the opportunity to determine — using his own brain — whether cracking could be avoided by using these intermediate temperature storage conditions. But for that, his brain had to be removed so it could be observed, because you can't see cracks by scanning technologies that we have today. You have to look at them visually, and if cracks are going to happen, our experience with large systems is that they show up on the surface of an organ. So by looking at the surface of Dr. Coles' brain, we could tell if it cracked or not. That was the other objective of the research — to find out if we could avoid the cracking.

For that, Hugh Hixon, one of the authors on the paper, was instrumental, because he had to devise a system for cooling Dr. Coles' brain under very controlled conditions to –140°C, which was the storage temperature, so that we would not artificially crack it. That way we could see the full potential of avoiding cracks. That was one of the other elements of the study.

Daniel: Okay, we've been teasing it for a while, but now let's just jump into the results. After all this, what did the evidence actually show?

Greg: Let's start with the cracking aspect first. So — Hugh actually screwed up. Sorry about that, Hugh. There had been some evidence that maybe you could cool a brain to maybe –130 or –138, but not to –140. So he was supposed to cool very slowly to –140 and then stop. But somehow the system got neglected and not watched carefully, and so the slow cooling process continued to –146, which raises really great risks of cracking — that extra six degrees.

When this was pointed out, Hugh very, very slowly rewarmed Dr. Coles' brain back to –140 and then transferred it into intermediate temperature storage. In the process of making that transfer, the brain was paused in the vapor phase of the storage unit so it could be photographed. The great news was that even though it went to –146 — even colder than the –140 we think is necessary for very, very long-term preservation — the Alcor team was not able to detect any cracks on the surface of the brain, which is hallelujah. That's a really great discovery.

Unfortunately, tragically, the photographs that were taken were lost by computer failure. We can't prove that at this point. We just have the assertions that were made at the time, and I don't think the photos were copied to anybody, so we just don't have them anymore. But still, at face value, that was a great triumph and a great revelation.

Now, it's unclear if the same result would happen if the brain had not been removed from the skull. It's possible contact with the skull would induce greater liability to cracking. But at least we know it's possible under some conditions to prevent the cracking of the brain, even at temperatures that are colder than necessary. That's a great thing.

The other thing was the structure. Dr. Coles' brain structure, from what we could see, was intact. As a matter of fact, it was more intact than the rabbit brains we'd been studying for many years before that. I was satisfied with the rabbit brain results, but Dr. Coles' brain was even better than the rabbit brain results.

One of the things we did is we looked at the brain both at the histological level, which gives you long-range structure, and the electron microscope level, which looks at more fine structure. I have photograph after photograph after photograph after photograph of Dr. Coles' brain at the histological level, showing the same result time after time after time, field after field after field. There are no ice holes anywhere, there are no rips or tears in the tissue anywhere, there's no loss of substance from the brain — the staining intensity is dark everywhere. That was very reassuring.

But you can be misled at the light microscope level, so you have to go to the electron microscope level to confirm it. On the other hand, if you see something good at the electron microscope level, you could be fooling yourself because you may pick a good area that's not representative. The histology — the light level — eliminated that drawback. Now we look at the electron microscope level and we see that everything is there. The cell membranes are there, the synapses are there, the organelles are there — everything is there. It's really hard to see presynaptic vesicles because of the great amount of shrinkage, but there are hints here and there of structures that could be presynaptic vesicles.

That was a complete revelation and a breakthrough. This provides the direct evidence that the human brain can be cryopreserved by vitrification with structural preservation. Without that demonstration, everything else about human brain preservation by vitrification is speculative. You can daydream about molecular repair technologies all you want, but if there's nothing there to repair, it's all a pipe dream, it's all a bunch of hot air. If you now have proof that you actually can preserve all the structures, then everything is transformed, because now you know that those hypothetical repair devices will actually have something to repair — that you haven't lost information beyond recognition, that if you can rehydrate and stabilize the biological systems that are already there, there is a reasonable scenario for restoring their function. I think it really transforms everything about this whole field of endeavor in a way that's never been possible before.

To take that just one step further: the shrinkage distorts the tissue, and you can look at it at high magnification and see that the structural elements of the tissue are still there — but you're still not quite sure what happens when you rehydrate it. So we were able to do a limited number of studies on rehydrating Dr. Coles' brain tissue. If you took a biopsy piece from Dr. Coles' brain and diluted it to 66% of full-strength M22 for a few minutes before you fixed it — so you could see how it responded osmotically — then you get a better idea about whether the structure is preserved even when it's rehydrated.

The beautiful results we saw were that the normal shape of the neurons in the cerebral cortex of Dr. Coles' brain was restored by this rehydration process. Before rehydration, the neurons, which are normally shaped like pyramids in the cerebral cortex, look like balls — they were shrunken down into marble-like structures. They're kind of black in the light microscope and hard to visualize. But after rehydrating them, they actually returned to their normal shapes. You could see axons and dendrites coursing away from and to them in a normal fashion with no sign of any interruption of neuronal processes or anything like that.

This suggests that yes, the shrinkage of the brain is reversible structurally — and even in terms of membrane permeability, because this return of cellular shape requires the cells to take up a certain amount of water as the cryoprotectant inside the cell is diluted. If the cells were chemically fixed by the cryoprotectants, for example, they would be osmotically unresponsive. Or if the membranes were completely permeabilized by the cryoprotectant, so that it didn't matter whether you were changing the concentrations outside the cell, the cell would not be able to respond to that. That would be a different story. But it seems that the cell membranes were actually physiologically intact, so that the cell was still able to respond to changes in its environment. That was a very exciting aspect of our results.

I think these results basically change the whole discussion about brain preservation and what it means and what the implications might be.

Daniel: Would it go too far to say that this now puts the onus on critics to prove that it doesn't work?

Greg: Well, I wish you could say that, but the burden of proof is always on the person proposing a certain proposition. The skeptics — all they have to do is remain skeptical. But they can't remain skeptical about as much as they could before. You've raised the bar for the skeptics. What the goal has to be now is to continue to raise the bar higher and higher until you transform brain preservation from a possibility into an accomplished fact. And when you get there, then there's no debate, there's no discussion — you've proved your point, and then you move on from there.

Daniel: In terms of the fixation-versus-cryopreservation debate we briefly touched on earlier, does this change the lines of scrimmage at all on that?

Greg: I think it does, because one of the arguments the aldehyde fixers have made is that you need to do aldehyde fixation because you can't be sure that if you don't use it, you can preserve the structure. Well, that argument is now gone as far as I'm concerned. Yes, you can preserve the structure. There may be circumstances in which aldehyde fixation still makes sense, but for people who want to have their brain preserved under good conditions, I think that aldehyde fixation is not necessary to preserve the structure. It may actually destroy important things that we're not able to define yet. Whereas if you preserve the structure as it is, there's more of a guarantee that you're preserving everything that's essential.

Daniel: Overall, do you feel like this research answered the questions you set out to answer?

Greg: Yes, I think we got the answers we were seeking. The answers were much better than I was afraid they might be. The answers are very encouraging, very positive, and hopeful. I think they set the stage for much more to come that will further refine the methodologies — maybe reduce shrinkage using better methods of blood-brain barrier opening, which we're working on in our lab. We have new cryoprotective agents that may reduce brain shrinkage and improve brain viability. So to get closer and closer to reversible brain cryopreservation — I think we've taken a step that takes the discussion in that direction, and that is actually beginning to match up with complementary results in other labs, such as the German lab. I think it's a very exciting time, and the future looks very exciting and bright, and we're just happy to be here.

Daniel: Looking ahead, what experiments are you currently doing that build directly on the results of this research?

Greg: This research was kind of a prerequisite for other studies. There's kind of two levels to where we go from here. One of them is to continue clinical research, like Dr. Coles' research, and the other is the research laboratory approach again.

On the clinical side, we have another human brain that was donated from a woman in Argentina. It was sent to us and biopsied and is now in storage at Alcor also. This brain was preserved under much worse conditions than Dr. Coles' brain. We're trying to figure out how to get this brain analyzed in the same way that we did for Dr. Coles, so we have a comparison. An organization like Alcor needs to know not only what the best-case scenario results in, but also what some of the lesser-best-case or worst-case scenarios result in. We can build up a kind of reality check on the whole spectrum of what happens in this practice. We want to see much more of that. And I know in Europe there's interest in applying this same kind of biopsying and examination technique to people who are having their brains preserved over there. We'll see how that turns out. This is going to ultimately result in far more knowledge about what human brain preservation results in under various conditions, so that we're going to see a great enrichment in our knowledge of this whole subject over the next few years. I think that will be wonderful in many different respects.

On the research level, we want to go beyond what we can do right now. Opening up the blood-brain barrier is part of that, and so we're looking at new molecules that may do that more effectively than what's been looked at in the past. Just to touch on the meaning of that: the brain shrinkage we see is caused by two things. It's caused by shrinkage of brain cells, which is an osmotic effect. Water moves across cell membranes quickly, and cryoprotectants move slowly. If you put cryoprotectants next to a brain cell, the brain cell has more water in it than the cryoprotectant solution, so the water diffuses down its concentration gradient and the cell shrinks. Then the cryoprotectant goes in, but it's a slower process. That's one of the reasons.

But you have an analogous situation in the brain as a whole, because you have the vascular system, and you load that with the cryoprotectant, and everything outside the vascular system is more dilute — so water comes out of that into the vascular system, which causes the brain to shrink as a whole. If you can overcome that second problem of making the vascular system permeable to the cryoprotective agents, then at least it gets out there in contact with the brain cells, giving them an opportunity to take it up, which may mean less brain shrinkage. What we find is that yes, if you open up the blood-brain barrier, you do get less brain shrinkage — therefore less distortion, therefore less changes that may limit viability later on. That's one avenue.

Dr. Spindler in our lab is now studying brain shrinkage in detail by quantifying it. He has video cameras monitoring the course of brain shrinkage and re-expansion under different conditions, so we can see how it responds to various things we're trying to do to modulate it. That's potentially very exciting.

Then we have new cryoprotective agents coming along. We're beginning to look at how much cryoprotective agent a brain can tolerate. Brains tolerate one-molar M22 pretty nicely, and they tolerate our new cryoprotectant at that level pretty nicely. When you go up to five-molar M22, you start seeing a pretty severe effect on brain viability. But we can go up to two-molar in a pig brain and not have any problem with viability. So we're marching our way up. As we go higher and higher in concentration, we'll encounter problems that exist but are small enough to be solved. When we figure out how to load the low concentrations, then we go to higher concentrations and gradually work our way all the way up to the full strength that we need to vitrify — just as we're doing with kidneys already. Brains are a little behind kidneys, but we can follow the same procedures to eventually get to reversible brain cryopreservation. Those are the main approaches right now.

There is one other thing I want to mention. We showed in 2012 — 14 years ago — that you can vitrify rabbit-brain hippocampal slices and warm them back up and have perfect long-term potentiation responses. So the molecular mechanisms for encoding memory, at least as you measure by long-term potentiation, are preserved by vitrification. German has shown that in his mouse brain as well, although not as well as we did 14 years ago. But once you get to that level, then you can start looking at brain preservation in a different way. You can start looking at whether you can preserve not just viability and structure, but also memory functions.

We have done studies in our lab in which you stimulate a vitrified, rewarmed hippocampal slice and you watch the waves of electrical depolarization across the surface of the slice. We made movies of that, and we show no difference between vitrification and warming. But that doesn't tell you if you've remembered anything — it just tells you the physiology is still there.

So we just made a proposal called Total Recall, in which we hope to show that you can teach brain cells — and maybe even hippocampal slices — how to do various tricks, and then vitrify them, warm them up, and show they still remember how to do those tricks. If we can do that, that will take this whole discussion to yet another level. We're really excited about the possibility of being able to show that mammalian memory can be preserved too. We know there's good prospects of it, because Natasha Vita-More and Daniel Barranco showed that you can restore olfactory memories in C. elegans, but that's not mammals. We want to show really high-level functions in mammals. For example, you can teach brain cells in a dish how to fly an airplane, or how to play pong, or how to play the game of Nim. If those memories can be preserved after cryopreservation, that really will mean something.

Daniel: That's similar to the brain shrinkage question — one you hear quite often from critics. "How do you actually know the memory will be there?" I think that would create a lot of excitement and take another layer off the overall skepticism.

What would you say to scientists in cryobiology or adjacent fields who might be encountering this podcast or your work for the first time, and who are maybe a bit skeptical overall?

Greg: Well, that's very natural. This is a very alien concept — we don't talk about brain cryopreservation at cryobiology meetings because we deal with the normal real world that's out there. You need a certain cell type or you need a certain tissue for medical or research purposes, and so you focus on that. As far as organs are concerned, transplantable organs make sense to bank because you can save people's lives that way. But brain preservation has fairly esoteric applications in neurobiology and that sort of thing, and they're not usually the focus of attention of cryobiologists. Cryobiologists have different fish to fry. There hasn't been a lot of thought about this sort of area before.

But now you have papers getting published in Proceedings of the National Academy of Sciences about brain cryopreservation and electrical activity coming back, and it's beginning to broaden the horizons that are out there. I'm pretty excited about the prospects that lie ahead in that direction. I actually plan to discuss this at a cryobiology meeting and introduce some of these concepts to the cryobiologists, so that we can have more informed discussion about this whole topic.

So far there has been pretty good reception to these things. Some of the cryobiologists have discussed the results of the paper we're discussing today. There was a review of our results in MIT Technology Review that just came out, I think yesterday. Of course, topics were discussed as to the fact that Dr. Coles' brain was not clinically alive at the time it was cryopreserved, but there was a general understanding that this is a successful experiment and that it has many applications in neurobiology. I think that's a really great start. It begins to open the discussion for deeper possibilities. As we refine brain preservation methods going forward, that discussion will become deeper and deeper, and more people will join it, and more people will begin to do research and be stimulated to get involved. We're at a pretty exciting time in that whole scenario.

Daniel: Final question. Looking back on the whole journey up to this point — the process, the years of work — not just specifically the results of this research, what would you say is your biggest takeaway personally?

Greg: I think the biggest takeaway is: dare to do things that are risky. Take chances. Do the impossible. Try to do things that other people think may not be doable, because you never know what's possible until you try. I think it was maybe Arthur Clarke or somebody who said that the only way to find the limits of the possible is to go beyond them.

Dr. Coles is a great case in point. He's kind of an exemplar of that whole concept: take a risk, stick your neck out, don't play it safe, because that doesn't change anything. If you want the future to be better than the present, you have to make changes, and it takes courage to do that. Don't take the easy path — dare to be different. If you are diligent and conscientious and you do your hardest work, you can succeed, and maybe be very satisfied at the results that you get.

Daniel: Love it. "Dare to be different" — the podcast title practically writes itself at the end.

Thank you, Greg, so much for taking the time to sit down and talk us through this. This has been a very special conversation. Before we close out, I want to take a moment to acknowledge some of the other people who made this possible. Obviously, thank you, Greg, and the team at 21st Century Medicine. Hugh Hixon, Steve Graber, and all the Alcor staff who were hands-on making this happen behind the scenes. And also Alcor management for agreeing to this experiment, allowing it to be done, agreeing for it to be presented, being open to this whole thing. That was a very broad-minded thing to do, and I really appreciate it.

Greg: Absolutely. And yes, we have many people at 21st Century Medicine who don't get the credit they deserve. There was a lot of microscopy involved, and a lot of analysis, and a lot of mental gymnastics that had to be done to get some of the results we published in the paper. My hat is off to the 21st Century Medicine team as well.

Daniel: I'd also like to thank Linda Chamberlain for helping put this together, and Fred — the co-founder of Alcor, who is now cryopreserved with Alcor. This research, alongside everything Alcor has accomplished, is a testament to his legacy.

Greg: Oh, so true. Yes, I want to definitely thank Linda for helping us pull together all the details of the Coles case. They had been unattended to for many years before we decided we wanted to write this up, and so a lot of people had to work pretty hard to pull together all the facts we needed to publish this paper. I really appreciate that.

Daniel: And we had talked about them before, but again — Stephen Coles and Steven Harris, who are not with us now. They dedicated their lives to this.

Greg: And let us not forget Natalie Coles as well.

Daniel: Yes, Natalie Coles. None of this would have happened without her, and so we'll always be grateful to Natalie for this as well.

To listeners: if you want to read this new research yourself, we'll have a link in the show notes. Thank you, Greg, and thank you everybody for listening. Until next time.

Greg: Thank you, Daniel. Take care.