X-Message-Number: 7415 Date: 03 Jan 97 23:56:01 EST From: "Kent, Saul" <> Subject: SCI.CRYONICS Brain Vitrification Bob Ettinger writes in Cryonet #7398: >1. WHY WAIT FOR VITRIFICATION? >According to relatively recent published and unpublished word >from Greg Fahy--if I understand it correctly--rabbit kidneys (and >perhaps other specimens) can already be preserved, using his >methods, by vitrification, and stored at liquid nitrogen >temperature without significant damage. The main problem >remaining is the RF rapid thawing required, which has also >already been solved in theory but not yet in practice. >From the standpoint of near-future patients, why not use these >vitrification methods (if applicable to brains)? A very good question indeed (although a bit puzzling given that CI has until now promoted the Visser method as more promising than vitrification). The suggestion of preserving cryonics patients by vitrification instead of freezing is at least 15 years old, having been written about by Mike Darwin, Jerry Leaf, Thomas Donaldson and others since the first seminal papers by Greg Fahy appeared in the early 1980s. The current state-of-the-art in vitrification is perhaps best summarized in the technical library of the Prometheus Project Web site at: http://www.prometheus-project.org/prometheus One clarification that should be made is that while recovery of vitrified organs from deep sub-zero temperatures is indeed theoretically possible with RF rewarming, substantial technical problems may still have to be overcome. For instance, injury to the vascular endothelium of the kidney as a result of _chilling injury_ (quite distinct from _freezing injury_) was a major obstacle for some years to recovering kidneys which had been loaded with a vitrifiable amount of cryoprotectant (i.e., VS41A) and cooled to, and rewarmed from, relatively high sub-zero temperatures without vitrification or freezing. Thus, it remains to be seen if rates of rewarming of between 100 and 300 degrees C per minute and the many associated problems, will pose additional obstacles to recovering viable rabbit kidneys following vitrification. Ettinger continues: >The thawing >problem (large licensed RF transmitters etc.) can be solved at >leisure. We would really then HAVE brain cryopreservation that is >reversible to a near-certainty. This exact approach has been the focus of 21st Century Medicine's (21CM) and BioPreservation's (BPI) brain cryopreservation program for that past 4 years. Careful readers of Cryonet will note that Mike Darwin has posted that such research has been ongoing, and has further stated that he is cautiously optimistic "that BPI has perfused its last optimally stabilized patient with glycerol." In fact 21CM and BPI have recently commissioned the construction of a sub-zero operating theatre for the express purpose of deep sub-zero introduction of cryoprotectants into human cryopreservation patients with the objective of vitrifying patients, instead of freezing them. (Construction of this room is now complete, and delivery is awaiting an improvement in L.A. area weather conditions.) The creation of a sub-zero perfusion capability for whole humans raises a number of interesting issues which bear on Ettinger's post, many of which cannot, regrettably, be discussed in detail here. However, a general discussion is possible and falls under the heading of "Obstacles to application of Fahy's vitrification solutions to human cryonics patients." Ettinger notes: >From the standpoint of public relations, of course there would >be very limited favorable reaction to e.m. photos showing no >discernible damage to the vitrified brains. Unfortunately, brain vitrification is not a simple matter, for direct toxicity to the central nervous system of vitrifiable cryoprotective solutions such as VS41A means that methods developed for kidneys cannot not be expected to be immediately translatable to the brain. For instance, the application of VS41A and a large number of other vitrification mixtures to brains with subsequent EM examination has yielded results inferior to those achieved by BPI with glycerol applied under optimum conditions and in the proper concentrations. Even the Visser agent (dimethyl formamide) and other peralkylamides, while more penetrating than glycerol, were found to be intensely hemolytic (membrane toxic) at vitrifiable concentrations. (Peralkylamides were also found to be poor cryoprotectants at lower concentrations, including 25%, in a variety of relevant systems.) Application of vitrification agents and technology to the brain and especially to the whole organism with a high degree of ultrastructural preservation is a complex proposition. Indeed, it is a proposition which has occupied 21CM and BPI for years and is currently consuming nearly $50K per month in resources (and has been for sometime). To the extent that Greg Fahy has had success with VS41A (a mixture of formamide, propylene glycol, DMSO, and a colloid), it has been due to the technologically demanding and costly careful control over the introduction and removal parameters for these comparatively toxic agents. It is necessary, for instance, to terminate loading of rabbit kidneys with VS41A at nearly 30 degrees below zero C! But, beyond this, it is also necessary to carefully control the shape of the cryoprotectant introduction "ramp" and to carefully control (via computer) many other parameters as well. As 21CM's and BPI's work shows, failure to do this results in severe membrane toxicity; in other words the cryoprotectants being used to achieve vitrification dissolve the cell membranes and other lipid containing structures of the brain. This injury is quite apart from any damage inflicted on cellular metabolic machinery and is of _primary_ concern to cryonicists, or should be anyway, since cells without membranes are not cells anymore and are unlikely to yield recovered cryopreservation (cryonics) patients with any technology we can now foresee. These observations notwithstanding, 21CM and BPI have made what we believe to be very significant progress in the application of vitrification to whole brains and intact humans. Many problems with membrane toxicity, temperature control, the need for sub-zero introduction of cryoprotectants, and the development of novel cryoprotectants suited to achieving excellent ultrastructural preservation of the brain, have now been largely overcome. The associated technologies and cryoprotective agents are proprietary, and patents are pending. Application of this technology to whole dogs has demonstrated the feasibility of its use in human cryopreservation patients, and technological tooling for application to humans is underway. The sub-zero operating room/perfusion capability is but one example of this-- there are many more. Mike Darwin, Biopreservation, Inc. Steven B. Harris, M.D., 21st Century Medicine Brian Wowk, CryoCare Foundation Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=7415