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maxikov comments on Even better cryonics – because who needs nanites anyway? - Less Wrong
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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
Thanks so much for the detailed review and lots of useful reading!
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.
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?
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.
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).
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:
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.
Thanks!
Huh after copying the link to my own post, it works! The link in the above post still does not. Weird!
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).