Cryonics 101

A deep dive into deep freeze

Since this is a 101, let’s start with the basics. What is cryonics? Speaking from a consumer’s point of view, I view cryonics as life insurance. Not the usual euphemism for a payout to one’s loved ones after his death, but a method to maximize one’s own survival. Or a way to hedge against catastrophic depreciation of your most valuable asset — your life.

From a technological point of view, cryonics is a method of putting an organism on pause (into a state of suspended animation), in order for it to be restored back to life in the future. To achieve this pause, cryonics employs cooling of the organism to very low temperatures. Note that cryonics is not the only possible method, just one of them. Maybe in the future science will discover ways of inducing long-term suspended animation without the need to cool the body. But it hasn’t yet, and cryonics is presently the only viable (or commercially available) method.

Why do we need cryonics? The answer is simple. Because our bodies have one unpleasant feature. They die. Nobody likes it, but most people prefer to gloss over the unpleasantness by coming up with various excuses why they need to accept it. A brave few, however, refuse to do so. Instead, they acknowledge the problem and attempt to use science and technology to eradicate it or at least mitigate it. Cryonics is one of the tools such brave people have invented for the benefit of Humanity. Other tools you may be more familiar with — defibrillators, heart-lung machines, pacemakers, organ transplants, vaccines and antibiotics. They all stem from the same motivation — increase every human’s odds of survival.

Of course, at the present level of scientific and technological advancement, there is no guarantee that anyone cryopreserved today will be restored back to life (or reanimated, as per cryonics parlance) in the future. To claim that cryonics guarantees anything would be intellectually dishonest. But what cryonics does provide, unlike any other technology, is a chance of revival. A nonzero chance. Varying, according to most scientific proponents of cryonics, between 1 and 70 percent.

By the way, cryonics can be viewed not only as life insurance, but also as an “ambulance to the future”. That characterization might not be of relevance for most of us today, as we are still in decent health, but for some people the question of life and death is a much more urgent issue — people who only have weeks or months to live. They already know that modern medical technology will not be able to save them, so their only hope for survival is medicine of far future, and cryonics is their only chance to get it.

I am sure no reasonable person doubts that in 50 or 100 years medicine will be extraordinarily more advanced than today. After all, science has been tirelessly pushing death back farther and farther for centuries: terminal diseases of 100 years ago have largely been eliminated or tamed. Moreover, I am certain that one day Humanity will slay the dragon tyrant once and for all.

But enough purple prose, let’s dive deeper into the science behind cryonics. What evidence is out there that cryonics can work? Broadly, it can be subdivided into two categories: what we see in nature, and what we see in experiments.

Nature

A great number of our planet’s inhabitants can survive subzero temperatures for months. The most numerous are, of course plants, most of which can safely tolerate temperatures from -4°C to -12°C.

Many animals too have evolved the ability to periodically endure exposure to subzero temperatures. I won’t list them all, just mention a few survival champions. The most notable is the Siberian salamander (Salamandrella keyserlingii) who regularly encounters temperatures as low as -50°C and some claim is even able to be revived after 90 years in the permafrost (source1, source2). Here he is:

The second place in Colder Games goes to another frost-resistant amphibian, wood frog (Rana Sylvatica), which can spend many months in a half-frozen state. Here’s a cool video of it thawing:

Another remarkable animal capable of surviving for months at -20°C as pupae is Cecropia moth (Hyalophora cecropia). It is a large insect, with a wingspan of up to 16 cm. To achieve this feat, Cecropia has developed its own mixture of cryoprotectants, namely a combination of glycerol and sorbitol.

Of course, let’s not forget about our much closer warm-blooded mammal relative, arctic squirrel (Spermophilus parryii), which can spend weeks in torpor while its body temperature fluctuates between -2 to 5°C. It was shown that it completely shuts off its electric brainwave activity (as do humans who are cooled below 18°C). By the way, this supports the hypothesis that long-term memory, which is the basis of our identity, is encoded in the structure of the brain — neurons, synapses, etc., rather than in its electrical activity.

Humans, by the way, although lacking in cold-resistance ability when compared to the above champion cryonauts, can also successfully survive deep hypothermia. Here are a few well-known examples:

1. Two-year-old Michelle Funk spent more than an hour under ice-cold water. When rescuers found her, her body temperature was 19°C and she showed no signs of life. However, doctors were able to quickly revive her, and were surprised to find that her brain did not show any signs of damage.
2. 1-year-old Erika Nordby spent several hours in the snow at a temperature of -24°C. Her body temperature dropped to 16°C. After 6 weeks in the hospital, she fully recovered and was discharged.
3. American Justin Smith spent 12 hours in the snow at a temperature of -5°C. Paramedics who found him declared him dead, but the ER doctor did not agree and began to carry out resuscitation. After half an hour, Jusin’s heart resumed beating. Doctors believed that Smith’s brain is severely damaged, but that proved not to be the case, as Justin fully recovered and returned to normal life.
4. Canadian Tayab Jafar spent several hours at a temperature of -11°C. His body temperature fell to 21°C. After 10 weeks in the hospital, he was discharged in good health.
5. Swedish skier Anna Bagenholm spent half an hour under the ice, cooled to a record 13,7°C. After several weeks in the hospital, Anna fully recovered.
6. Jean Hiliard was found in the snow at a temperature of -30°C, where she spent more than 6 hours. After several weeks in the hospital, she fully recovered.
7. Russian tractor driver Vladimir Kharin was successfully revived in 1960 after being found frozen in the steppe where he had spent several hours in subzero temperatures.
8. Japanese ice cream truck driver Masaru Saito got locked inside his refrigerator and was found frozen several hours later. He fully recovered.
9. One of the earliest described cases in the medical literature occurred in 1951, when a 23-year-old American from Chicago spent 12 hours at temperatures from -18°C to -24°C. At the same time her body temperature dropped to 16°C. Although doctors had to amputate her fingers and legs below the knees, the rest of her organs were not injured.
10. The earliest documented case of recovery after deep hypothermia occurred with a Swedish farmer in 1756 and has been described in the publication of the Swedish Academy of Sciences in 1757: NAUCLER, S. Berichte van einem Mannes welcher dem Anschein nach efroren war denn aber wie der zum Leben verhollen war . K. Schwed. Akad. Wiss. 18: 107.
11. There are 6 cases of successful survival after several hours of being in the cargo hold of the aircraft at temperatures below -40°C:
    http://www.bbc.com/news/magazine-33222405
12. Probably the most famous, although not the most applicable example is that of Beck Weathers, who in 1996 fell asleep on top of Mount Everest, and then woke up and came down to camp.

As we see above, nature has many examples of organisms able to withstand deep subzero temperatures. Therefore, it is only reasonable to try to unravel the biological mechanisms that enable this, and try to adapt them to humans with the goal of pausing our biological processes.

Experiments

First, let’s very briefly go through the main cooling problems of living organisms. The main problem is certainly the formation of ice crystals that can damage cells, as crystals (a) are prickly and (b) take up more space than the original water. But this is not as scary as some opponents of cryonics make it out to be, painting pictures of exploding cells and other anti-scientific nonsense.

First, as tissue cools, water from cells exits into the extracellular space, so the cooling cells actually shrink rather than explode. Secondly, ice has only 9% more volume than the source water, while cells have a much greater margin of elasticity, due to which they can safely tolerate the increase or decrease in volume, as well as the presence of a certain amount of ice in the intracellular space. Thirdly, cryobiology has learned to saturate cells, tissues, and whole bodies with cryoprotectants — substances that minimize the formation of ice crystals during freezing. Coupled with optimal protocols of controlled temperature reduction, this enabled scientists many decades ago to successfully master the technology of freezing (or vitrifying, to be precise) and thawing back embryos and whole organs.

Here is a table of organs or tissues that as far back as 1980 scientists already knew how to freeze to -79°C and thaw back to viability (source):

Among other cooling problems I should also point out denaturation (unfolding) of proteins, but, fortunately, denaturation from lowered temperature is often reversible. In contrast to denaturation from increased temperatures — a boiled egg can not be unboiled. Also, since as temperature lowers, all chemical reactions (and hence biological processes) are slowed down and ultimately stop at all, damage from such denaturation for quickly frozen organisms is minimal.

Finally, among problems arising form deep low temperatures, I should mention thermal macro-cracks, in particular those that happen below -140°C. Just how dangerous they are remains an open question, but there are opinions that their danger is low to moderate. Also on the horizon there might be some new technology with which these cracks may be avoided — such as not allowing the temperature of the patient to fall below -140°C:

Systems for Intermediate Temperature Storage for Fracture Reduction and Avoidance

Patent US7278278 — Cryogenic storage system

Patent US7299641 — Cryogenic storage system with improved temperature control

Let’s move on already from theory to practice and look at the experimental data — when scientists were trying to freeze organisms that do not normally enjoy being frozen. Humans, for example. But first, let’s take a look at animals.

A variety of insects were successfully being frozen and thawed as far back as 100 years ago, so insects no longer surprise anybody. Therefore, I will mention only a couple of insect examples.

Many have heard about the indestructibility of tardigrades (Tardigrada) — they were sent into space, and frozen to -196°C without any cryoprotectants. And -196°C was no problem for these guys, as they were thawed and lived on.

Nematode worms are a favorite model organism of biologists. They were used to perfect vitrification technology, achieving a 100% survival rate. Moreover, it was also shown that their long-term memory is retained after many days of being frozen at -80°C! That was a very important result providing evidence that cryogenic freezing is able to preserve the personality of the patients.

In other experiments, Alaskan beetle (Upis ceramboides) was successfully thawed after cooling to -75°C, and this is a much larger insect than nematodes or tardigrades:

Some of the first but highly important experiments on freezing mammals were carried out as early as 1951 (the very first was performed in 1912 by Porphyry Bakhmetyev when he induced anabiosis in a bat, here is his original 1912 (!) article). In these studies, rats were cooled without any cryoprotectants, and it was found that even as the temperature is lowered to 0°C (but not below), it is possible to achieve almost 100% survival. Moreover, some rats were frozen and thawed repeatedly — some as many as 10 times (poor bastards). The researchers also found that during rapid freezing (supercooling), when the water has no time to turn into ice, some rats can survive even after cooling to -3°C:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1365903/pdf/jphysiol01396-0138.pdf

In the same 1950, other researchers were frozen hamsters, bringing them to a temperature of -1°C, and ranged freeze time to establish how much of the ice of the body can tolerate. In these experiments, it was shown that as much as 60% of water in the brain can be transformed into ice with no visible effects on animal behavior after thawing (source1, source2).

Incidentally, in some 1954–6 studies the hamsters survived even after cooling below -3°C. Some of them were cooled to body temperature between -3°C and -5.5°C, at which they were held for 16 to 38 minutes, and then rapidly heated back up and revived, after which those hamsters lived for many months without any apparent health issues:

Experiments on primates (Galago crassicaudatus) in the same 1950 studies were less successful. After freezing below 0°C (without cryoprotectants!) the primates initially recovered, but none survived for more than a day, dying either from pulmonary edema, or from intraperitoneal bleeding (seemingly caused by the gastric juice that diffused during freezing from the biliary glands or stomach into abdominal tissues). But their cooling graphs are still quite impressive:

Twenty-first century, too, has something to boast about. For a long time cryopreserving a kidney remained an unattainable goal — upon defrosting, both its structural integrity and function were critically impaired. But in the early 2000s, the star of cryobiology, Gregory Fahy, was able to finally achieve it. By the way, it is quite symbolic that this was done under the auspices of a company called 21st Century Medicine.

In his experiments, Fahy would remove a kidney from a rabbit, vitrify it and cool to a certain temperature, then thaw it and transplant back to the donor, while also removing the second, healthy kidney. In 2003, Fahy was able to find a successful combination of cryoprotectants and a rather complex vitrification protocol, which allowed him to successfully cool the kidney to -22°C or -45°C, and for two rabbits, he was able to achieve cooling to as low as -130°C. Notably, while 30 rabbits survived various cooling protocols to between -22°C or -45°C, only 2 rabbits had their kidneys cooled to -130°C. Of those one died nine days after backward transplantation, but the second lived for 48 days after which it was sacrificed for histological analysis.

By the way, this famous photo, that demonstrates a striking difference between a frozen and vitrified kidney originates from a 1984 (!) work by Fahy et al.:

In 2008, a group of Israeli scientists reported of successful preservation of murine and porcine livers, as well as rat hearts, but their method of verifying viability was quite limited and did not include transplantation back into living animals to verify actual organ function.

Compared to the kidney, the brain is considered by cryobiologists to be more friendly to cryopreservation. Many studied showed its full or partial freezing or vitrificaton without subsequent structural damage. Moreover, several studies have even demonstrated preservation of some of its functions.

The most intriguing such studies were done by a Japanese cryobiologist Isamu Suda. In 1966, Suda published a paper in which he claimed that he was able to detect electrical activity in cat brains after they have been frozen at -20°C for several months. Here is an excerpt of EEG graphs from his work:

And here is how his perfusion apparatus looked like:

In 1974, Suda published another, more detailed paper. It it, he showed that even after 7 years of being frozen at -20°C cat brains exhibited synchronized electrical activity for a few hours after thawing, although of lower quality than that of control brains not subjected to freezing. He also compared EEG of “fresh” brains and brains after 5 days of storage at -20°C; their results were virtually identical.

Here I must mention that to date no one has managed to reproduce Suda’s results. At the same time, no one has really attempted an identical step-by-step reproduction. Before his retirement, Suda sent a copy of all his experimental notes and data to Gregory Fahy, who did not see in them any signs of falsification.

Fahy, too, has done a lot of research in pursuit of an optimal protocol for cryopreservation of the brain. In 2016, along with Robert McIntyre and other colleagues from 21st Century Medicine, he was awarded the Small Mammal Brain Preservation Prize for technology that enabled perfect preservation of a rabbit brain’s histological structure. Unfortunately, that technology involved certain irreversible processing of such brain (or fixation) with aldehyde, rendering impossible any biological brain function upon thawing, but this does not negate the importance of this achievement for cryobiology.

Long before that, in 2006, Fahy along with Yuri Pichugin et al. demonstrated that with a certain combination of cryoprotectant and vitrification protocol, slices of rat brains could be perfectly preserved even after cooling to -130°C: more than 90% of their samples retained their structure and even the potential for electrical activity (as measured by the proportion of sodium and potassium ions as compared to the control).

Moreover, in 2007, 21st Century Medicine announced that it was able to directly confirm the preservation of basic electrical “learning ability” (long-term potentiation, LTP) in rabbit brain slices after vitrification, and later elaborated on those results in a 2012 book chapter Cryopreservation of Precision Cut Tissue Slices:

Figure 6B. Lack of effect of vitrification on the long term potentiation (LTP) response, a form of neurophysiological “memory” which consists of a permanent increase in the magnitude of the response to a given CA3 cell stimulation (recorded in this case as the amplitude of the excitatory post-synaptic field potentials at the Schaffer collateral-CA1 dendrite junction) as a result of prior “training” (intensive stimulation) of the involved synapses. Control brain slices increased their field EPSP response to about 30% above the baseline response amplitude (LTP ratio of about 1.3) in response to prior “training”. The same basic result was also seen after loading and unloading of VM3 (LU); after loading of VM3, vitrification, rewarming, and unloading of VM3 (VIT); and after storage of vitrified slices for days to months below the glass transition temperature (STR; storage time had no effect on the results obtained). n values represent the number of independent experiments represented by each bar. Previously unpublished data of 21st Century Medicine.

Fahy and Pichugin’s research helped establish an optimal cryoprotectant composition and perfusion protocol for cryonics patients. Today, anyone can look at the perfused brains of some of these cryonics patients thanks to the miracles of computer tomography and Youtube:

In the frame below, we see that the patient’s brain contains almost no ice (blue), and is well-saturated with cryoprotectant (green, purple and orange):

Here are two other patients:

In 2016, Suda’s cat brain experiments got upstaged by an even stranger paper when several Canadian scientists from Sudbury published significantly more incredible (or actually, less credible, in the view of many) results. They claimed that they were able to detect electrical activity in human brains that have been stored in formalin for over 20 years:

When Is the Brain Dead? Living-Like Electrophysiological Responses and Photon Emissions from…

Formalin — is a well-known tissue fixative that is widely used in the storage of various biological samples precisely because it irreversibly arrests the state of the tissues at the cellular level. Roughly speaking, it “glues” cells shut, turning them into jelly. Thus, no one expected aldehyde-fixed tissues to maintain any biological function — if only because fixation perforates cell membranes making them unable to pump protons, which is a prerequisite for transmission of electrical impulses. That is why virtually everyone is highly skeptical of the Canadians’ claims. At the same time, there are no grounds to suspect outright falsification, so it would be very helpful if some seasoned cryobiologists, especially those with experience in studying viability of brain tissue, tried to reproduce that work.

What is the importance of experiments demonstrating the brain’s ability to restore its function after an interruption in its functioning (even a short one, and even without freezing)? Because they show that cryonics can work: after all, almost all neuroscientists agree that long-term memory, and therefore our personality, is encoded in physical structures of the brain, and not in its electrical activity. Moreover, the electrical activity of the brain naturally ceases below +18°C, but with proper recovery that does not have long-term negative consequences. We see evidence of that claim in humans undergoing profound hypothermia or brain surgery (source1, source2), as well as in experimental animals — from nematodes and hamsters to primates.

In fact, various medical procedures that involve stopping brain activity with subsequent full recovery have been repeatedly validated in dozens of experiments on animals, and are even presently undergoing clinical trials in humans in the United States — see the EPR CAT trial of suspended animation, which is evaluating cooling gunshot victims to 10°C. Here is a scientific paper describing the study:

Emergency preservation and resuscitation for cardiac arrest from trauma. — PubMed — NCBI

And here is a popular article in New Yorker:

Can Hypothermia Save Gunshot Victims?

Finally, here’s another ER doctor who uses cooling to assist in the recovery of heart attack victims. By the way, he believes that within 20 years medicine will be able to restore patients 12 or even 24 hours after the onset of “clinical death”:

Back from the Dead: Resuscitation Expert Says End Is Reversible — SPIEGEL ONLINE — International

Prior to use in humans, the above suspended animation technology has been tested in our very close relatives — pigs. Just as in human EPR CAT patients, those pigs had all their blood drained and replaced by a cooling saline solution while bringing their body temperature to 10°C. In total, more than 200 pigs have undergone this procedure with successful recovery:

https://www.ncbi.nlm.nih.gov/pubmed/18404056
https://www.ncbi.nlm.nih.gov/pubmed/16456447
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4166101/

Before pigs, a similar protocol was tested in dogs:

Survival without brain damage after clinical death of 60–120 mins in dogs using suspended animation…

Moreover, Mike Darwin, a cryonics star and former Alcor CEO, was even able to prolong the duration of bloodless anabiosis to 5 hours. That means a dog spent 5 hours completely without blood, and then was fully restored, including its long-term memory — recognizing familiar people and responding to commands. And it was not an isolated experiment. Together with Jerry Leaf, Mike had performed a whole series of “total body washouts”:

Alcor’s Pioneering Total Body Washout Experiments

Also, I want to give honorable mention to a cat that in 1980s managed to recover after an hour of heart stoppage — and that was without any cooling:

Recovery of integrative central nervous function after one hour global cerebro-circulatory arrest…

All these data allow us to hypothesize that, even with a delay of several hours between the onset of clinical death and the beginning of perfusion of the cryonics patient, the latter still retains reasonable chances of future restoration of his brain functions. First, because even without cooling, irreversible brain changes begin to occur only after 1–2 hours, and neuronal necrosis begins only after 4–6. And secondly, because the brain is a highly plastic organ that is able to recover from serious injuries.

Let me mention just a couple of cases of such recovery.

Here is US Senator Gabrielle Giffords, who in 2011 was shot right through the brain, and six months later returned to work in the Senate. The left photo is of her in the hospital, and on the right one she is skydiving a few years later.

Here’s another case of a successful recovery from a bullet wound to the brain, Rachel Barezinski. The bullet passed through the brain, but Rachel survived and recovered:

Moreover, some people undergo removal of an entire hemisphere:

Hemispherectomy — Wikipedia

And some people survive even worse brain injuries:

7 People Who Lost Half of Their Brain but Survive

And even if all other bodyparts of the cryonics patient will not be suitable for recovery, as long as the brain remains viable, there remains a chance to restore the patient’s identity. After all, several head transplantation experiments have been successfully performed by the fathers of transplantology, Vladimir Demikhov and Sergei Brukhonenko, as far back as the 1930s. The photo below shows a living dog’s head separated from its body:

There is even archive video footage of several two-headed dogs, some from Demikhov, some from Brukhonenko (in Russian):

Here’s a longer, English-language original clip from 1940:

By the way, it was Demikhov’s pioneering work that paved the way to successful human kidney and heart transplants, as was confirmed by Christiaan Barnard himself — the first surgeon to successfully transplant the human heart in 1967. Demikhov also inspired Robert J. White to perform his head transplant research that culminated in a successful monkey head transplant. In his research, White also found that the brain is not rejected by the recipient, unlike other organs.

All of the above supports the idea that the main task of cryonics is to ensure maximum viability of the brain for future revival.

About that revival

Proponents of cryonics like to joke that there already are thousands of revived cryonics patients among us. The only caveat is that they were frozen and revived while they were still embryos. Still, that is quite a successful achievement of cryobiology. Especially considering that some of these cryonauts were frozen for decades:

Baby born from embryo frozen 20 years ago

Embryos are great, but they are only 4 or 8 cells large. However, there are more significant examples. For example, some women with cancer undergo removal of their ovaries, to protect them from chemotherapy, have them frozen and then transplanted back. And these ovaries resume their function: women who have undergone these procedures have given birth to more than 70 children:

Ovarian tissue cryopreservation and transplantation: scientific implications

Unfortunately, cryobiology can not yet boast of something more significant — for example, of recovering a mammal after cooling it to temperatures below 0°C. It is rumored that someone was trying to do something similar with pigs, but public confirmation of this has yet to surface.

The bottom line

• no need to turn cryonics into a cult, all problems and issues must be recognized and not swept under the rug
• many areas of improvement still remain
• but we are already reasonably good at brain preservation

Sources (other than those linked to throughout):

• Cryonics: Reaching for Tomorrow by Brian Wowk and Michael Darwin
• Scientific Justification of Cryonics Practice by Ben Best:
  https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4733321/
• Low Temperature Biology of Insects by D.L. Denlinger
• Igor Artukhov’s lecture:
  https://www.youtube.com/watch?v=7f5KNsXSeFQ
• Cryopreservation of Precision Cut Tissue Slices by Gregory M. Fahy, Na Guan, Inge A.M. de Graaf, Yuanshen Tan, Lenetta Griffin, Geny M. M. Groothuis. Xenobiotica (2012).
• Ben Best’s Cryonics website: http://www.benbest.com/cryonics/cryonics.html