X-Message-Number: 15222 From: Date: Sun, 31 Dec 2000 11:24:49 EST Subject: Vitrification Instalment 2 U.S. PATENT 5,952,168 Wowk, Federowicz, Russell, & Harris (21st Century Medicine) Sep. 14, 1999 Method for vitrification of biological materials using alkoxylated compounds This is instalment 2 of my sometime series of background pieces. What follows is my summary and commentary, not the wording of the patent itself, except where quotation marks are shown. The reports and recommendations in this patent seem related to the Alcor reports of the Asilomar conference, but are not necessarily closely related to current Alcor procedures, which have not been disclosed publicly, as far as I know. As previously noted, the current Alcor "vitrification" procedures seem to be based on guesswork, there having been (to my knowledge) not a single mammalian brain reported anywhere, formally or informally, as vitrified to long term storage temperature and then rewarmed and studied. This is not necessarily a criticism, since life's decisions must often be based on one's best guess. ----- About 2-1/2 pages are devoted to "background," but the first paragraph of this really summarizes the invention as centering on temperature control, rapid internal cooling and rewarming of organs and tissues with an inert fluid, and use of a "class of new cryoprotective agents" (CPAs) used for "organ cryopreservation and cryonics." (Actually, the agents are not new--only their use for vitrification, rather than freezing, is new.) It is explained that suitable CPAs should be highly penetrating, non-toxic, strongly inhibit ice formation, and should vitrify at low concentration. In the couple of paragraphs devoted to cryonics, it says that "all the other agents" (than glycerol) have proven too toxic, too prone to cause edema, or result in worse ultrastructural preservation. (This is not necessarily completely correct--more on that another time.) Disadvantages of glycerol include viscosity and poor permeation, too much dehydration, poor preservation of myelinated areas of the central nervous system, and poor glass-forming characteristics. "The present invention discloses a class of new glycol ether cryoprotectant agents with excellent overall properties." After perfusion with CPA, rate and uniformity of cooling are crucial--several degrees per minute, with minimal temperature gradients to avoid fracturing. Rewarming must be greater than 100 deg C per minute (!) to avoid devitrification (formation of ice crystals). Radio frequency (RF) heating systems have been developed for this, but my impression is that this is still not reliable, if available at all. However, it is later claimed that use of an inert fluid, in a procedure more or less opposite to the cooling phase (see below) is a "possible alternative" to RF heating. After perfusion with CPA, the vascular system is flushed with an inert fluid that remains liquid at low temperature, with low viscosity and low toxicity. These might include fluorocarbons, perfluorocarbons, siloxanes, or silicones. The possible CPAs include many compounds, the "new" feature (for vitrification) being the inclusion of "alkoxyl" instead of hydroxyl groups (e.g., "methoxyl" would be OCH3 instead of OH). It is very important to wash out the CPA completely with the inert fluid; otherwise, after further cooling, CPA may block parts of the vasculature. A carefully controlled process is required, with a series of steps. In the reported dog experiments to support the claims, the lowest temperature reached does not appear to be reported--at least, I didn't find it. However, in the reported rewarming, the "initial temperature" of the dogs is reported as - 80 C. This agrees with the recent report in Alcor's journal showing a microphoto of brain tissue rewarmed from -80 C. ---------- Now, a further word about toxicity. The patent claims that low toxicity is one of the virtues of the alkoxylated compounds. Well, first, on a clinical level, after seeing the recommendations in the patent, we at CI have done a bit of our own research, and so far (freezing, not vitrification) have found no improvement over glycerol. But we did find that we had to use special equipment to protect our personnel against the fumes. For that matter, we all know that some ethers can be used as anaesthesia in surgery, but without great care the patient can be killed by the anaesthetic. I have had surgery more than once with ether anaesthesia, and you wake up sick as a dog. The patent of course relates mainly not to toxicity at the level of the organism, but at the cellular level. In the 4th quarter 2000 issue of CRYONICS, Alcor's magazine, Fred Chamberlain's article says that Alcor, through BioTransport, has licensed a variant of 21 CM's "low toxicity vitrification formulas." It also says that, as evidence of this low toxicity, a LESS concentrated formula resulted in 53% viability--presumably meaning of cells. This test apparently used only a single criterion of viability, the potassium/sodium ratio--with rat hippocampal slices, not whole brains. If almost half the cells are killed, even by the single criterion used, then "relatively low toxicity" clearly must emphasize the "relatively." Again: None of this is meant to imply criticism of the work done--only of exaggerated claims. The work itself is remarkable, even if it is not yet of the Hallelujah variety. It is especially interesting that the inventors, employees of 21CM, were not professional cryobiologists. Brian Wowk, as I recall, has a recent Ph.D. in biophysics, and had little or no previous experience in cryobiology. Federowicz has no academic credentials whatever, and also, as far as I know, no previous professional publications. I am not acquainted with Sandra Russell. Steve Harris is a young physician, also, I believe, without previous experience in cryobiology. So this new blood was able in short order to do things that the whole "community" of cryobiologists had not done in previous decades. That is encouraging. As I have said many times, once the evidence is clear and verified, we expect to make any and all useful procedures available to our members, directly or indirectly, subject to decisions by our Board of Directors from time to time. Robert Ettinger Cryonics Institute Immortalist Society http://www.cryonics.org Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=15222