The Cryobiological Basis to Cryonics
Posted by Jeriaska on September 2nd, 2007
Brian Wowk presenting at the 6th Alcor Conference in Scottsdale
Brian Wowk is a Senior Scientist at 21st Century Medicine, Inc. where he studies the low temperature preservation of tissues and organs for medical use. He was a co-founder with Dr. Gregory Fahy of technology permitting successful cryonic temperature preservation of the mammalian kidney. Cryobiology studies show steady progress in the quality with which brain information can be preserved under ideal conditions. However the absence of demonstrable reversibility, and the vast variety of conditions under which cryopreservations can take place, introduce uncertainty in the “information theoretic” paradigm of cryonics. At the 6th Alcor Conference in Scottsdale, Arizona, Dr. Wowk gave a talk on the basis of cryonics in the science of cryobiology, while deconstructing several popular myths of cryobology.
The following transcript of the 2006 Alcor conference presentation by Brian Wowk has not been approved by the author. DVDs of the 6th Alcor conference are available for purchase at the Alcor website.
The Cryobiological Basis to Cryonics
In the movie Ice Age, this hapless character is frozen in a solid block of ice and then effortfully thaws out a thousand years later. In the real world of cryobiology, things aren’t that simple. When tissue is slowly cooled, ice crystals first form between cells, and then eventually, as cooling continues, the cells become squashed into pockets of concentrated salt solution. This brings us to the first myth of cryobiology, which is the common belief that freezing bursts cells. In fact, the complete opposite is true. Under conditions of slow cooling, freezing squashes cells. It does not burst them.
Squashed frozen cells versus those frozen in cryoprotectant
More than fifty years ago, it was first discovered how to use cryoprotectants to reduce the damage that occurs during freezing cells. Some of the common cryoprotectants are glycerol, dimethyl sulfoxide (DMSO), ethyl glycol, and propylene glycol. When tissue without cryoprotectant is cooled, the salt water inside the tissue converts almost completely to ice. But if you first add just a dash of cryoprotectant before you cool your cells, the freezing process concentrates the anti-freeze cryoprotectant in the unfrozen water outside the ice crystals, lowers the melting point, and limits the extent of ice formation that occurs, so that there is a larger volume of unfrozen solution in which cells can continue to survive. So, instead of having a situation where cells are squashed at very low temperatures, you have a situation where there is more room for the cells to survive.
There are a large number of different types of cells and even tissues that can be successfully cryopreserved by freezing. Still a problem with freezing, if you want to adapt it to large organs, although individual cells can survive in between these ice crystals, the ice crystals break up the delicate connections between cells. For most organs, that prevents recovery of function of the whole organ. A solution to this problem was proposed by my colleague Greg Fahy more than twenty years ago when he proposed vitrification as an approach to cryopreservation. The idea with vitrification is you begin by loading tissue is so much cryoprotectant that freezing cannot occur at any temperature, so during the whole process you have a liquid solution. And as you continue cooling, the liquid becomes thicker and thicker like syrup, then like molasses in January. Then finally, as you go below minus 100 degrees Celsius, the solution converts to what is called a glass, which is a solid substance where everything is stopped, but there are no ice crystals in it. That is the state of vitrification.
Frozen liquid with ice crystal formation versus vitrified glass state
On the level of the tissue, you start with adding cryoprotectant to cells. Then you begin cooling, and even as you reach very cold temperatures the state of the tissue is unchanged, because there is no ice crystal formation leading to structural damage of the tissue. It can remain in this stage practically indefinitely. This is what happens during freezing when you have a dilute solution of cryoprotectants. The water has become filled with millions of tiny ice crystals. But if you have enough cryoprotectants in the solution to vitrify, you can cool it to extremely low temperatures and everything still looks like a liquid. Although, in fact, it’s not a liquid, it’s a solid. The temperature that is being read here is minus 124 degrees Celsius. At that temperature, what was formerly a liquid is now a solid, even though it has no crystals in it.
Now this photograph shows what vitrification looks like on the scale of organs. On the left you have a frozen rabbit kidney, and on the right you have a rabbit kidney that’s been protected by a virtification solution and then cooled to -140 degrees Celsius. There are no visible signs of damage. Time has essentially stopped for that organ. And what has now been shown to be possible just a few years ago is you can rewarm an organ the size of a rabbit kidney, unload the cryoprotectant, and implant that kidney back into a rabbit, and have it support the animal’s lifelong term. This brings us to Cryobiology Myth #2, sometimes stated in media, that cryoprotectant can’t penetrate into organs. In fact, it can, because you can circulate cryoprotectant through organs just as blood circulates through organs. Essentially you can replace the blood with cryoprotectant, thereby introducing cryoprotectant to all the cells in the organ. This process is called “perfusion.”
This is an organ perfusion machine used at 21st Century Medicine to prepare organs for vitrification. Inside is an organ chamber with a rabbit kidney. A tube called the cannula is attached to the renal artery, pumping cryoprotectant into the organ, preparing it for vitrification. Just as for freezing, there is also a very large list of tissues that can now be successfully cryopreserved by vitrification. The most sophisticated of which is the rabbit kidney at this point. We hope to eventually have success with larger organs as well. As you attempt to scale up vitirification to large organs, there are a few problems you run into. The two most serious are the slow heat transfer in large systems which limits the rate at which you can cool large organs. And the time it takes to cool large organs allows for the toxicity of the concentrated cryoprotectants to accumulate and, under some circumstances, poison the organ. So, with vitrification you are always walking a tightrope between avoiding ice formation and toxic effects of the vitrification solution. That is the tightrope we are attempting to lengthen in the field of vitirification research in medicine.That brings us to cryonics. Cryonics can be defined as the cryopreservation of a person with medical needs that cannot be met by available medicine until resuscitation and healing by future medicine is possible. Note that cryonics correctly defined is not the cryopreservation of dead people because obviously if the process were to ever succeed, then that person was never really dead. So, that is not a correct way to look at it. Cryonics includes cryopreservation by methods not currently reversible. It includes medical problems not currently addressable. And also includes problems so severe they may not even be recognized as problems by contemporary medicine. An example of that would be the kinds of injuries that happen to a person when their heart stops for a prolonged period of time, such as a couple of hours. What do cryobiologists say about cryonics? Probably the most famous expert opinion about cryonics is a quote that’s been circulating in the media for more than 20 years now. And it is credited to my colleague Arthur Rowe. He has stated that “Believing cryonics could reanimate somebody who has been frozen is like believing you can turn hamburger back into a cow.”
Well, I’ll do you one better than that. Not only can hambuger be turned back into a cow, I will tell you how you can turn hamburger into a person. You eat it. With sufficient molecular manipulation, hamburger can be turned back into healthy tissue. This tells us that whether one form of matter can be manipulated into another form of matter is the wrong cryobological question. What do other cryobiologists say about cryonics? Here is a quote that you will never see in the media because it’s too boring. “Cryobiological issues of cryonics are complicated.” The most complicating factor is that cryopreservation is time travel. This fact is not in dispute. If you ask any cryobiologist, they will tell you that if you cool tissue to a sufficiently low temperature, it can be preserved essentially indefinitely in an unchanging state for centuries or possibly even thousands of years. The problem is that this fact is seen as largely academic in the cryobiology community because in medical cryobiology as it now exists there is no need to cryopreserve things for centuries and people just don’t spend a lot of time thinking about the technological implications of keeping things stored for centuries. But the fact that you propose to store for centuries does mean that the feasibility of cryonics must be evaluated based on technologies that don’t yet exist, but that can exist according to physical law.
Injury reversibility plot
To think about these things, I think it is helpful to consider a plot of injury reversibility versus injury severity. I think we can all understand intuitively that as the severity of an injury increases, it becomes less and less likely to be reversible. There is also a time dependence to this plot. In the year 2000 we can reverse injuries that are more severe than in the year 1900. The classic example that is used of this is cardiac arrest. If someone’s heart stops, there wasn’t much that could be done in the year 1900, but that particular injury can be reversed often in the year 2000. Unfortunately, with human cryopreservation, or cryonics, you are dealing with an injury during the cryopreservation process that is so severe, it is way off the scale of injuries that are currently reversible. The general consensus is that the injury is so severe and the status quo in medicine is so eternal that this situation will never change. That cryopreservation of humans is worthless because the injuries are essentially permanently irreversible.
But what people who have looked at this problem hard and seriously have found is that that’s not quite the case. If we look to the limits of what medicine is able to do decades or even centuries in the future, there are going to be large classes of injuries that are presently irreversible that will be reversible in the future. That means we really have to consider a third class of injury which is future-reversible. What are some of the technologies that we have to take a serious look at if we are to consider the medicine that is going to be available decades or centuries from now? One of the most important parts of foreseeable medicine is going to be advanced tissue rejeneration technology. Within this century we will see technologies developed for teaching the body to regrow lost limbs, for example. And at the farther frontiers of medicine, it is possible to foresee technologies where patients who have suffered severe trauma can be placed on artificial life support until vital organs can be regenerated. In fact, according to the laws of physics and principles of biology, there is no reason why it would not be possible to program cells on the surface of a brain, or even just one cell on the surface of the brain, to initiate a growth process that would eventually regrow a whole body around just a brain if that were all that was able to be recovered from a victim of trauma.
In case this seems fantastic, let me remind everyone that generation of whole bodies starting from a single cell is a technology already demonstrated by nature. This is not molecular nanotechnology. The production of tissue and organs is a normal business of biology that living things have been doing for hundreds of millions of years. It is just a matter of us learning to steer these processes in a direction of therapy and not just reproduction. An area where nanomedicine becomes very important, in fact indispensable, is for repairing the brain in cases of severe injury. The brain is obviously the one one organ that we cannot casually replace. It has been very gratifying to see over the past 20 years that nanomedicine, at least in some of its forms such as organic conceptions of it, is becoming almost a mainstream view. The National Institute of Health’s website on nanomedicine makes reference to “synthetic biological devices” that could heal diseases and “fix the ‘broken’ parts inside cells.” So there is an emerging awareness that these kinds of capabilities are coming downstream in medicine.
What we have to look at in terms of the ultimate limits in nanomedicine are extremely advanced technologies for analyzing the molecular content of a brain and performing arbitrarily complex molecular repairs inside the brain. The implied ability from such nanomedical capabilities is the ability to extract all molecular content of arbitrarily injured tissue, and reconstitute an inferred healthy tissue state. We already said that with sufficient molecular manipulation, we can turn hamburger back into healthy tissue, as that’s proven in nature. The extension of that is that with sufficient molecular manipulation any injured tissue can be turned back into healthy tissue.
But the question is, How much information can be missing about the original state of tissue before a repaired person is no longer the original person? That brings us to the information theoretic criterion for death. A person is dead according to the information theoretic criterion if their memories, personality, hopes, dreams, etc. have been destroyed in the information theoretic sense. That is, if the structures in the brain that encode memory and personality have been so disrupted that it is no longer possible in principle to restore them to an appropriate functional state, then the person is dead. If the structures that encode memory and personality are sufficiently intact that interference of the memory and personality are feasible in principl, and therefore restoration to an appropriate functional state is likewise feasible in principle, then the person is not dead.
So, clearly, the brain is the key to reversibility of injury in future medicine. And this brings us to the key cryobiological question of cryonics: Does cryopreservation preserve sufficient brain information to permit recovery of the original person? At this point I have to digress onto a side issue because it is such a common misconception. That is the question, Does stopping a brain kill it? This is a quote from a standard textbook of medical physiology. “We know that secondary memory does not depend on continued activity of the nervous system, because the brain can be totally inactivated by cooling, by general anesthesia, by hypoxia, by ischemia, or by any method, and yet secondary memories that havebeen previously stored are still retained when the brain becomes active one again. Therefore, secondary memory must result from some actual alterations of the synapses, either physical or chemical.”
This means that our brains are not like random access memory in a computer that is lost when power is turned off. Our brains are more like a computer’s hard drive in terms of the way they store long-term memories. There are clinical examples of brain inactivation that many people have survived. If your heart stops more than thirty seconds, all your brain electrical activity will go silent. Barbiturate coma can turn off brain activity for days at a time. And hypothermia, at less than 18 degrees Celsius, will also generally silence all brain activity. The first study attempting to cryopreserve brains was conducted by Isamu Suda in the 1960s. He froze cat brains in glycerol and re-perfused them with warm blood. He found he could recover normal electrical activity after five days frozen at -20 degrees Celsius, but when he attempted to measure electrical activity after seven years of storage at -20 degrees Celsius, he only found partial return.
This tells us that -20 degrees C is just too warm for long-term storage. He found however partial return after another period of time at -60 degrees C, and no return of integrated electrical activity at -90 degrees C, which means those temperatures were cold enough to injure the brain beyond the point of recovery of electrical activity. And we can understand why that is by looking at our concept of what happens to cells when they freeze. If you don’t have enough cryoprotectant around the cells, when you get to very low temperatures the cells will become separated from each other and integrated organ function will no longer be possible. And I should mention that Suda did find that when he cooled brains to very low temperatures he could still recover what he called “unit electrical activity,” activity in single cells. It’s just that they weren’t able to communicate with each other because the freezing damage was so severe.
One way to deal with this problem is to increase the cryoprotectant concentration. Instead of 15% cryoprotectant you can start with 40%, and then you get less volume converting to ice. In fact you can increase it to even greater concentrations such as 60% glycerol, and then you get very little ice. Most of the tissue volume is going to be preserved in a vitreous, glassy state. This brings us to the largest study of brain cryopreservation published to date, which was “Effective Human Cryopreservation Protocol of the Ultrastructure of the Canine Brain,” performed by Mike Darwin and collaborators at the cryonics company BioPreservation, Inc., back in 1995. And he showed after brains were treated with 60% glycerol and cooled to deep subzero temperatures, and then rewarmed, that there was remarkably good structural preservation.
This is an electron micrograph at 6700 times magnification. These holes here are actually capillaries, blood vessels, so they are supposed to be there. They have nice smooth walls, looking generally uninjured. The round blobs with dark brown boundaries are axons. And the myelin, which is the black outline around the axons, is generally intact. There are just a few spots where it turns just a bit foggy. And also you have a problem of chromatin clumping in cell nuclei, which is very commonly seen in cryopreservation.
This is a closer up view. Here we are looking at synapses, which are the points where nerve cells communicate with each other. You can actually see that they are very well preserved. This is an extreme close-up of a synapse, and what is really gratifying to see here is that not only can you see the junction between the cells, but you can actually see these tiny circles in here. These are neurotransmitter vesicles, which is a very fine structure and an important part of that aspect of brain anatomy, and it’s very suggestive that the essential biochemical nature of the synapses being preserved. Here, we have a close-up of some axons, again, and the myelin around them. You can see that there is some damage to the myelin, where the boundary becomes indistinct here. Here, more damage to the mylelin.
In some parts of these brains there is ice damage. There are holes apparently produced by ice crystals, and this is not unexpected because at that concentration of glycerol we know ice has got to form somewhere. And if you look every 20 or 30 microns or so, there is a hole there. So there is still freezing damage occurring. What we would like to do is eliminate that freezing damage completely. The technology I showed you of perfusing approximately 60% glycerol was a technology used in cryonics during the 1990s. And into the 21st century, we moved to true vitrification solutions, which are approximately the same concentration as the old 60% glycerol but a different composition that is more resistant to ice formation and that allows the entire tissue volume to be cooled without ice formation.
This brings us to Cryobiology Myth #3, which is the belief that only tiny things can be vitrified. In fact, any size tissue can be vitrified, even at slow cooling rates if you use a sufficiently high concentration of cryoprotectant. Here you have five pounds of vitrification solution (M22) that is completely ice-free. M22 is used both to cryopreserve kidneys experimentally and used as Alcor’s vitrification procedure. There is a catch, though. And that catch is, you can vitrify things as large as you want, but the vitrification of large organs with current technology will often result in loss of viability. Viability being defined as the ability to spontaneously recover due to biochemical injuries caused by the cryoprotectant solution.
But as far as structural preservation goes, that was studied in a publication in the annals of the New York Academy of Sciences two years ago. The title was “The Arrest of Biological Time as a Bridge to Engineered Negligible Senescence.” It documented the use of M22 to cryopreserve rabbit brains and found on extensive transmission electron micrographs taken through vitrified, rewarmed rabbit brains that there is no evidence of ice formation anywhere in the brain. In an electron micrograph shot of the hippocampus of the brain, there is extremely dense preservation of the grey matter of the brain, no sign of ice crystal damage anywhere. If you zoom in, you do see signs of other damage. In particular you have white streaks caused by dehydration, where this concentration of cryoprotectant, which only partially penetrates the blood-brain barrier, causes a lot of water to flow out of the tissue. This causes cells to contract away from each other, producing these lines of dehydration artifact between the cells. And that seems to be, at least on a structural level, reversible if cryoprotectant is removed and the brain is rehydrated.
This is a scanning electron micrograph, part of the same study. The use again is to look for the formation of ice crystal formation and what was seen in all the scanning electron micrographs of this brain was holes that are blood vessels. There are no signs of tearing or the kinds of damage that would be expected if ice crystals were present. What about cryopreservation under poor conditions? Poor conditions can include long ischemic time, which means time without blood flow. That, in turn, can lead to limited or no cryoprotection. If cardiac arrest lasts too long and blood clots it becomes difficult or impossible to perfuse cryoprotectant. A study of a canine brain cryopreserved with a lower concentration of glycerol, 30% as opposed to the 60% glycerol concentration showed in the previous micrograph, and here we can see more obvious signs of damage because a larger volume of the tissue is being converted to ice, causing greater loss of structure.
Now I’m going to show you something really amazing. This is a type of image called a free substitution image, meaning you take tissue that is still in the frozen state and you treat it with chemicals, making it possible to electron microscopic images of the tissues in their frozen state. On the left you have a scale of millimeters. You have some rabbit brain slices here perfused with 3.72 molar glycerol, which is probably around a 35% concentration of glycerol. What you see is extensive ice damage. In fact, when I look at that slice it looks to me like frost on a windowpane when I used to live in cold Canadian winters. There’s more ice there than tissue. If you zoom in to a higher magnification you can again see the devastating effect of the ice crystals. In fact, it’s not even recognizable as tissue. Between the ice crystals there is actual recognizable biological tissue, it’s just terribly squashed in between all those ice crystals.
Now I’m going to show you the really amazing thing. This is what it looks like in the frozen state [left]. But if you then rewarm it and allow the ice to thaw, you get something that looks like that [right]. Where most of those terrible gaps and obvious damage from the ice apparently disappears, at least on a light microscopic level. In fact, this is a section of brain tissue that was thawed after freezing and it did not have any cryoprotectant in it at all. This is straight frozen tissue. There is still damage evident even on the light microscopic level, but it’s remarkable how the tissue somehow seems to remember the state it’s supposed to be in when the ice goes away.
Recently Dr. Sergey Sheleg at Alcor has also begun to explore the question of straight freezing without cryoprotectant because it is relevant to certain classes of cryonics cases where cryoprotectant perfusion is impossible. And he’s also finding that although there is extensive damage visible on these electron micrographs, you can at least usually see what is damaged. You can still generally recognize the damaged structures. It’s not a process of massive obliteration of information, which is gratifying because there has been a lot of controversy over the years about exactly how damaging freezing without cryoprotectant is. Lately the pendulum is beginning to swing back in the direction that it may not be as damaging as once thought for cases that are subjected to that kind of freezing.
So, obviously, this is not hamburger. Even if you freeze without cryoprotectant, there is a lot of structure that is still preserved. But still, if enough damage occurs, it will be permanently irreversible by any technology. So, what does that mean clinically and for the outcome of cryonics? We’ve already stipulated that with sufficient molecular manipulation any injured tissue can be turned back into healthy tissue. So what happens when information encoding memory and personality is incomplete or non-existent? One would expect that the patient would wake up with partial or total amnesia. So that means that for medicine capable of completely general biological repairs, survival is not a binary question. In medicine today we are used to thinking of the patient either making it or not making it. But for really advanced medical technology, cases are rarely going to be like that. There will be a lot of ambiguity about exactly whether an original patient really survives after extreme biological repairs.
No talk on the cryobiology of cryonics would be complete without mentioning the fracturing problem. This is a problem that occurs not just in vitrification, but in frozen tissues as well. It’s just more obvious in vitrification because it can be so easily visualized. The problem is that as you cool tissue below the glass transition temperature to vitrify it, thermal stresses build up once the tissue becomes solid. And if you cool too cold, the solution and tissue will fracture. Generally, I think the feeling is of the people who have encountered this problem is that this is not a process that is destroying much information. But it does greatly complicate the problem of repair because the vascular system is disrupted. It sort of pushes you from the realm where simpler biotechnologies were needed for repairs into the realm where more hardcore molecular nanotechnology will be needed for repair while keeping the tissues still at very cold temperatures. So there is incentive to reduce or eliminate this problem in cryonics because of the extra burden it places on requirements for future repair.
All right, after showing you so many horrible looking micrographs under non-ideal conditions, I just want to remind you that under ideal conditions we are dealing with excellent structural preservation. Under ideal circumstances, it appears possible to cryopreserve a human brain with little or no ice crystal damage. But current brain cryopreservation methods still aren’t reversible. The remaining issues of brain cryopreservation are unknown effects of cryoprotectant toxicity, unknown effects of highly concentrated solutes on the other side of the blood-brain barrier that cryoprotectants don’t penetrate, structural damage caused by dehydration, limited blood-brain barrier penetration of cryoprotectants complicating questions of toxicity. Of course, it has been shown possible, thanks to the support of Ben Best and the research of Dr. Pichugan and Dr. Fahy, to cryopreserve brain slices. And that’s important because it shows that the process of cryopreserving brain tissue in a living, viable state is possible. And if we can do it in slices, with more work we should eventually be able to succeed in that with whole brains. But we have to realize that because of higher cryoprotectant concentrations, longer exposure times and the blood-brain barrier, cryopreservation results obtained in slices are not generally applicable to whole brains. So we can’t be too excited that we’ve reversibly cryopreserved just parts of the brain. We still have a lot of work to do before we can reversibly cryopreserve the whole brain.
Another extremely important question: Is memory preserved by cryopreservation? And I think there are powerful arguments that it is. This micrograph here shows tremendous amounts of structure that are preserved. I think it’s hard to look at these kind of data and argue a mechanism by which memory would not be preserved that’s consistent with known neuroscience. However, it’s not proof. I have seen people that are absolutely adamant that it is not satisfactory to make a handwaving argument about memory preservation, you really should do experiments to show it. The best proof that a cryopreservation method is reversible in theory is to reverse it in practice. And I think that’s what Aubrey was saying, and that’s what I’m going to say again today. It is an extremely important message.
Short of reversible suspended animation, I think the best proof cryonics can work will be the demonstrably reversible cryopreservation of the brain in a suitable animal model. Until that’s done, to be perfectly honest to our critics, we cannot say for certainty whether cryonics is today adequately preserving memory and personality using current cryonics methods until we do the research to absolutely prove it, in my belief. So therefore I propose what I consider to be a logical development sequence for a cryobiological study of cryonics, and it constitutes a continued improvement of structural and biochemical preservation of the brain, the explicit demonstration of memory preservation, reversible cryopreservation of the whole brain, continued improvement of structural and biochemical preservation of the rest of the body, and eventually whole body reversible suspended animation as sort of the ultimate goal of both cryobiology and cryonics.
There are great benefits to be had from this process of continuing to improve cryopreservation methods and push them closer and closer to contemporary reversibility. The greatest benefit is that it makes cryonics what you could call a last in, first out process. For instance, a cryopreservation method in 2006 may leave a patient out in a more severe zone of injury, whereas if we continue developing the technology, the cryopreservation methods of 2030 could result in much less injury. This could mean the difference between getting revived in the year 2100 versus getting revived in the year 2200 if you are cryopreserved with cruder methods. There is one final thought I would like to leave you all with, and that is, because of an idea like cryonics the distinction between reversible and future-reversible injury is really rather arbitrary. If you have the means of stabilizing the condition of a patient indefinitely the proper way to think about injury reversibility is just on one scale. It’s ultimately either reversible or it isn’t. And that is the ultimate limit that defines life and death. The way to think about cryonics is that it is a way of potentially keeping people on the life side of that spectrum. And, in fact, I suggest that even in cases that we are not sure how much of a person we are preserving, life should still be given the benefit of the doubt. Thank you very much.
Related articles: A Precursor to Cryonic Revival: Aubrey de Grey explores how implementing Strategies for Engineered Negligible Senescence might be viewed as a precursor to the prospect of reviving patients from cryonic suspension.