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.
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:
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.
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.
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
Next up for discussions is the issue of "hyperonconicity." Just as cells require a certain "tonicity" (electrolyte concentration) to maintain their normal volume, tissues with capillaries require a certain concentration (and type) of large (macro-) molecules (colloid) to avoid accumulating water between the cells and becoming swollen, or edematous. Hyperonconicity refers to any solution that has more ability to hold water in the circulatory system (circulating blood or perfusate) than would be the case under NORMAL conditions. The key word there is NORMAL. The macromolecules that comprise colloids can be thought of as molecular sponges that hold water in the capillaries and prevent it from accumulating in between cells as a result of the hydrostatic pressure of perfusion.
This water holding ability is quite complex and nuanced and depends upon the condition of the junctions between the cells in the capillary, the charge of the colloid, the unique chemical properties of the colloid (poorly understood), the configuration of the colloid molecule, and so on.
Onconicity and hyperonconicity are thus in actual practice, relative terms - relative to the condition of the capillary membrane. It is quite possible to have a markedly hyperoncotic perfusate and still have massive edema due to accumulation of water and of the colloid in between the cells! This is so because injured capillary membranes do not behave the same way as healthy or intact ones do - they leak! They leak colloid and with the colloid goes water. Simply cooling the organs (or bodies) of non-hibernating animals results in increased capillary permeability and the leakage of colloid and water into the spaces between cells. There is currently not a complete understanding of why this happens, or why some colloids do not leak as much in the cold as do others. In fact, only a very few species of colloid have been shown to leak less in hypothermia.
Capillary injury and consequent leakage of colloid from ischemia is vastly worse than that induced by hypothermia alone, and no colloid has been identified which is effective at inhibiting this leak, or even reducing it enough in clinical settings to meaningfully change outcome. In the setting of serious ischemic injury in the presence of high concentrations of cryoprotectant, NO COLLOID OR OTHER MOLECULAR SPECIES HAS BEEN SHOWN TO SIGNIFICANTLY REDUCE EDEMA - INCLUDING the PEGs OF VARIOUS MOLECULAR WEIGHTS. CI's own research associates had reported this to CI prior to Ben's decision to do an ad hoc experiment on a human patient with absolutely no prior laboratory animal or even bench testing of the perfusate. The only thing more unconscionable than such an uninformed and reckless action is the continued denial that it was such, and that his "mistake did not have the disastrous consequences implied by Mike Darwin."
Here is what Ben Best says about the outcome for Curtis Henderson in terms of cryoprotective concentration at the end of perfusion:
" The refractive index of the effluent was 1.366 after six liters of VM−1 had been perfused, and was 1.3586 at the end. Intermediate values were as low as 1.3586 and as high as 1.3651, but this was a small range with no trend, and is indicative of random variation. These values are well below the values of 1.416 for 60% VM−1 and 1.4275 for 70% VM−1 — and they showed no trend."
Based on the reported refractive indexes of the venous effluent, I would estimate that Curtis Henderson had approximately 20% to 20% cryoprotectant in his brain - most of which was ethylene glycol. That would (again roughly estimating) equate to about 1.5M to 2.0 M glycerol in terms of colligative cryoprotective effect. I have not yet posted the electron micrographs (EMs) of the damage incurred when 3M glycerol is used as a cryoprotectant in the cat brain, but the histology is posted here: http://wp.me/p1sGcr-lt . I can tell you that the EM's are vastly worse than are the light micrographs.
I have provided a detailed, and I believe accurate, scientific rebuttal to Ben Best's claims. For onto a decade I have privately urged CI to either stop advertising that they are perfusing human patients under conditions which yield 86.1% viability +/- 5.8% (for a 55% concentration CI-VM-1 at - 20 deg C FOR TEN MINUTES, followed by cooling to and rewarming from -130 degrees C at 0.3 degrees C/min) brain tissue viability and ultrastructure, when in reality they are treating patients with 70% VM-1 delivered at +7 (or higher) to -7 deg C (rarely) over far longer periods of time (hours) and in the presence of ischemic insults that typically run to many hours, or even days!
This isn't about elegance of writing, it's about facts, most of which are derived from CI's own website.
As you can see from the CI data above and below, patient temperatures never come anywhere near -7 degrees, let alone the -20 degrees C called out in either the original animal research, or in CI's own publicly posted protocol for how cryoprotective perfusion is to be administered. In fact, it is necessary to look a number of case reports to even document that CI is perfusing its p atients with VM-1 chilled in a mechanical freezer: "Perfusion with CI−VM−1 vitrification solution began at 3:04 A.M. The CI−VM−1 was at freezer temperature (about −20ºC) in contrast to the ethylene glycol, which was at refrigerator temperature (about 3ºC)" see: http://www.cryonics.org/reports/CI110.html In fact, this patient was one of the very few who achieved any subzero temperature during cryoprotective perfusion with VM-1:
Refractive Index values only taken during CI−VM−1 perfusion CI Patient 110: http://www.cryonics.org/reports/CI110.html
TIME (AM) Nasopharyngeal temperature (ºC) Flow rate(liters/minute) Pressure mm Hg RJVRI
3:07 8.25 1.07 102
3:08 6.9 1.06 101
3:09 5.3 1.07 100 1.3700
3:11 3.6 1.3769
3:16 4.3 1.39 101 1.3670
3:19 2.0 1.37
3:20 0.8 1.00 1.62 1.367
3:20 Perfusion Halted/Surgery
3:30 0.4 0.35 134 1.4166
3:33 −1.4 0.29 135
3:37 −2.6 0.26 120 1.42
3:40 −3.6 0.24 111 1.424
3:41 −1.4 0.29 135
3:43 −3.7 0.26 127 1.42
3:40 −3.0 0.28 126 1.454
3:45 −3.7 0.26 118
3:48 −3.9 0.28 128 1.4346
3:53 −5.3 0.28 125 1.4281
3:57 -5.6 0.27 122 1.4285
4:00 −5.8 0.26 120 1.4296
4:03 −5.8 0.26 117 1.4276
4:05 −5.8 0.26 117 1.4276
4:10 −5.7 0.26 115 1.4284
4:15 −4.6 0.26 114 1.4284
4:20 -3.8 0.26 109 1.4250
4:23 −3.0 0.27 86 1.4181
4:07 −2.3 0.34 82 1.4204
Since it is standard CI operating procedure (and a biological imperative to reduce toxicity) to pre-cool VM-1 in a freezer before use, and since PEG-VM-1 solutions invariably undergo gel formation/precipitation under such conditions, then how is it possible to say, as Ben Best does, "There is no incompatibility between DMSO and PEG"? In fact, there is, because PEG solutions with glycerol or ethylene glycol do NOT undergo this kind of transition - at least they didn't in my laboratory. Even more to the point, Aschwin & Chana deWolf, two researchers who work with CI reported this phenomenon to Best some weeks or months (as I recall) before he decided to conduct this ad hoc experiment on Curtis Henderson. I know this because i was a party to the correspondence.
Continued....
This Mickey Mouse operation results in perfusate that is at some (variable) subzero temperature when it is pumped through the perfusion circuit and delivered to the patient. While CI case reports are chaotic and inconsistent - some report temperature data during perfusion (http://www.cryonics.org/reports/CI97.html), some do not (http://www.cryonics.org/reports/CI75.html) - it is clear that even with the practice of pre-cooling the VM-1 perfusate in a freezer before perfusing it, CI patients never (so far as I can determine from published case reports, see: http://www.cryonics.org/refs.html#cases) reached subzero temperatures of -7 degrees C throughout VM-1 administration and in fact rarely reach subzero temperatures at all. This despite what CI says in its own description of how its patients are to be perfused with VM-1:
http://www.cryonics.org/research/CI-VM-1.html
"The Cryonics Institute protocol for perfusing the heads (brains) of cryonics patients is a 4-stage stepped open circuit perfusion:
(1) blood washout with carrier solution (4ºC) (2) 10% Ethylene Glycol (4ºC) (3) 30% Ethylene Glycol (4ºC) (4) 70% CI−VM−1 (−7ºC)"
I would also note that in the same document, it is stated that the positive research results achieved with VM-1 in rats were achieved only under these conditions:
*"To test the toxic effects of CI−VM−1 (with or without ice blockers) hippocampal slices were saturated with increasing concentrations of ethylene glycol at 0ºC and −7ºC before cooling to −20ºC for ten minutes of saturation with CI−VM−1 (with or without ice blockers). The DMSO in CI−VM−1 is less toxic at lower temperatures, and is least toxic when introduced at −20ºC. Adding the ethylene glycol first and cooling at 0.3ºC/minute ensured that the solution would not be frozen at −20ºC when the CI−VM−1 (with or without ice blockers) is introduced. The results of the toxicity test were as follows:
86.1% viability +/- 5.8% for 55% concentration CI-VM-1 without ice blockers 89.6% viability +/- 6.2% for 52% concentration CI-VM-1 with ice blockers*
Refractive Index values only taken during CI−VM−1 perfusion
CI Patient 97: http://www.cryonics.org/reports/CI97.htm
TIME (AM) TEMP (ºC) Flow rate(liters/minute) Pressure mm Hg RJVRI LJVRI
1:11 3.2 1.13 127
1:14 3.8 1.06 131
1:20 5.5 1.36 120 1.3976
1:26 7.0 1.07 117 1.3986
1:30 5.6 1.32 103 1.4017 1.4167
1:35 4.9 1.4048
1:37 4.1 1.4258 1.4242
1:40 3.5 1.4043 1.4183
1:45 2.5 1.4137 1.4209
1:47 2.0 1.4153 1.4224
1:50 1.6 1.15 139 1.4207 1.4236
1:52 Upper Body Perfusion Halted
2:00 Lower Body Perfusion Begun
2:00 0.5 0.42 121
2:03 0.5 0.32 136
2:05 0.5 0.32 134
2:10 0.5 0.31 143
2:13 0.5 0.40 200
2:15 0.5 0.46 185
2:20 0.5 0.46 175
2:25 0.5 0.48 191
2:33 0.5 0.48 174
Lower Body Perfusion Halted
Dry Ice Slurry Added to Head
2:37 −2.0
Refractive Index values taken during CI−VM−1 perfusion CI Patient 91: http://www.cryonics.org/reports/CI91.html
TIME (am) TEMP (ºC) RJVRI RBHRI LBHRI
9:35 7.0 1.4084
9:38 5.4 1.3655
9:40 4.2 1.4169
9:42 3.7 1.4198
9:46 2.1 1.4041
9:48 1.7 1.4138
9:50 1.8 1.4194
9:53 1.5 1.3721
9:55 1.1 1.4239
9:57 0.6 1.4206
10:00 0.4 1.3809
10:02 0.4 1.3830
10:07 0.7 1.4229
10:09 0.7 1.4233
10:11 0.6 1.3959
10:15 0.6 1.3971
10:16 0.8 1.4046
Continued....
This is a remarkable statement from Ben Best, and one that perhaps speaks best as to why CI is not a cryonics organization being run on a rational, scientific,or evidence based basis. When Ben Best writes: "There is no incompatibility between DMSO and PEG. The PEG make the solution hyperoncotic as the expected. My big mistake, and it was a bad one, I acknowledge, is that most of the vitrification solution was ruined because I was not aware that PEG would come out of solution when placed in a freezer," he is making a statement that has the following outright errors, misunderstandings or distortions in it:
First, DMSO and PEG are incompatible in that they cannot be used either safely or effectively under the conditions required to carry out cryoprotective perfusion in a clinical (or research) setting AS PRACTICED BY CI. The first fact to consider is that DMSO-PEG solutions will often undergo gel formation when cooled to temperatures above freezing if left under refrigeration long enough. This phenomenon has a variable time course and is akin to nucleation and freezing in supercooled solutions - such mixtures may remain clear for days, or undergo precipitation/gel formation within hours of cooling.
Second, the perfusate in question, VM-1, is designed to be administered at a SUBZERO temperature (-7 degrees C) in order to minimize toxicity. The final concentration of cryoprotectants in VM-1, a roughly equal mixture of DMSO and ethylene glycol (the latter is the principal ingredient in automotive antifreeze) and has a total concentration of these two agents of ~ 70%!
In the brain tissue slice experiments performed by CI's researcher Dr. Yuri Pichugin who invented VM-1, this very high concentration of agent was not introduced until the temperature of the brain tissue was -20 degrees C! CI's own protocol for human cryonics patients calls for the introduction of VM-1 at the lowest possible temperature that they can achieve (~ -7 degrees C), given that they have no heat exchanger in their patient perfusion circuit. The way CI attempts to get the temperature of the final pass of VM-1 below 0 degrees C, and as close to the ideal of -20 degrees C as possible, is by the expedient of placing bottles containing the perfusate into a standard household-type freezer. The pre-chilled bottles of perfusate are then loaded into picnic chests and the perfusate is dispensed from there.
Continued....
Brian, when you say: "Mike, let's be fair about this. Veterinary surgeons for thoracic surgery (after loss of Jerry Leaf) and chemists for running perfusion machines were also used during your tenure managing biomedical affairs at Alcor two decades ago. You trained and utilized lay people to do all kinds procedures that would ordinarily be done by medical or paramedical professionals, including establishing airways, mechanical circulation, and I.V. administration of fluids and medications. Manuals provided to lay students even included directions for doing femoral cutdown surgery," you are either not reading what I wrote or are not being fair yourself. I not only acknowledge that this was so, I go so far as to say it is completely acceptable with the caveat that such people are instructed, vetted and mentored properly. I'll go even further (as I have repeatedly, elsewhere) and state that the most highly qualified medical personnel can be dangerous, or even worse than useless unless they have been trained and mentored in human cryopreservation as a specialty. There's nothing remarkable about this; no reasonable person would want a psychiatrist or a dermatologist doing bowel or brain surgery.
Some of the same people who performed very well in the past, and who are not medically qualified, are still at Alcor. The individual people, per se (in this instance), are not the problem. Rather, it's the absence of the paradigm of cryonics as a professional medical undertaking that's missing. The evidence for that is present in Alcor's own case histories where highly qualified medical personnel do things like discontinue cardiopulmonary support on still warm patients in order to open their chests for cannulation (http://alcor.org/Library/pdfs/casereportA2435.pdf) or drill burr holes without irrigating the drilling site with chilled fluid to prevent regional heating of the brain under the burr. We are in complete agreement on these issues, as far as I can tell. Where we apparently differ is on how to resolve them.
The most interesting thing to me about this post from Brian is information it communicates for the first time. I follow Alcor's announcements, read its magazine and track its public blog, as I necessarily must, so I am surprised to learn that "In Alcor's O.R., Alcor is presently evaluating and training two board certified general surgeons to supplement the veterinary surgeon and neurosurgeon who have been used by Alcor for the past 15 years." This is the kind of information that I would expect to see showcased in the organization's literature and on its website, not disclosed here. This is the kind of thing that happens over and over and which degrades member confidence in the transparency of the organization. The next question is, who what, where and how? What are the details of this training? What kind of model is being used? What are the results to date?
Yes, SA does use pigs for training, but they use them in a non-survival mode - they get no robust feedback about errors, and no new insights. In fact, Brian might have mentioned that Alcor has used both animals and human cadavers in this manner, but I think he understood that the point I was making was about vetting your skills in an outcome driven fashion. That is not being done.
What's even more disturbing is that there is virtually no visibility into the outcome from even these training operations. SA and Alcor are both essentially black boxes - there is no data, no performance reports, not even any reports or internal scoring of how well simulated cases proceeded. There's at least one reason for this, and that is that there is no scoring system, internal or external. When things go wrong, well, it's oops, we shouldn't do that next time. And if that isn't the case, then I'd love to hear it and I want to see the data to document it. That is an eminently reasonable request.
It's great that Alcor can sometimes mount skilled perfusionists and highly skilled emergency vascular surgeons. But that isn't the issue. The issue is the framework of knowledge, understanding and consistent performance that is absent. A surgeon or a perfusionist are, absent mentoring (internship), TOOLS to be used by and within that framework. If a man tells me he has the best glass cutting tool money can buy, but he doesn't know how to cut glass, well, I'm going to be underwhelmed.
Alcor patient case reports are disorganized, inconsistent and erratic narratives that make objective evaluation impossible. No great genius is required to consistently collect and organize the key data that define how well a case went - or didn't. The first cryonics case report was done by a 17 year old and a 22 year old graduate student:
http://www.lifepact.com/images/MTRV3N1.pdf
Examples of competently executed cases and case reports are available on Alcor's own web site and the data captured, reduced and presented in these case reports was achieved using a tiny fraction of the financial and personnel resources Alcor currently has available:
http://www.alcor.org/Library/html/casereport8511.html
http://alcor.org/Library/html/fried.html
http://alcor.org/Library/html/casereportC2150.htm
http://alcor.org/Library/html/casereport8504.html
LOOK AT THESE CARE REPORTS CAREFULLY and then look at those on the Alcor website from 1997 forward: http://www.alcor.org/Library/index.html#casereports
I'm not trying to be contrary, difficult, or unreasonable. What I am asking for is core competence, not perfection. There is nothing either exotic or impossible in that. For example, Alcor has a Novametrix CO2SMO capnograph and respiratory function analyzer. The device can effortlessly capture and write to disk over 60 different respiratory parameters and it measures the end-tidal expired carbon dioxide (EtCO2) in the patient's breath. The EtCO2 is the gold standard for determining how effective cardiopulmonary support (CPS) is. And if CPS is not effective, than that is both additional ischemic time the patient is experiencing and it is an opportunity to intervene and fix the situation. Or at worst, it offers the possibility of learning what caused inadequate CPS so that it might be avoided next time. The only skill required to use the device is to put the walnut sized sensor in line between the patient's airway and the ventilator on the LUCAS CPR machine: http://frankshospitalworkshop.com/equipment/documents/pulse_oximeter/user_manuals/Novametrix_8100_-_User_manual.pdf That should make it easily possible to produce graphic data like this:
http://i293.photobucket.com/albums/mm55/mikedarwin1967/EtCO2inCPSgraph.png
THAT kind of data speaks definitively to how that patient was stabilized and transported, and in aggregate it provides a statistical dataset that speaks to the overall performance of the organization. It should be accompanied with graphic data for the patient's TEMPERATURE, mean arterial pressure (until the time of arrest), the SpO2 (pulse ox) and other relevant data. This was done in the past by stressed out, sleep deprived, mostly volunteer people who were trained in-house. If that kind of data collection and accountability are considered "perfectionist," or some kind of golden past no longer to be achieved, then I restate my opinion that something is terribly wrong.
Paramedics are taught that the single most important and most critical indication of the efficacy, or lack thereof, of CPR is the EtCO2 of the patient over time. Where is this data???? This is only one of countless examples I could use - but it is especially relevant because it is simple data to collect, and I know from Alcor's recent case reports that they have a CO2SMO and they are actually using it on patients during the peri-arrest hospice period. Again, where is the data? That data is the ONLY way anyone has to evaluate the quality of cryonics cases because the patients cannot speak to us.
If you want to stop my criticisms, you need only show me the data and offer me and everyone else the opportunity to be reasonably certain it is valid and representative.
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...