New Cryopreservation Paper Published

[jordansparks]

https://www.biorxiv.org/content/10.6489 ... dium=email
This paper seems to have been eagerly anticipated by some in the cryonics community. At the end of the abstract they describe this paper as the first evidence that ultrastructural integrity is preserved in vitrification without the need for prior aldehyde fixation. OK, so now there is one paper that shows that cryopreservation is possible. Science is not based on individual papers. I want to be very clear about what this does not mean. It does not mean the quality is as good as with aldehyde fixation. I can't think of any situation where the quality would be equivalent. Instead, the quality will vary between slightly worse and significantly worse.

The first figure has four of my favorite images ever. They show the consequences of ice damage. Every single Alcor patient has imperfect perfusion and many have bad or no perfusion. This means that every Alcor patient probably has some of the damage shown in those images. That damage is unnecessary and is never present in aldehyde fixation.

Even assuming no ice, as their paper tries to claim, delay or elimination of aldehyde will always result in inferior structural quality. This is the strong consensus of the entire scientific community. It's not the slightest bit controversial to flatly state that aldehyde results in better quality and it's frustrating to have to keep repeating this as if it's some sort of new information.

I really can't figure out why they are still so enamored by cryo. On the Biostasis Substack,
https://biostasis.substack.com/p/biosta ... =146726813
Max (actually Aschwin) says the reason is:

Previously, the best whole-brain ultrastructural preservation appeared to require aldehyde fixation before cooling. While effective for structural studies, fixation prevents meaningful investigation of biological function and is less relevant to future medical applications.

I really don't understand this explanation. We're not preserving brains so that we can investigate biological function or to be relevant to future medical applications. We're preserving brains in order to survive.

The paper itself gives the reason as:

We conclude that both animal and human brains can be cryopreserved by vitrification with predominant retention of ultrastructural integrity without the need for prior aldehyde fixation. This observation has direct relevance to the feasibility of human cryopreservation, for which direct evidence has been lacking until this report. It also provides a starting point for perfecting brain cryopreservation.

This explanation is also baffling. You may not strictly need aldehyde to retain some structure, but the quality would sure be a lot better. If the quality is better, why on earth wouldn't you use it? The authors seem to have an emotional attachment to cryo because there just isn't any rational explanation.

[MaxMore]

FYI: That was written by Aschwin, not me.

[carrie_radomski]

I think the reason cryonics organizations and researchers have approached this problem differently is that they came at it from the organ-preservation paradigm.

The intuition was not irrational: if one could vitrify and recover a rabbit kidney, and other mammalian organs, then perhaps the remaining problem was partly one of scaling, perfusion quality, toxicity management, and thermal stress. The brain is obviously more difficult, and vitrification is damaging and imperfect. But aldehyde perfusion is not perfect either, especially in real-world cases with any post-mortem interval.

One important distinction is perfusion versus immersion. If a brain is only partially perfused and then immersed in cold fixative, penetration to the center can take days. In a good cryonics case, by contrast, the goal is rapid convection cooling, followed by cryoprotectant perfusion with internal cooling and descent to subzero temperatures within hours. In the best attended cases, that may mean less ongoing ischemic degradation than an approach that depends heavily on slow fixative diffusion from the outside inward.

That does not mean cryonics produces better ultrastructure. I think most people would concede that prompt, high-quality aldehyde fixation often gives better structural preservation, especially for electron microscopy. That is why aldehyde fixation is so powerful as a preservation method.

But there is a tradeoff. Aldehyde fixation chemically locks tissue into place. That is precisely why it preserves structure so well, but it also means viability assays and meaningful functional recovery tests are no longer possible. Cryonics researchers are often willing to accept some loss in ultrastructure because they are preserving within a paradigm where some future biological repair, functional testing, or viability-relevant recovery is an option. Cells can also be functional while not having the shapes they typically appear in.

So I do not think the attachment to vitrification is simply emotional. It reflects a different priority. Aldehyde-based preservation is optimized for structural fidelity. Cryonics is trying, however imperfectly, to stay closer to the organ-preservation and biological-revival paradigm.

The difficult question is which kind of damage creates the harder future repair problem: the mechanical, osmotic, ischemic, and ice-related damage of cryonics, or the chemical crosslinking and molecular alteration produced by fixation. I do not think that question is settled.

That is why I think the vitrification approach remains rational, even if aldehyde fixation currently has clear advantages for structural preservation.

[jordansparks]

Aldehyde perfusion is not perfect, but the quality is always better than cryopreservation. Every single time. Your logic is just disturbingly wrong. You literally compared a poor aldehyde case with an ideal cryonics case.

Here's a side by side comparison of a poor perfusion with aldehyde compared with a poor perfusion with cryoprotectant. In both cases, the best possible perfusion was attempted, but in both cases, it was largely unsuccessful. In both cases, a small amount of perfusate got into the major arteries, but then it just couldn't get into the capillary beds for complex reasons. The aldehyde brain is then removed and submerged in formalin. The cryo brain is then straight frozen. In this scenario, some small amount of fixative will have reached the deepest parts of the aldehyde brain within hours (not days). Yes, this is terrible, but there is nothing better. We would consider injection, by the way, in that scenario, so that it wouldn't take nearly as long. The cryo brain, meanwhile has suffered a catastrophic straight freeze. That's 70,000 psi of shearing forces, like a mortar and pestle. It "smears" the molecules. I would also point out that when I claim that aldehyde is better in a poor case, I'm not making that claim myself. I'm instead stating the scientific consensus of mainstream scientists. There's a reason scientists use aldehyde. The reason is that there is no better alternative. It's not like they haven't tried.

Here's a side by side comparison of a good perfusion with aldehyde compared with a good perfusion with cryoprotectant. In both cases, the best possible perfusion was performed, and in both cases it was very successful. In the aldehyde case, metabolism was halted within about one minute everywhere. In the cryonics case, the internal cooling slowed metabolism gradually over the course of about 30 minutes. But metabolism was not halted. Degradation actually continued for the next 6 hours until it finally reached the glass transition temperature.

In the real world, cases are somewhere between those two extremes. In all cases, aldehyde works much faster and much better than cryoprotection and cooling. Yes, attachment to cryonics is emotional, not rational.

Both aldehyde and cryo create damage that is equivalent for future repair. You mention mechanical, osmotic, ischemic, and ice-related damage of cryonics. I think that's over complicating it. The damage is simpler than that: First you have the damage caused by about 6 hours of delay in halting metabolism. It's different at different parts of the brain and in different scenarios, but that's really about what we're looking at. The second kind of damage is from ice, which is really nearly unavoidable and is very common in cryonics. It's unacceptable.

Look at https://www.sparksbrain.org/revival.html about 3 images down. The gray image with red dots shows aldehyde fixation. Does that look "damaged" to you?

[carrie_radomski]

Hi Jordan, thanks for the response.

It is misleading to say cryonics involves “six hours of degradation” until glass transition. In a good cryonics case, the patient is being cooled continuously from the start. Metabolism slows dramatically as temperature falls; a common rule of thumb is that biochemical reaction rates drop by about half for every 10°C decrease.

In the case I’m referring to, the patient was around 20°C and ready for surgery about 1.5–2 hours after legal death. After aortic cannulation, internal cooling through washout and heat exchange brought the patient to about 5°C roughly an hour later. Cryoprotectant perfusion was completed about an hour after that, and the patient reached about −15°C around six hours after legal death. That is not the same as six hours of warm ischemia.

On the aldehyde side, we need to distinguish between fixative reaching tissue and fixation actually being complete. Some fixative may reach deep tissue earlier, especially with vascular perfusion or injection, but adequate fixation is a chemical process that can take much longer. With immersion fixation, full stabilization of deeper brain tissue can take days or longer.

Formalin reaching part of the brain is not the same as the brain being chemically stabilized. If fixation takes days to become adequate in deeper regions, then the question is what happens to that tissue during the interval before it is fully fixed.

This is not just my concern: “In human postmortem brain tissue, it has been estimated that it can take 20 to 46 days for a sufficient amount of formaldehyde to diffuse to the innermost parts of a brain hemisphere and begin fixation. During this time, tissue in the inner regions of the brain will undergo microbial degradation, autolysis, breakdown of cellular membranes, and stochastic diffusion of molecules.” https://link.springer.com/article/10.11 ... 019-0799-y?

So the fair comparison is not “aldehyde stops everything instantly while cryonics allows six hours of degradation.” The fair comparison is: how quickly does each protocol actually stabilize the deep brain in the case being discussed?

In the 2026 Fahy paper, the vitrified human brain tissue reportedly did not show ice damage, despite being from a real patient with prolonged agonal hypoxia and being stored for years at cryogenic temperature before unloading and EM preparation. So I do not think it is fair to treat cryonics as if it always means catastrophic straight freezing.

A poor cryonics case with failed perfusion and straight freezing is obviously terrible. But an aldehyde case relying on partial perfusion plus immersion has its own problem: the visible structure may look better, but the deep brain may not have been chemically stabilized quickly. That is the comparison I am trying to make.

Anyway, I think immersion vitrifixation is probably better than straight-freezing so new biostasis organizations like Tomorrow Biostasis will default to brain removal with immersion vitrifixation if they cannot perfuse a patient in time. When they do have SST they do stabilize patients rapidly.

[jordansparks]

You just verified that traditional cryonics takes about six hours until the molecules actually start to get locked in place. I was pretty clear that the rate of deterioration varied dramatically over that time, but it's still 6 hours. In a good case like that, it would be under 5 minutes for aldehyde. How many synapses are you willing to lose in 5 hours and 55 minutes? Sure, you'll lose them slower toward the end, but still... You really think that's ok? Yes, just a little bit of formaldehyde locks many molecules in place and abruptly stops metabolism. "Complete" fixation just means it's firmer. But I really don't know why you keep bringing up immersion fixation as if that's anything close to our standard protocol. Remember that we perfuse immediately after death in an ideal case. That does get the formalin to many cells and immediately stops metabolism. For the inevitable poorly perfused areas, even in a good case, the remaining diffusion distance is quite small and it really does only take a very small amount. If you look at the history of CT scans from Alcor, every single one of them show some degree of inadequate perfusion. That means ice. You complain that the deep brain in an bad aldehyde case might not get stabilized quickly, but remember that there is no better option. Traditional cryonics doesn't stabilize quickly either in a bad case unless you are willing to put up with massive ice and you seem to not want massive ice. Immersion fixation is terrible. That's why we perfuse. That's why we inject. We work very hard at all of that, but the results in traditional cryonics will always be worse for a given case, every single time, for every single possible scenario.

[jordansparks]

The molecular damage from traditional cryonics is known to be worse. You’ll have entire synapses that have degraded to some degree and might not even be inferable anymore. That’s real damage. But you instead seem to be concerned about hypothetical damage on an extremely small scale. In fixation, the entire synapse will be intact with all its glorious complexity, but many of the proteins will have an extra carbon attached. That's not damage. You instead want to sacrifice the entire original architecture of the synapse? That wouldn’t make sense to any neuroscientist. Remember that the traditional cryonics scientists to whom you pose this question are self selected to prefer cryopreservation because that has been their entire life's work. They are in the less than 0.1% of the scientific consensus. They are the outliers and need to be ignored. That’s how science works. But even they are straddling both worlds and are ambivalent about which one is better. They want traditional cryonics to be better, but they frequently reluctantly agree that fixation quality is better. Those very few scientists are wrong to lean away from chemical fixation at all. The very very clear scientific consensus is that chemical fixation is better. This is not up for debate even though it might sound like we are having a debate. Its just a fact, and I'm just informing you that you are wrong. You yourself have already repeatedly admitted that the quality of fixation is better, yet you still can't let go of the alternative that makes you feel better. I realize that my bluntness is rude, but your life is at stake here. I'll take my chances.

[carrie_radomski]

I think there are two separate issues here.

First, I don’t think “scientific consensus” can be used that broadly. There is probably a mainstream consensus that aldehyde fixation is excellent for preserving tissue for histology, EM, and connectomics.

But there is not a mainstream scientific consensus that preserving a brain today is equivalent to rescuing a person, preserving identity, or preserving enough memory-relevant information for future revival. That is a speculative biostasis claim. Cryonicists, ASC advocates, and brain-preservation advocates are all a tiny minority relative to mainstream medicine and neuroscience.

So the real question is not “what do scientists use for microscopy?” It is “which preservation method best preserves the information needed for possible future survival?” That question is not settled by the ordinary scientific use of aldehyde fixation.

Second, I think you are conflating fixative reaching tissue with tissue actually being fixed. Those are not the same thing. Some formalin reaching a region may start chemical reactions, but that does not mean the tissue is adequately stabilized or that all meaningful degradation has stopped. Fixation is a time-dependent chemical process.

I don’t know exactly how long adequate fixation takes in each scenario. At Vitalist Bay, I asked Andy about this. He said it would not take two weeks for fixative to reach the center of the brain, but we discussed in the order of days. The paper I cited, which Andy co-authored, discusses estimates of 20 to 46 days for sufficient formaldehyde to diffuse to the innermost parts of a human brain hemisphere and begin fixation. I agree that a good fixation case with vascular perfusion would be better than that but it's unclear how long it takes.

So when I ask about timing, I am not asking whether any formalin got there. I am asking when the deep brain is actually stabilized enough that postmortem change is meaningfully halted. That is the fair comparison to cryonics, where cooling begins immediately and biochemical activity slows as temperature falls.

My concern with the “scientific consensus” argument is that it seems to conflate consensus about tissue preparation for microscopy with consensus about personal survival through brain preservation. If mainstream neuroscientists generally believed aldehyde fixation today was enough to preserve their identity for future revival, we would expect that to be reflected in their own choices. But it is not. Almost everyone in this discussion is already far outside the mainstream scientific consensus by taking brain preservation for survival seriously at all.

[jordansparks]

In a fixative case where perfusion is used, formalin acts deep in the brain within seconds to minutes. This is not at all unclear. The other numbers you are throwing out are nothing but a red herring aka logical fallacy.

Fixative being present is more than adequate to arrest autolysis. I'm actually quite surprised that this detail is something that you are hung up on. It's a pretty bland ordinary fact. Pose the following question to your AI of choice: "If fixative is perfused into tissue, how long until autolysis mostly stops?"

The scientific consensus is regarding structural preservation. There is indeed strong scientific consensus.

[jordansparks]

Max More just posted an interview video with Dr. Fahy about his cryopreservation paper.
https://biostasis.substack.com/p/dr-gre ... r-evidence
Most of it was not relevant to what we do, but there were a few interesting moments:
46:26-Max brought up his visit to our facility a week earlier. Dr Fahy's responses were about what I expected they would be. They were far enough off base that I was motivated to start a new topic here in the forum called "Lay People Must Ignore Certain Scientists" and to add a similar section to this page: https://www.sparksbrain.org/scientificBasis.html. Because no lay person can discern which of his statements are accurate and which are biased, he's the wrong expert to ask about brain preservation. The scientific process requires lay people to exclude all statements from scientific experts who are obvious outliers. With all of that in mind, he made a series of statements that seemed reasonable to him, but which were biased and inaccurate. I'll quote some of them here. I think I've already addressed all these issues within this topic. Now that I've identified Dr Fahy as a biased outlier, it becomes easier for me to simply ignore the noise. He can debate with other scientists, but we lay people can simply ignore him.

...with chemical fixation, we don't know how that information is going to be used... My ideas about what that's good for would be entirely different... We do know that if you could cryopreserve the brain and have it viable, you could definitely reconstitute the individual... It's also less of a stretch technologically... We don't have to impose this extra level of damage on top of everything else that nobody knows how to reverse... It seems unnecessary to destroy everything by fixing it, turning you into a block of plastic... except under some conditions...

54:06-He read a question posed by Andy McKenzie, MD, PhD, our research scientist. The question was regarding storage of fixed tissue at -20.

We need more evidence... even if that would theoretically be good... it may just be that in most cases, the brain is not going to be thoroughly fixed...

That was a nonsense answer. He was criticizing the execution of the protocol, not the protocol itself. It's not plausible that we would put a brain in permanent storage without properly fixing it first. Storage in fixative is based on mountains of existing evidence. This is a common storage strategy in neuroscience.
56:37-Talked about Nectome research. Remember that they do chemical fixation like us. They were critical of the Nectome paper. I agree with that criticism.

[jordansparks]

At 22:54

...membrane properties are preserved also. The membrane acts like a normal membrane. When water goes across it, the cell volume is able to change in response to that. If, for example, the cells had been fixed chemically, the cells wouldn't care what you did to the environment. They'd stay in the same shape. But these cells have properties that are more similar to living cells. They can actually respond to changes in their environment. so that's very exciting also.

I'm going to push back on that a little bit. I'm not a cryobiologist, so he might be getting at some nuance that escapes me, but that doesn't sound quite right. Even if the membrane was shredded, the cell volume would tend to return to normal shape because it's a passive process. The water would move in response to osmotic gradients, and the cell would swell back to normal shape regardless of membrane integrity. Someone feel free to correct me, but I think he misspoke.

[carrie_radomski]

In a fixative case where perfusion is used, formalin acts deep in the brain within seconds to minutes. This is not at all unclear. The other numbers you are throwing out are nothing but a red herring aka logical fallacy.

Fixative being present is more than adequate to arrest autolysis. I'm actually quite surprised that this detail is something that you are hung up on. It's a pretty bland ordinary fact. Pose the following question to your AI of choice: "If fixative is perfused into tissue, how long until autolysis mostly stops?"

The scientific consensus is regarding structural preservation. There is indeed strong scientific consensus.

This is the crux of the issue and I am not understanding this. Can you show me the paper where structures deep in the brain are fixed within hours rather than several days? Also how do you evaluate how well perfusion has occurred using fixation perfusion? Are there CT scans of current patients? If there is a link to the SBP website that point to that I would go there and check it out.

[jordansparks]

Really? We're talking about perfusion, not immersion. You still don't understand that when fixative is perfused, that it quickly reaches the center of the brain? Isn't that the exact same mechanism that cryonics uses that so impresses you?

[AndyMcKenzie]

Hi Carrie,

Here are some articles on the topic:

- https://pmc.ncbi.nlm.nih.gov/articles/PMC10076536/
- https://pubmed.ncbi.nlm.nih.gov/4596479/
- https://pmc.ncbi.nlm.nih.gov/articles/PMC12557960/
- https://pmc.ncbi.nlm.nih.gov/articles/PMC12189018/