X-Message-Number: 33162 From: Date: Thu, 30 Dec 2010 05:02:30 EST Subject: Cryopreservation since 1990 Content-Language: en Date: Wed, 29 Dec 2010 11:21:21 -0800 Subject: Cryopreservation since 1990 From: Brian Wowk <> Mike Darwin wrote: >>Finally, the quality of cryopreservation most patients are now receiving is dismal and has, on average, deteriorated since 1990.>> I think Brian's response fails to account of two short, but very important words in my post:"on average." I'm not going to spend the time required to tot up the statistics, but you can go here: http://www.cryonics.org/patients.html, and you can go here: http://alcor.org/cases.html, and then do the math yourself. I am in complete agreement with Brian, that increasing the degree of cryoprotection (ice suppression), by increasing CPA concentration, trumps everything else that could be done to improve the quality of care for cryopatients. But that assumes that this is actually being implemented. And yes, I know it is being implemented occasionally, and I hope it will be implemented more consistently in the future. And no, I do not believe that patients who have experienced days of cold ischemia, absent any stabilization beyond being packed in ice hours or minutes post-cardiac arrest, will achieve even, let alone good equilibration with CPA, or that CPA perfusion will be as "benign' as is the case in patients with vastly less warm and cold ischemia. I don't think these are either controversial or unsupported statements. >>Nor are the effects of incomplete perfusion with vitrification solution as bad as Mike fears. Attempting to vitrify small tissue pieces with sub-vitrifiable concentrations of cryoprotectant is very bad because rapid cooling can cause them to freeze intracellularly. However human heads are so much larger than tissue pieces that even when cooling a head at the maximum possible rate, that rate in the brain is not much faster than the canonical 1 degC per minute that allows an average cell to dehydrate in response to growing extracellular ice and thereby avoid intracellular freezing. In other words, it appears plausible if not probable that the effect of poor perfusion or incomplete equilibration with vitrification solution in human cryopatients is freezing of those tissues in a manner similar to that seen with low concentration glycerol in the 1980s.>> This statement surprises me, and maybe Brian can add some clarifying detail that will resolve my puzzlement. I don't know anything about trying to rapidly cool small samples of tissue with sub-vitrifiable concentration(s) of CPAs in them. It's not an experiment I would do to try to resolve this issue, for exactly the reason that Brian subsequently points out: the maximum core cooling rate of a human brain (in situ in the head) is quite low. From the ata I've seen, the maximum achievable rate for an average sized human head is ~0.3 - 0.4 deg C/min, which is even slower (and therefore likely more favorable in the case of intact organs) than the value of 1 deg C/min, which Brian gives. So, as near as I can tell, this is not a particularly relevant experiment (though I admit it is an interesting and worthwhile one). What we are really interested in is what happens when we cool tissue loaded with a high concentration of CPAs, *and* ice inhibiting molecules, SLOWLY to the glass transition temperature (Tg). This is a very different situation than usually pertains when tissue is frozen in the presence of cryoprotectants for several reasons. First, tissue loaded with very high concentrations of CPA (but still not sufficiently high enough to permit vitrification) will have a strong tendency to undergo supercooling. Supercooling occurs when ice does not form at the "true' (thermodynamically prescribed) freezing point of the solution or tissue, and the system remains in a liquid state well below this point. When freezing does occur under such conditions, it happens with extreme rapidity, and there is usually insufficient time for the extracellular ice mass to osmotically extract water from the cells. The result is intracellular freezing. This is a posited, if not proved problem, in cryopreserving large masses of tissues that have been treated with cryoprotectant, where it is thought that regions of supercooling in may occur resulting in patch damage from intracellular freezing. In particular, it is posited (quite reasonably) that this will occur most often under conditions where there is minimal extracellular space and/or where the small volume of extracellular space is comprised of high viscosity fluid. This is a perfect description of a brain with regions of sub-vitrifiable CPA present, or worse still, where most or all of the brain has failed to reach the concentration needed to vitrify at the cooling rate the patient actually experiences. The brain has virtually no extracellular space with even the briefest exposure to ischemia. In fact, there was a great deal of debate for many years as to whether the brain had ANY non-vascular extracellular space, because of the difficulty of fixing brains under conditions that do not "abolish' the extracellular space. In addition, CPA perfusion can result in severe cerebral dehydration which will further increase tissue and intravascular fluid viscosity, and reduce/eliminate non-vascular extracellular space. There is also the issue of the ice inhibiting molecules X-1000 and Z-1000, that are present in 21st Century Medicine vitrification solutions (such as B2C and M-22 which have been used on human patients). Ice blocking molecules are wonderful things in the context of vitrification. They slow or stop ice formation during cooling and re-warming at rates slow enough that extensive freezing would normally occur. However, a consequence of this suppression of ice growth is that they, in effect, act as agents for the induction of supercooling under conditions of freezing. Finally, Arav, et al., the inventors of "directional freezing,' state that: "when crystallization starts at low temperatures, water will not leave the cells because dehydration occurs only in a certain range of high subzero temperatures, and at lower temperatures the membrane becomes impermeable. Therefore, supercooling must be avoided and crystallization should start at atemperature as close as possible to the freezing point of the solution." (Arav A, Natan Y. Directional freezing: a solution to the methodological challenges to preserve large organs. Semin Reprod Med. 2009 Nov;27(6):438-42. Epub 2009 Oct 5. Review. PubMed PMID: 19806511.) The freezing point of tissues loaded with a sub-vitrifiable concentration of CPAs could be quite low (i.e., -10 deg C, or even considerably lower) and if Arav, et al., are correct, then this would be yet another reason for concern over the possibility of intracellular freezing under these conditions. Despite the long road to reach it, my point is fairly simple: absent experimental evidence demonstrating that the occurrence of freezing in mammalian brains under conditions likely to attend sub-vitrifiable cooling and freezing of human brains (e.g., brains in heads), there should be no presumption that the attendant freezing damage is less than, or equivalent to, that which would occur under conditions of "conventional' slow freezing in the presence of cryoprotectant with adequate nucleation of the tissue. Indeed, it would seem (at least to me) that the presumption would favor intracellular freezing. The point is, the experiment must be done, and the results disclosed. On a different, but perhaps related topic, all these many years I have been deeply troubled by Audrey Smith's hamsters, and by my own red-eared slider turtles. Both can tolerate truly incredible amounts of ice formation in their kidneys and brains. Since the kidneys are located in the retroperitoneal space, it is reasonable to presume that in Smith's hamsters they experienced at least 40-50% ice formation. In the case of the brain, it is probably reasonable to assume that approximately 60% of its water content is converted into ice. And yet, if as little as 8% ice is formed in the medulla of the inadequately vitrified rabbit kidney, it is lethally injured. How can these two seemingly contradictory facts be reconciled? An experiment I've long wanted to do, but could never figure out how to carry out, is to find out EXACTLY how and where ice is forming in frozen hamsters and frozen turtles. I can't help but wonder if the answer to this question may prove critical to achieving workable cryopreservation for some organs and tissues. Currently, rabbit kidneys are failing to survive mostly (leaving viscoelastic injury aside) because of the formation of a "trivial' amount of ice, compared to what is acutely tolerable in WHOLE RABBITS. Smith not only froze hamsters, she froze rabbits as well, and while none survived long term, some did recover acutely, and were ambulatory. I continue to wonder if a great deal of "load' could be taken off vitrification if it were only possible to control the locus and extent of ice formation. This is perhaps not realistic, but it is certainly an idea that deserves some additional thought. I'm not a cryobiologist, and I cannot evaluate the scientific rigor of directional freezing. But, that may be beside the point if other investigators demonstrate that it is much less injurious than conventional cryopreservation of complex (as well as simple) tissues, and/or explain the mechanics. I'd be very interested in Brian's take on this? Selected References: Elami A, Gavish Z, Korach A, Houminer E, Schneider A, Schwalb H, Arav A. Successful restoration of function of frozen and thawed isolated rat hearts. J Thorac Cardiovasc Surg. 2008 Mar;135(3):666-72, 672.e1. PubMed PMID: 18329491. Gavish Z, Ben-Haim M, Arav A. Cryopreservation of whole murine and porcine livers. Rejuvenation Res. 2008 Aug;11(4):765-72. PubMed PMID: 18729808. Hubel A, Darr TB, Chang A, Dantzig J. Cell partitioning during the directional solidification of trehalose solutions. Cryobiology. 2007 Dec;55(3):182-8. Epub 2007 Aug 10. PubMed PMID: 17884036. Robeck TR, Steinman KJ, Montano GA, Katsumata E, Osborn S, Dalton L, Dunn JL, Schmitt T, Reidarson T, O'Brien JK. Deep intra-uterine artificial inseminations using cryopreserved spermatozoa in beluga (Delphinapterus leucas. Theriogenology. 2010 Oct 1;74(6):989-1001. Epub 2010 Jun 8. PubMed PMID: 20570326. Mike Darwin Content-Type: text/html; charset="UTF-8" [ AUTOMATICALLY SKIPPING HTML ENCODING! ] Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=33162