Even better cryonics – because who needs nanites anyway?

post by maxikov · 2015-04-07T20:10:49.757Z · score: 49 (52 votes) · LW · GW · Legacy · 41 comments

Abstract: in this post I propose a protocol for cryonic preservation (with the central idea of using high pressure to prevent water from expanding rather than highly toxic cryoprotectants), which I think has a chance of being non-destructive enough for us to be able to preserve and then resuscitate an organism with modern technologies. In addition, I propose a simplified experimental protocol for a shrimp (or other small model organism (building a large pressure chamber is hard) capable of surviving in very deep and cold waters; shrimp is a nice trade-off between the depth of habitat and the ease of obtaining them on market), which is simple enough to be doable in a small lab or well-equipped garage setting.

Are there obvious problems with this, and how can they be addressed?

Is there a chance to pitch this experiment to a proper academic institution, or garage it is?

Originally posted here.


I do think that the odds of ever developing advanced nanomachines and/or brain scanning on molecular level plus algorithms for reversing information distortion - everything you need to undo the damage from conventional cryonic preservation and even to some extent that of brain death, according to its modern definition, if wasn't too late when the brain was preserved - for currently existing cryonics to be a bet worth taking. This is dead serious, and it's an actionable item.

Less of an action item: what if the future generations actually build quantum Bayesian superintelligence, close enough in its capabilities to Solomonoff induction, at which point even a mummified brain or the one preserved in formalin would be enough evidence to restore its original state? Or what if they invent read-only time travel, and make backups of everyone's mind right before they died (at which point it becomes indistinguishable from the belief in afterlife existing right now)? Even without time travel, they can just use a Universe-sized supercomputer to simulate every singe human physically possible, and naturally of of them is gonna be you. But aside from the obvious identity issues (and screw the timeless identity), that relies on unknown unknowns with uncomputable probabilities, and I'd like to have as few leaps of faith and quantum suicides in my life as possible.

So although vitrification right after diagnosed brain death relies on far smaller assumptions, and if totally worth doing - let me reiterate that: go sign up for cryonics - it'd be much better if we had preservation protocols so non-destructive that we could actually freeze a living human, and then bring them back alive. If nothing else, that would hugely increase the public outreach, grant the patient (rather than cadaver) status to the preserved, along with the human rights, get it recognized as a medical procedure covered by insurance or single payer, allow doctors to initiate the preservation of a dying patient before the brain death (again: I think everything short of information-theoretic death should potentially be reversible, but why take chances?), allow suffering patient opt for preservation rather than euthanasia (actually, I think it should be done right now: why on earth would anyone allow a person to do something that's guaranteed to kill them, but not allowed to do something that maybe will kill, or maybe will give the cure?), or even allow patients suffering from degrading brain conditions (e.g. Alzheimer's) to opt for preservation before their memory and personality are permanently destroyed.

Let's fix cryonics! First of all, why can't we do it on living organisms? Because of heparin poisoning - every cryoprotectant efficient enough to prevent the formation of ice crystals is a strong enough poison to kill the organism (leave alone that we can't even saturate the whole body with it - current technologies only allow to do it for the brain alone). But without cryoprotectants the water will expand upon freezing, and break the cells. But there's another way to prevent this. Under pressure above 350 MPa water slightly shrinks upon freezing rather than expanding:

Phase_diagram_of_water.svg

So that's basically that: the key idea is to freeze (and keep) everything under pressure. Now, there are some tricks to that too.

It's not easy to put basically any animal, especially a mammal, under 350 MPa (which is 3.5x higher than in Mariana Trench). At this point even Trimix becomes toxic. Basically the only remaining solution is total liquid ventilation, which has one problem: it has never been applied successfully to a human. There's one fix to that I see: as far as I can tell, no one has ever attempted to do perform it under high pressure, and the attempts were basically failing because of the insufficient solubility of oxygen and carbon dioxide in perfluorocarbons. Well then, let's increase the pressure! Namely, go to 3 MPa on Trimix, which is doable, and only then switch to TLV, whose efficiency is improved by the higher gas solubility under high pressure. But there's another solution too. If you just connect a cardiopulmonary bypass (10 hours should be enough for the whole procedure), you don't need the surrounding liquid to even be breathable - it can just be saline. CPB also solves the problem of surviving the period after the cardiac arrest (which will occur at around 30 centigrade) but before the freezing happens - you can just keep the blood circulating and delivering oxygen.

Speaking of hypoxia, even with the CPB it's still a problem. You positively don't want the blood to circulate when freezing starts, lest it act like an abrasive water cutter. It's not that much of a problem under near-freezing temperatures, but still. Fortunately, this effect can be mitigated by administering insulin first (yay, it's the first proper academic citation in this post! Also yay, I thought about this before I even discovered that it's actually true). This makes sense: if oxygen is primarily used to metabolize glucose, less glucose means less oxygen consumed, and less damage done by hypoxia. Then there's another thing: on the phase diagram you can see that before going into the area of high temperature ice at 632 MPa, freezing temperature actually dips down to roughly -30 centigrade at 209~350 MPa. That would allow to really shut down metabolism for good when water is still liquid, and blood can be pumped by the CPB. From this point we have two ways. First, we can do the normal thing, and start freezing very slowly, so minimize the formation of ice crystals (even though they're smaller than the original water volume, they may still be sharp). Second, we can increase the pressure. That would lead to near-instantaneous freezing everywhere, thus completely eliminating the problem of hypoxia - before the freezing, blood still circulated, and freezing is very quick - way faster than can ever be achieved even by throwing a body into liquid helium under normal pressure. Video evidence suggests that quick freezing of water leads to the formation of a huge number of crystals, which is bad, but I don't know near-instantaneous freezing from supercooled state and near-instantaneous freezing upon raising the pressure will lead to the same effect. More experiments are needed, preferably not on humans.

So here is my preservation protocol:

  1. Anesthetize a probably terminally ill, but still conscious person.
  2. Connect them to a cardiopulmonary bypass.
  3. Replacing their blood with perfluorohexane is not necessary, since we seem to be already doing a decent job at having medium-term (several days) cardiopulmonary bypasses, but that could still help.
  4. Submerge them in perfluorohexane, making sure that no air bubbles are left.
  5. Slowly raise the ambient pressure to 350 MPa (~3.5kBar) without stopping the bypass.
  6. Apply a huge dose of insulin to reduce all their metabolic processes.
  7. Slowly cool them to -30 centigrade (at which point, given such pressure, water is still liquid), while increasing the dose of insulin, and raising the oxygen supply to the barely subtoxic level.
  8. Slowly raise the pressure to 1 GPa (~10kBar), at which point the water solidifies, but does so with shrinking rather than expanding. Don't cutoff the blood circulation until the moment when ice crystals starts forming in the blood/perfluorohexane flow.
  9. Slowly lower the temperature to -173 centigrade or lower, as you wish.

 

And then back:

  1. Raise the temperature to -20 centigrade.
  2. Slowly lower the pressure to 350 MPa, at which point ice melts.
  3. Start artificial blood circulation with a barely subtoxic oxygen level.
  4. Slowly raise the temperature to +4 centigrade.
  5. Slowly lower the pressure to 1 Bar.
  6. Drain the ambient perfluorohexane and replace it with pure oxygen. Attach and start a medical ventilator.
  7. Slowly raise the temperature to +32 centigrade.
  8. Apply a huge dose of epinephrine and sugar, while transfusing the actual blood (preferably autotransfusion), to restart the heart.
  9. Rejoice.

 

I claim that this protocol allows you freeze a living human to an arbitrarily low temperature, and then bring them back alive without brain damage, thus being the first true victory over death.

But let's start with something easy and small, like a shrimp. They already live in water, so there's no need to figure out the protocol for putting them into liquid. And they're already adapted to live under high pressure (no swim bladders or other cavities). And they're already adapted to live in cold water, so they should be expected to survive further cooling.

Small ones can be about 1 inch big, so let's be safe and use a 5cm-wide cylinder. To form ice III we need about 350MPa, which gives us 350e6 * 3.14 * 0.025^2 / 9.8 = 70 tons or roughly 690kN of force. Applying it directly or with a lever is unreasonable, since 70 tons of bending force is a lot even for steel, given the 5cm target. Block and tackle system is probably a good solution - actually, two of them, on each side of a beam used for compression, so we have 345 kN per system. And it looks like you can buy 40~50 ton manual hoists from alibaba, though I have no idea about their quality.

cryoshrimp

I'm not sure to which extent Pascal's law applies to solids, but if it does, the whole setup can be vastly optimized by creating a bottle neck for the pistol. One problem is that we can no longer assume that water in completely incompressible - it had to be compressed to about 87% its original volume - but aside from that, 350MPa per a millimeter thick rod is just 28kg. To compress a 0.05m by 0.1m cylinder to 87% its original volume we need to pump extra 1e-4 m^3 of water there, which amounts to 148 meters of movement, which isn't terribly good. 1cm thick rod, on the other hand, would require almost 3 tons of force, but will move only 1.5 meters. Or the problem of applying the constant pressure can be solved by enclosing the water in a plastic bag, and filling the rest of chamber with a liquid with a lower freezing point, but the same density. Thus, it is guaranteed that all the time it takes the water to freeze, it is under uniform external pressure, and then it just had nowhere to go.

Alternatively, one can just buy a 90'000 psi pump and 100'000 psi tubes and vessels, but let's face it: it they don't even list the price on their website, you probably don't even wanna know it. And since no institutions that can afford this thing seem to be interested in cryonics research, we'll have to stick to makeshift solutions (until at least the shrimp thing works, which would probably give in a publication in Nature, and enough academic recognition for proper research to start).

41 comments

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comment by mikedarwin · 2015-04-09T06:54:23.565Z · score: 49 (51 votes) · LW · GW

I was asked by several people to comment on this post/proposal. Clearly, Maxikov put a lot of time and effort into this post and, at least in part, there's the pity. When you find you have an idea which seems at once compelling and obvious (in tems of the science) in an already well explored field, the odds are very good that you weren't the first to reach that conjecture. And that almost always means that there is someting wrong with your premises. Very smart and capable people have been trying to achieve cryopreservation of cells, tissues, organs and organisms for over 50 years now and the physical chemistry of water under very high pressures and very low temperatures has been understood for far longer. This should be a hint that some careful searching of the literature is in order before going public with a proposal to "fix cryonics," and especially before spending a lot of time/energy on proposal like this.

Attempts to use extreme hydrostatic pressure to mitigate or eliminate freezing injury go back at least 60 years, and probably longer. As your phase diagram above shows, when the pressure is sufficiently high during cooling the expansiuon of water is prevented, but ice formation is not. What happens is that other allotropes of ice form which do not require expansion. However, this turns out to be a bad thing, since, as opposed to any of these ices being formed first in the interstitial spaces, as happens with Ice I, freezing occurs both intracellularly and extracellularly at the same time in the presence of other ice allotropes. Crystal formation inside cells results in devastating ultrastructural disruption - far worse than would occur if ice formed outside cells first, grew slowly and dehydrated the cells, and finally resulted in a vitrified cellular interior (providing that cryoprotectant is present).

However, the problem with this approach doesn't stop there. Extreme hyperbaria itself is directly damaging by at least two mechanisms: denaturation of cellular proteins (including critical enzymes and membrane proteins) and damage to cell membrane lipid leaflets resulting in permeabilization of the membrane to ions (Onuchic LF, Lacaz-Vieira F., Glycerol-induced baroprotection in erythrocyte membranes. Cryobiology. 1985 Oct;22(5):438-45.) Irreversible membrane damage occurs in mammalian red cells exposed to a pressure of 8000 atm (~117,600 psi) applied for ~10 minutes. Exposure of more comnplex mammamalian cells to far lower pressures~20,000 psi, results in loss of viability due to protein denaturation, and perhaphs due to alterations in the molecular structure of membrane lipids,as well. Interestingly, the same compounds that provide protection cellular (molecular) protection against freezing damage also confer substantial protection against baroinjury. Fahy, et al., have extensively explored the use of hyperbaria to augment vitrification in the rabbit kidney (http://www.freepatentsonline.com/4559298.pdf) and have further extended work from the 1980s demonstrating that cryoprotectives are also substatntially baroprotective.

The first work that I'm aware of to attempt to achieve organ cryopreservation using hyperbaria was that of the late Armand Karow, in the late 1960s - early 1970s (Karow AM Jr, Liu WP, Humphries AL Jr. Survival of dog kidneys subjected to high pressures: necrosis of kidneys after freezing.Cryobiology. 1970 Sep-Oct;7(2):122-8. PMID: 5498348). Karow was able to demonstrate the brief tolerance of dog kidneys to pressures of about ~18,000 psi, however, kidneys subjected to isothermal hyperbaric freezing, even in the presence of of moderate cryoprotection, did not survive.

When I started research and experimentation in cryobiology nearly 40 years ago, there was no Internet, no (affordable) photocopiers and the only way to do a "literature search" was with something called the Index Medicus (http://en.wikipedia.org/wiki/Index_Medicus) which was a veritable wall of bound volumes. I used 3" x 5" index cards to write down possible cites to look up - which then required a trip(s) to the "stacks" to look for the journals. Today, I have the Internet, Pubmed, the international patent database and on line library for 30 million books available. I currently have a digitial library of 12,000 mostly scientific and technical books which, at its current rate of growth, should double in size within a few months. My computer is almost constantly reading a book to me with software that cost me just under $5.00. One of the books I "read" recently was The Shallows: What the Internet Is Doing to Our Brains by Nicholas Carr. Carr argues that the Internet is fundamentally altering the way most people today process information - and not for the better. I don't use the Internet the way most people seem to, today. I rely heavily on books, especially textbooks, to educate me about areas with which I have little or no familiarity, and my approach is pretty much what it has been since I started my intellectual life; namely to study intensively and deeply until I achieve basic mastery of an area, and only then use skimming and browsing over large amounts of material to advance my knowledge. The tools of the information-digitial age have thus been a nearly unblemished advantage to me. If you want to reads Carr's book, click on this link:

http://www.mediafire.com/download/5s4wdr554ia4axn/Nicholas_Carr-The_Shallows__What_the_Internet_Is_Doing_to_Our_Brains_(2010).epub and then click on the green Download button.

I'm also posting links to a number of full text books on cryobioolgy which you can download, as per above:

ADVANCES IN BIOPRESERVATION: https://www.mediafire.com/?raccqhv0rrqfhmh

ADVANCES IN LOW TEMPERATURE BIOLOGY: https://www.mediafire.com/?4i6v9qublf3l8q2

FUNDAMENTALS OF CRYOBIOLOGY: https://www.mediafire.com/?pxq6mxbxvfib41j

CURRENT TRENDS IN CRYOBIOLOGY: https://www.mediafire.com/?pxq6mxbxvfib41j

CRYOPRESERVATION... https://www.mediafire.com/?pxq6mxbxvfib41j

LIFE IN THE FROZEN STATE: https://www.mediafire.com/?ydx3a89m2f47r7y

THE FROZEN CELL: https://www.mediafire.com/?ydx3a89m2f47r7y

Cheers, Mike Darwin

comment by drethelin · 2015-04-09T07:06:51.069Z · score: 7 (7 votes) · LW · GW

Thank you very much for the effort involved in this post and that you put in in general.

comment by maxikov · 2015-04-09T09:36:39.594Z · score: 6 (6 votes) · LW · GW

Thanks so much for the detailed review and lots of useful reading!

comment by mikedarwin · 2015-04-09T21:32:23.263Z · score: 22 (22 votes) · LW · GW

My pleasure!

I have a few (hopefully helpful) comments to add. I am a huge advocate of trying things yourself on a do-able scale. For instance, many years ago I had pretty much the same idea you did and I decided to it out, directly. I lived across the street from a mechanical engineer from Eli Lilly, Inc., named Bud Riever. I asked Bud to figure how much prsssure would be developed if I simply cooled a closed steel container which was completely filled with water to well below the frrezing point? The answer was about 2,000 atmospheres, or about 24,000 psi. As it turns out, a piece of steel pipe of the right thickness threaded on both ends and capped with screw on galvanized steel pipe caps will hold that pressure. And, since it is hydrostatic pressure with no gas present, if the pipe fails (splits), it will not fail explosively. My test subject was to be Baker's yeast, reconstituted in a dilute sugar solution and placed inside of a twist tied sawdwhich bag (no air bubbles) which was in turn placed inside the section of pipe which was then capped on the open end.

It took me forever to figure out that the only way to close the pipe with the yeast inside, whilst excluding also all air bubbles, was to do so in a galvanized metal wash tub filled with water. The cap on the pipe was screwed shut under water in tub. I could then cool my self-pressurizing chamber with a slush of dry ice and acetone. I broke several pipes before I found a thickness of steel that would take the pressure. Alas, my experiment showed only a little better survival of yeast under pressure than that which was achieveable under the same conditions with a vented pipe; i.e., almost none.

Maybe two years ago, I got the idea that inhaled hydrogen gas might be profoundly radioprotective. H+ should be available to neutralize the OH- radicals produced by the interaction of gamma rays and water, thereby acting as an "instantaneous" neutralizer of the bulk of radiation injury (the bulk of the non-hydroxyl radical injury occurs when high energy particles directly impact and disrupt DNA). I did a literature search and found nothing. I also asked a medical physicist friend and several other scientists whom I respected. I was told that this approach would not work in large measure because the addition of dissolved hydrogen would not deal with the problem of the hydrogen radical that would remain after the hydroxyl radical was neutralized. My hypothesis was that the hydrogen radical would react with oxygen to form another hydroxyl radical, and then subsequently be neutralized by the abundand molecular hydrogen.

After some months, I couldn't stand not knowing anymore so I found an industrial X-ray service with powerful enough X- and gamma ray sources to deliver ~16 gray of radiation to half a dozen mice in a reasonable pewriod of time and I cobbled up a test apparatus. The next step was to expose mice to supralethal doses of X- and gamma rays. Hydrogen gas at 80% of the breathing air (balance oxygen) was indeed profoundy protective. When I passed this information along to my medical physicist friend he quickly found cites of other (pretty obscure) work showing the same effect:

http://cdn.intechopen.com/pdfs/35987/InTech-Hydrogen_from_a_biologically_inert_gas_to_a_unique_antioxidant.pdf

Qian LR, Cao F, Cui JG, Huang YC, Zhou XJ, Liu SL, Cai JM: Radioprotective effect of hydrogen in cultured cells and mice. Free Radic Res 2010, 44:275-282. PubMed Abstract | Publisher Full Text OpenURL

Qian LR, Li BL, Cao F, Huang YC, Liu SL, Cai JM, Gao F: Hydrogen-rich pbs protects cultured human cells from ionizing radiation-induced cellular damage. Nuclear Technology & Radiation Protection 2010, 25:23-29. PubMed Abstract | Publisher Full Text OpenURL

Alas, my dreams of a commercializable product that would render radiolgical exams effectively safe for children, young and middle aged adults vanished, well, as in a puff of hydrogen and oxygen igniting. But here (to me) is the really strange thing, despite the stunning degree of radiprotection inhaled hydxrogen gas proivides, as well as evidnce that it is pluripotent protect against ischemia-reperfusion injury, cancer and a variety of other free radical mediated pathologies (http://www.molecularhydrogeninstitute.com/studies/), no one I know has shown the slighest interest in it. So, even if you identify something that is workable and easy to implement, don't expect the world to beat a path to your door!

Nevertheless, DOING THINGS and actually carrying out experiments changes how you think, how you approach problem solving and how your brain is wired. These changes are, for the most part, empowering and make you better problem solver.

comment by maxikov · 2015-04-09T22:25:37.714Z · score: 3 (3 votes) · LW · GW

That's actually surprising: I thought yeast survives freezing reasonably well, and http://www.ncbi.nlm.nih.gov/pmc/articles/PMC182733/?page=2 seems to confirm that. What was different in your setup so that even the control group had a very low survival rate?

comment by mikedarwin · 2015-04-10T02:38:38.296Z · score: 12 (12 votes) · LW · GW

Most of my childhood notes and cryo-memrobilia were lost when my house burned down in September, of last year. So, regrettably, I can't consult my notes from those experiments. However, as best I recall, the mortality rate in yeast frozen in distilled water was ~90%. No special treatment was required beyond removing them from the incubating medium and resuspending them in distilled water prior to freezing. Viability was determined indirectly by adding the frozen-thawed yeast in water to culture medium in an Erlenmeyer flask connected to a water displacement set-up very much like this:

http://herbarium.usu.edu/fungi/funfacts/respiration.jpg

I later repeated this experiment with red cells (my own) which is much more sensitive and directly quantative of cell survival. You do, however, need a centrifuge and related equiupment to measure microhematocrit - things I could easily acquire back in the day (and in fact, still have).

If people did hands-on biology in the same way and to the same extent they do hands-onelectronics and programming, we'd all likely be either "immortal," or dead, by now.

Here is an experiment I am currently struggling to tool to do which may serve as an example. Recently, a very simple way was discovered to induce apoptosis in a significant fraction of senescent cells in vivo in rodents, and in human cell culture cells, as well: http://onlinelibrary.wiley.com/doi/10.1111/acel.12344/pdf. This results in partial rejuvenation of the animals because senescent cells release myriad toxic cytokines, chemokines and other pro-inflammatory and probably telomere shortening species. While there is as yet no evidence that eliminating senescent cells - or reducing their number - will increase lifespan, there is ample evidence that it will greatly increase healthspan. This new class of drugs has been dubbed the "senolytics" by their discoverers, Zu and Tchkonia. The nice things about these two drugs is that they are both small molecules which are readily available, FDA approved/GRAS and have very low toxicity. One is the OTC nutrient quercetin, and the other is the relatively exotic molecularly targeted antineoplastic agent dasitinib, marketed under the name of Sprycell by Bristol-Meyers-Squibb.

In mice, one dose of these agents in combination was effective at reducing the senescent cell burden dramatically, with benefits lasting for 7 months. The cost of a dose of dasitinib for an adult human is about $400 - eminently affordable (the cost of the quercetin required is a few cents). So, what's the problem? Well, if you are over 30, odds are that you have a significant burden of senesacent cells, and by the time you are 50, somewhere between 15 to 30% of your body mass may be senescent cells. In my days in ICU doing hemodialysis, I saw more than a few patients critically ill and in renal failure from something called "acute cell death syndrome" (ACDS) which most often resulted from chemotherapy given to lymphoma or leukemia patients too rapidly, resulting in a massive die-off of cancer cells. Large scale cell death is toxic and can be, and often is, lethal.

Animals treated with dasitinib+quercetin do not show signs of ACDS. However, careful monitoring of blood chemistrires during the treatment phase was not done and the animals so far studied were middle aged rodehts - not humans, and certainly not older, or elderly humans. Thus, additiional data are needed. In my opinion, dogs are ideal for such a study because they are available in abundance as old and very old (senile) animals, have large blood volumes which allow for harmless routine clinical laboratory evaluations, and have neurobehavioral faculties which are easily and reliably assessed by untrained humans. They also stand to benefit from the treatment if it does not prove lethal, or can be adjusted so that it is easily tolerated.

You have to "make" your own aged rodents and that takes years. And years are something many of us no longer have... Research begun now (or soon) will very likly yeild results that will be immediately clinically applicable to humans. Unfortunately, this research cannot practically be done anywhere in the West legally.

comment by EGI · 2015-05-26T17:44:24.045Z · score: 0 (0 votes) · LW · GW

I would be VERY interested in reading that http://onlinelibrary.wiley.com/doi/10.1111/acel.12344/pdf paper. Unfortunately the link does not work for me (page not found).

comment by gwern · 2015-05-26T17:54:57.002Z · score: 4 (4 votes) · LW · GW

When a link doesn't work, try googling a unique-looking prefix. In this case, 'acel.12344' looks like a unique ID. If I google "http://onlinelibrary.wiley.com/doi/10.1111/acel.12344/", the first hit is http://onlinelibrary.wiley.com/doi/10.1111/acel.12344/abstract which is the paper "The Achilles’ heel of senescent cells: from transcriptome to senolytic drugs", Zhu & Tchkonia et al 2015 in Aging Cell; note that the journal sounds relevant, both Zhu and Tchkonia were mentioned by Darwin, the keyword 'senolytic' is present in the title, and the abstract reads:

The healthspan of mice is enhanced by killing senescent cells using a transgenic suicide gene. Achieving the same using small molecules would have a tremendous impact on quality of life and the burden of age-related chronic diseases. Here, we describe the rationale for identification and validation of a new class of drugs termed senolytics, which selectively kill senescent cells. By transcript analysis, we discovered increased expression of pro-survival networks in senescent cells, consistent with their established resistance to apoptosis. Using siRNA to silence expression of key nodes of this network, including ephrins (EFNB1 or 3), PI3Kδ, p21, BCL-xL, or plasminogen-activated inhibitor-2, killed senescent cells, but not proliferating or quiescent, differentiated cells. Drugs targeting these same factors selectively killed senescent cells. Dasatinib eliminated senescent human fat cell progenitors, while quercetin was more effective against senescent human endothelial cells and mouse BM-MSCs. The combination of dasatinib and quercetin was effective in eliminating senescent MEFs. In vivo, this combination reduced senescent cell burden in chronologically aged, radiation-exposed, and progeroid Ercc1−/Δ mice. In old mice, cardiac function and carotid vascular reactivity were improved 5 days after a single dose. Following irradiation of one limb in mice, a single dose led to improved exercise capacity for at least 7 months following drug treatment. Periodic drug administration extended healthspan in Ercc1−/∆ mice, delaying age-related symptoms and pathology, osteoporosis, and loss of intervertebral disk proteoglycans. These results demonstrate the feasibility of selectively ablating senescent cells and the efficacy of senolytics for alleviating symptoms of frailty and extending healthspan.

Hence you can be immediately confident that this must be the paper Darwin was linking. (Or if the link heuristic didn't occur to you, you could have tried googling the buzzwords in Google Scholar; "senolytics senescent cells in vivo in rodents, and in human cell culture cells" would have turned up that paper as #5, and the preceding papers all look relevant too. And if that didn't work, you could have searched "author:Tchkonia", since it's a highly unusual surname, and it would be #9 in Google Scholar.)

The paper can be downloaded from Wiley right now, but if it couldn't, you could have still gotten a copy from Libgen.

comment by EGI · 2015-05-26T18:11:36.489Z · score: 0 (0 votes) · LW · GW

Thanks!

comment by EGI · 2015-05-26T17:54:57.922Z · score: 0 (0 votes) · LW · GW

Huh after copying the link to my own post, it works! The link in the above post still does not. Weird!

comment by Vaniver · 2015-05-26T21:23:31.973Z · score: 1 (1 votes) · LW · GW

It's the period at the end of the link, which Darwin included to end the sentence and which you did not (because your sentence continued).

comment by Capla · 2015-04-09T15:50:58.512Z · score: 4 (6 votes) · LW · GW

With which of those books should I start?

comment by mikedarwin · 2015-04-09T20:36:27.196Z · score: 7 (7 votes) · LW · GW

I'd say FUNDAMENTALS OF CRYOBIOLOGY, followed by Baust's ADVANCES in BIOPRESERVATION. However, you may find another starting point better. I recently felt the need (out of self defense) to learn about dentistry. That's a bit like saying I decided to learn about neurosurgery:that covers a lot of ground. However, mostly what I was interested in was plain old restorative dentistry and the much more exotic implant dentistry. There are easily half a dosen textbooks on basic, restorative dentistry... After perusing a number, I settled on one as a proper "read through" introduction. All were adequate, but only that one really communicated in my style. The good thing about most modern textbooks is that there are now study and review guides and, of course, mock-up Board exams. This kind of learning allows me to get a good basic grasp of what is being done to me and to overcome the "condescension" factor when speaking with the dental professionals treating me. Please note, it does NOT make me a dentist! I wish I could recommend the same thing vis a vis cryobiology or cryonics. But I can't. I've tried to get support to start a formal training college for cryonics professionals (I actually have some funding), but have been laughed at, or dismissed out of hand - perhaps justifiably so. Nevertheless, that is what needs to be done and textbooks, study guides, testing and certification do not occur until a discipline is professionalized and formally taught.

comment by Joshua_Blaine · 2015-04-07T22:08:00.177Z · score: 18 (20 votes) · LW · GW

First off, I love that you're actively pursuing alternative methods of human preservation. That's awesome, and I hope you manage to find some useful ideas in your search. However, I fear that this approach in particular doesn't really solve the problem that cryoprotectants successfully do (toxicity briefly aside).

without cryoprotectants the water will expand upon freezing, and break the cells.

This line in particular is my biggest point of contention. I am by no means an expert in this field, and my understanding may be moot in this context, but the expansion of water-ice crystals isn't the central concern for frozen biological cells. A quickly found source claims that:

Since ice is essentially pure H2O, ice formation can increase the concentration of minerals in the remaining cytosol to a toxic level. The increased mineral concentration in the cytosol will cause water to be drawn in from the surrounding cells by osmosis, which can cause the cell to swell and burst.

Alcor's official FAQ also says that:

When tissue is slowly cooled, ice first forms between cells. The growing ice crystals increase the concentration of solutes in the remaining liquid around them, causing osmotic dehydration of cells.

Your method doesn't prevent the formation of ice crystals, it merely changes the structure of the crystals, and at what temperature they form, so I suspect harmful cell osmosis can still occur. Of course, I could be insufficiently understanding why ice crystals effect the mineral concentration of cytosol, or the order in which certain biological areas freeze under variable conditions, and your smaller ice/lower freezing temperature would successfully prevent this issue. I don't believe this is necessarily the case, given your explanation, but if anyone who's more studied in these fields could speak up, I'd be happy to defer to their expertise.

comment by maxikov · 2015-04-07T22:59:03.378Z · score: 10 (10 votes) · LW · GW

It seems like the approach of cooling the organism to -30C at 350MPa, and then raising pressure further to ~600Mps to freeze it could actually solve that. As far as I understand, the speed of diffusion in water it far slower that the speed of sound (speed of sound at 25C is 1497 m/s, while diffusion coefficient for protons at 25C is 9.31e-5 cm^2/s, which corresponds to 1.4e-4 m/s - 8 orders of magnitude less), which is the speed of pressure gradient propagation. So if we use raising pressure as a way to initiate phase transition, it will occur nearly simultaneously everywhere, and the solutes won't have time to diffuse anywhere.

ETA: I just realized that since diffusion propagates according to inverse square law, while sound is linear, they should be compared to each other at the shortest distance possible. So I checked the time it takes for a proton to cover 0.1nm (hydrogen atom diameter) in water - 5.37e-13s, which gives us 186 m/s. It's far greater than the original number, but still an order of magnitude smaller than the speed of sound. And if we take 4nm (the thickness of a cell membrane) we have 8.59e-10s - only 4 m/s, so it decreases very quickly, and we're pretty much safe.

comment by Joshua_Blaine · 2015-04-08T01:38:50.755Z · score: 9 (9 votes) · LW · GW

That's a very sound (pun partially intended) insight, and I don't immediately see a significant reason for why that shouldn't be the case.

However, humans aren't perfectly uniform spheres of water (to borrow from a common physics joke), so some concerns do still exist. Namely: Pressure might propagate through them less predictably/quickly than just water, and different areas of the body might begin freezing at different pressures/in different orders (which can, however, be countered by raising pressures quickly).

I have updated significantly in the direction of "This idea might actually be very valuable to cryonics proponents," for sure.

comment by [deleted] · 2015-04-08T09:37:01.145Z · score: 3 (3 votes) · LW · GW

Which is why, I think, it's better to start from sea mammals and not shrimp; imagine if, for some reason, tiny ice crystals damage blood vessels - not even due to bloodflow - and upon thawing all those clotting factors are immediately, chaotically released. It can even happen in the brain.

comment by maxikov · 2015-04-08T20:14:38.277Z · score: 2 (2 votes) · LW · GW

I'm sure I'm following why mammals should be less susceptible to this problem, can you elaborate?

Doing this with mammals has a lot of challenges though, which it'd make sense to bypass in initial experiments. The deepest dive (aside from humans in DSVs) is only 3km, which accounts for 30 MPa. I guess it's safe to say that no mammal can withstand 350 MPa with air or any gas in its lungs, so total liquid ventilation is required, which is just as challenging to do with sea mammals as with land mammals. Also, mammals are warm-blooded, and usually experience asystole at abnormally low body temperatures, which are nonetheless far above freezing. So there's the issue of making it survive the time it takes to go form cardiac arrest to freezing, which is also probably just as hard to do with sea mammals as with land mammals. So although the ultimate goal is to develop a protocol for humans, it'd the much easier to start with an animal that's already capable of surviving 100 MPa of ambient pressure and +4C of its own body temperature.

comment by [deleted] · 2015-04-08T20:56:00.650Z · score: 2 (2 votes) · LW · GW

I meant that in mammals of comparable sizes, you have brains with comparable sizes - and, ultimately, if you salvage a brain all is not lost. Also, they have definable behaviour, which (as you approach more harsh experiments, like the ability to recognize kin after being thawed) might tell you something useful. How would you interpret a shrimp's ability to move after thawing? And all that blood chemistry - the closer it gets to human, the better. Starting with shrimp is useful at the very beginning, to see if it can be done at all, maybe.

As to mammals, perhaps mice are better to begin with, because they are smaller than we. I just thought - without checking - that sea mammals are tougher when it comes to oxygen depletion combined with evenly distributed heightened pressure. I can be wrong.

BTW, what do you think of Tardigrada, water bears?:)

comment by maxikov · 2015-04-08T21:47:01.003Z · score: 1 (1 votes) · LW · GW

Ah, that's true. I guess going back to normal vitals and motion is good enough for preliminary experiments, but of course once that step is over, it's crucial to start examining the effects of preservation on cognitive features of mammals.

Tardigrada and some insects are in fact known to survive ridiculously harsh conditions, freezing (combined with nearly complete dehydration) included. Thus, it makes sense to take a simple organism that isn't known to survive freezing, and make it survive. I suspect though that if you can prevent tardigrades from dehydrating before freezing, the control group won't survive, which means that some experiments can possibly be done on them too.

comment by passive_fist · 2015-04-08T03:15:54.919Z · score: 11 (11 votes) · LW · GW

This is definitely an interesting idea but there may be a lot of unforeseen problems. At pressures as high as 350 MPa, chemical reactions that are energetically unfavorable under normal pressure suddenly become favorable (this is why, among other things, air becomes toxic). I have no idea how this would mess with the various chemical processes that go on in the body, but my guess is that unwanted reactions could lead to development of fatal toxins. Also, just as such high pressures cause phase changes in ice, they could cause phase changes in plenty of other molecules.

I'm sure someone who is more knowledgeable on this could weigh in, but googling around a bit revealed some studies on subjecting human cells to high pressure. This study subjected human amnion cells to 70 MPa and found significant changes in cell activity involving blebbing (this is when the cell membrane disassociates from the cytoskeleton), although they did point out that the effects of pressure were reversible, which is promising.

comment by maxikov · 2015-04-08T19:58:54.731Z · score: 7 (7 votes) · LW · GW

Hmm, I wonder what the exact biochemistry that prevents life forms (including, apparently, vertebrate fish) in Challenger Deep at 111 MPa from experiencing these problems is, and whether it can be replicated in mammals.

They also mentioned that blebbing first appears at 90-120 seconds, but that's way too short even for the fastest protocols possible. Theoretically, it's not unthinkable to cool the body to just above 0C, and then go straight to 632 MPa and above, to make it instantly freeze, before blebbing occurs. And then, if total liquid ventilation allows one to drop the pressure that quickly as well, just go from solid directly to a non-dangerous pressure range. But for any protocol that involves temperature changes under pressure, tens of seconds is positively too short to allow the temperature to stabilize.

As for toxicity though, I though it was entirely due to the increased partial pressure of oxygen (which thus creates too strong of an oxidizing environment) and having too many nitrogen atoms dissolved in tissues, physically messing with fine-grain biochemistry like ion channels. Is there another chemical component of toxicity beyond that?

comment by JenniferRM · 2015-04-09T07:31:29.517Z · score: 5 (5 votes) · LW · GW

9 . Slowly lower the temperature to -173 centigrade or lower, as you wish.

If I'm reading the chart correctly, the additional cooling would send the ice III through the zone marked as ice II and then... wait for it... into the zone of ice nine!!!

If the secret of eternal life involves the non-fictitious version of ice IX... I mean... that seems like "the author" would be clubbing us over the head with the implication that we're living in a post-modern novel :-P

On a less metaphysical note, it seems like there is a technical question about whether additional cooling might cause problems due to transitions between different kinds of ice? From Le Wik on the real ice IX (not the fictional ice-nine):

Ice IX is a form of solid water stable at temperatures below 140 K and pressures between 200 and 400 MPa. It has a tetragonal crystal lattice and a density of 1.16 g/cm³, 26% higher than ordinary ice. It is formed by cooling ice III from 208 K to 165 K (rapidly—to avoid forming ice II). Its structure is identical to ice III other than being proton-ordered.

It looks like if you were in the ice IX zone, and then heated up from LN2 temperatures, you would necessarily go through ice II on the way to liquid water (see this awesome site):

Ice-nine (ice IX) is the low-temperature equilibrium, slightly denser, structure of ice-three (Space group P41212, cell dimensions 6.692 Å (a) and 6.715 Å (c) at 165 K and 280 MPa [385]). It is metastable in the ice-two phase space and converts to ice-two, rather than back to ice-three, on warming. The change from proton disordered is a partial process starting within ice-three that is only completed at lower temperatures, but with a first order transition near 126 K[1087]. The hydrogen bonding is mostly proton-ordered as ice-three undergoes a proton disorder-order transition to ice-nine when rapidly cooled in liquid nitrogen (77 K, so avoiding ice-two formation, see Phase Diagram); ice-three and ice-nine having identical structures apart from the proton ordering [389].

From what I can tell, if you start at ice III and cool things way down from there, you'll have to spend some time in the ice II zone, at the very least while being re-heated up from ice IX and perhaps as the state to be kept in for very long term storage. Luckily, ice II appears to also have a density of ~1.16 g / cm^3, so it is also denser than normal water and presumably would also not pop cellular membranes due to expansion :-)

comment by oge · 2015-04-08T06:58:26.156Z · score: 5 (7 votes) · LW · GW

Thanks for taking the time to write this up and putting numbers to things: it makes it actually possible to evaluate your idea critically.

The thing that jumped out at me was the amount of pressure required for human preservation. What kinds of devices can generate 100KBar of pressure?

Edit: changed GBar to KBar

comment by TrE · 2015-04-08T08:35:52.203Z · score: 8 (8 votes) · LW · GW

Materials science undergraduate student here (not a mechanical engineer, my knowledge is limited in the area, I did not go to great lengths to ensure I'm right here, etc.).

A typical method to generate high pressures in research are diamond anvils. This is suitable for exploring the behavior of cells and microorganisms under high pressure.

For human preservation, however, you'd need a pressure vessel. As the yield strength of your typical steel is on the order of 100, maybe 300 MPa, you're really up against a wall here, materials-wise. I don't doubt that suitable alloys for human-sized pressure vessels at 350 MPa exist, however, such vessels will be expensive, and controlling processes within will be difficult. In any case, generating such pressures will probably not involve a moving piston.

I can't really tell whether or not the procedure you've outlined is viable, but I'm quite sure it's far from trivial, just from an engineering point of view.

The concerns of user passive_fist are also valid.

comment by maxikov · 2015-04-08T18:47:39.559Z · score: 3 (3 votes) · LW · GW

That's an interesting observation! When I was looking into this, I found several suppliers[1][2][3][4] that claim to produce pressure vessels, tubing, and pumps all the way up to 150'000 psi (1GPa). If 300MPa are already pushing the boundaries of steel, do you know what they could use to achieve such pressures?

comment by passive_fist · 2015-04-09T01:23:38.869Z · score: 4 (4 votes) · LW · GW

One common technique is composite construction with carbon fibers wound concentrically around an alloy core.

comment by maxikov · 2015-04-09T02:51:46.453Z · score: 1 (1 votes) · LW · GW

Is that done to convert shear force to tension?

I wonder, how much can be achieved by merely increasing the thickness of the walls (even to such extremes as a small hole in a cubic meter of steel)?

comment by passive_fist · 2015-04-09T03:23:20.886Z · score: 1 (1 votes) · LW · GW

To my understanding it's because of the higher tensile strength of carbon fiber, although I could be wrong.

I wonder, how much can be achieved by merely increasing the thickness of the walls (even to such extremes as a small hole in a cubic meter of steel)?

In a round vessel containing pressure, a pressure gradient is set up from the inside wall to the outside. You can think of such a vessel as a series of concentric shells of increasing radius, each of which only has to support the pressure differential acting upon it. At some pressure level, this pressure differential itself becomes so high that it tears the material apart, regardless of how thick the walls are or how tiny the interior radius is. The physics of this isn't terribly complicated but I don't have any links at the moment, sorry.

comment by maxikov · 2015-04-09T03:39:20.989Z · score: 0 (0 votes) · LW · GW

Sure, I can easily imagine that by mentally substituting steel with jello - at some point you're tear it apart no matter how thick the walls are. However, that substitute also gives me the impression that most shapes we would normally consider for a vessel don't reach the maximum strength possible for the material.

comment by TrE · 2015-04-09T04:37:55.640Z · score: 0 (0 votes) · LW · GW

Most vessels are spherical or cylindrical, which is already pretty good (intuitively, spherical vessels should be optimal for isotropic materials). You might want to take a look at the mechanics of thin-walled pressure vessels if you didn't already.

It's important to note that the radial stresses in cylindrical vessels are way smaller than the axial and hoop stresses (which, so to say, pull perpendicular to the "direction" of the pressure). This is also why wound fibers can increase the strength of such vessels.

comment by passive_fist · 2015-04-09T01:14:29.842Z · score: 2 (2 votes) · LW · GW

350 MPa is about 3.5 KBar, not 100 GBar.

comment by DanielLC · 2015-04-07T21:59:27.289Z · score: 4 (4 votes) · LW · GW

While we're looking at high pressures, supercritical drying doesn't look like a bad idea. It has no phase change, so it might be less damaging than even what you're suggesting. It's not something you could just wake someone up from, but it might work better than normal cryonics. Once they're dried, you don't need the pressure, so it's easier to sustain. And even if the temperature does rise for a short time, the lack of water will probably help preserve them longer.

comment by RolfAndreassen · 2015-04-09T04:46:32.920Z · score: 3 (3 votes) · LW · GW

I'll observe that cold vessels fail gradually; pressure vessels may fail catastrophically.

comment by mikedarwin · 2015-04-10T03:27:01.578Z · score: 6 (6 votes) · LW · GW

Actually, cryogenic vessels do not really fail, in the sense I think you mean, over time - with the notable exception of liquid helium and liquid hydrogen storage vessels. Liquid helium has bizzare effects of metal (in addition to quantum tunneling) causing high strength steel to embrittle over time. It is thoought that this occurs due to the presence of helium in solid solution in the metal subjected to loading, and being present at a temperature sufficiently low to form grain boundary cracks as a result of sliding along grain boundaries (which contain steps developed as a result of prior intragranular shear).

Hydrogen "embrittlement" is due to migration of lone hydrogen atoms into the metal where they re-combine in sub-micron sized voids in the metal matrix to form hydrogen molecules. In so doing, they create pressure from inside the cavity where they are located which can increase in vulnerable areas of the metal (e.g., where it has reduced ductility and tensile strength) to the point where the metal develops first micro-cracks and then a large, macro-fracture resulting in castastrophic failure.

Liquid nitrogen storage containers kept dry and free from liquid oxygen accumulation, and which remain stationary, can and do last "indefinitely." They will require periodic re-hardening of the vacuum, but this is not due to structural failure, but rather is due to outgassing of materials from the reflective/convective barrier wrap and of hydrogen from hydrogen inclusions in the welds. If the units are not man-handled and are well cared for, there is essentially no work-hardening of the welds, or of the structural metal itself, and they may well last for many decades, or even centuries. If the nitrogen gas boil-off were used to create a dry nitrogen sheild around the exterior of the vessels, their lifespan would likely be in the range of many centuries. Work-hardening, hydrogen ingress into the metal from water condensed from the air and corrosion from atmospheric oxygen and water at the neck-tube are the three principal causes of structural cryogenic dewar failure. If the dewar is not moved about, and if water is eliminated from the environment, stainless steel dewars should last indefinitely. I've seen dewars in semen storage facilities that are 50 years old and have not yet required rehardening of their vacuum. Conversely, I've seen vessels in lab use and used to haul industrial gases fail after a few years, or even a few months of use. TLC is almost everything when it comes to liquid nitrogen dewars.

Probably the best example of how robust ultra-high pressure vessel engineering can be is to look to long range guns on battleships. These tubes are about 2" in diameter shy of being big enough to hold an average human and can withstand pressures in the range Maxikov is talking about. These "vessels" also have a breech and operate under horrible conditions wih respect to heat and corrosion. And yet, failure is almost unheard of. When failure means the loss of a battle ship, failure is not an option; consider that one turret on a 20th century battleship, exclusive of the guns, cost ~$1.5 million, U.S. These guns were made with mid-1920's technology and remained in service until the last decade of the past century. Then, there was the Paris-Geschütz (http://en.wikipedia.org/wiki/Paris_Gun) of Krupp, but that's another story...

comment by gjm · 2015-04-07T23:41:12.849Z · score: 3 (3 votes) · LW · GW

Is "natines" in the title meant to be "nanites"?

comment by maxikov · 2015-04-08T00:14:50.916Z · score: 2 (2 votes) · LW · GW

Yep, fixed that, thanks.

comment by [deleted] · 2015-04-08T09:21:13.046Z · score: 2 (2 votes) · LW · GW

Very neat idea, but what kind of energy demands would be required to maintain that level of pressure over the long term? I also think the structural demands of maintaining pressure would be substantial.

comment by passive_fist · 2015-04-09T01:25:48.627Z · score: 1 (1 votes) · LW · GW

Theoretically, zero. However you're right that the structural demands of maintaining pressure over long term (and, especially, maintaining cryogenic temperatures and high pressures at the same time) are high and there is a large risk of unintended pressure release.

comment by TrE · 2015-04-09T04:50:00.979Z · score: 1 (1 votes) · LW · GW

There's also leakage by diffusion of gasses, which might be non-negligible due to the high pressure gradient, although the diffusion coefficient e.g. of water through steel should be low. Not sure how that works out.

comment by CronoDAS · 2015-04-08T02:08:53.657Z · score: 0 (2 votes) · LW · GW

Alternatively, one can just buy a 90'000 psi pump and 100'000 psi tubes and vessels, but let's face it: it they don't even list the price on their website, you probably don't even wanna know it. And since no institutions that can afford this thing seem to be interested in cryonics research, we'll have to stick to makeshift solutions (until at least the shrimp thing works, which would probably give in a publication in Nature, and enough academic recognition for proper research to start).

Maybe we could turn to crowdfunding? (A list of crowdfunding sites for scientific research)