X-Message-Number: 5096 Date: 02 Nov 95 12:55: EST From: Mike Darwin <> Subject: SCI.CRYONICS:hyperbaria VITRIFICATION: Bob Ettinger responds to Ben Best's claim that Greg Fahy's current vitrification approach does NOT require hyperbaria by saying that he is unaware of this, if it is true. I wish to confirm that Ben is *correct.* Greg has not needed to use hyperbaria for at least two years in order to recover viable kidneys following loading and unloading with a 1 atmosphere vitrifiable concentration of cryoprotectants. This is a BRIEF and very compressed status report: 1) Rabbit kidneys have been loaded with a 1 atmosphere vitrifiable solution, cooled to -30 C or -40 C (I think Greg recently went from -30 C to -40 C, but am not sure), been reimplanted into the same animal, and supported the animal as the SOLE kidney indefinitely. This work has been repeated many times. 2) WHY then have not kidneys been vitrified since all that is required is further cooling from -30 or -40 C to -135 C or thereabouts and, since no ice will form, there will be no cause for added injury except for possible thermal effects on membranes, proteins, or other cell components (known to be minimal)? 3) The answer to #2 above is that it is easier to fall down a well than to climb out of one. While cooling to and vitrification at -135 C (no ice formation) using a nontoxic 1 ATM vitrifiable solution can be done in rabbit kidneys TODAY, what cannot be done is to rewarm them without freezing. During cooling, the system steadily loses energy. It also forms microscopic and biologically insignificant amounts of ice which are scattered through the organ. These nidi or crystals are few, far between and do not propagate during cooling over reasonable time intervals and are inhibited completely by the glass transition of the solution (VS4 in this case). Unfortunately, rewarming at rates achieveable with traditional conductive methods in something as big as whole kidneys results in the system FREEZING. The alternative is to rewarm very rapidly at a rate of 300-400 C/min. This is theoretically possible using appropriate frequencies of RF (*not* conventional microwaves as used in kitchen ovens) even for whole human bodies. It is certainly possible for masses the size of rabbit kidneys because it has been done with a previous solution (VS2). 4) WHY THEN HAVE KIDNEYS NOT BEEN VITRIFIED? The answer here is as simple as it is perverse. The expertise and equipment required to do this are controlled by a researcher employed by the US FDA named Paul Ruggera. For the past 5 YEARS Dr. Ruggera and Dr. Fahy have been seeking to get FDA permission to collaborate (their early collaboration was aborted by bureacuratic red tape after initial experiments showed the feasability of the system). Just recently the CRATA allowing the collaboration was approved. However, by this time Greg had moved on to the Naval Medical Research Facility and left the Red Cross. The first CRATA between the FDA and Red Cross took intervention by a Congressman to facilitate plus the personal approval of FDA Commissioner David Kessler. As I understand it, efforts are underway to get the approved CRATA transferred to NAMRI. 5) WHY CAN'T SOMEONE ELSE FILL THE BILL FOR THE TECHNOLOGY RUGGERA IS NEEDED FOR? a) Patents on the technology. b) This work requires extreme expertise and physical proximity of the investigators. c) Money; a radio station transmitter, complete with FCC license is required to rewarm rabbit kidneys (and humans kidneys) fast enough to avoid freezing upon rewarming. These things don't come cheap. Last I heard the transmitter was sitting in a crate somewhere. It has been in the crate for YEARS. Any Questions? As you can see from the above, ice free and ultrastructurally injury-free cryopreservation now exists for rabbit kidneys. Greg has long ago completed TEM studies showing tissue at fixed at -30 C in the liquid state and after loading and unloading with VS4: there is no visible mechanical or ultrastructural disruption and the tissue at this temperature is virtually undistinguishable from control (there are some changes to intracellular structures which appear to be fully reversible on rewarming). By way of example, our dogs, whenm cooled to 4 C lose all the intra-axonal microtubule structure. It just falls apart upon simple cooling. It also snaps back together via self-assembly upon rewarming. The dogs seem to be no worse for wear. Bob asks about the speed of recrystalization. It is fast. So fast that it is one of the major barriers to recovery of slowly cooled cells and tissues in the presence of modest amounts of cryoprotectant. What constitutes slow cooling? Temperature descents slower than 0.5 to 1.0 C/min. The fastest you could reasonably cool a humam brain would be about (5-10 C an hour). The rapid loss of viability of sperm frozen with low concentrations of cryoprotectant to dry ice temperature is due almost exclusively to recrystalization. It has for over a decade been a working assumption that humans cooled at the slow rates their bulk and the laws of thermodynamics require will be fully or near fully recrystallized by the time they reach -79 C, with our without cryoprotectant. Modest concentrations of colligative cryoprotectants slow the rate of recrystalization but do not stop it or inhibit it sufficiently over the time scales required for cooling large masses of tissues to stable temperatures. Thermohysteriesis proteins do not seem to be a viable solution to this problem either, much to the chagrin of ice cream manufactuers who face the same problem at -20 C with loss of "mouth feel", loss of texture, and the development of "gooiness" in ice cream products stored over time or rewarmed a little in transit, especially cheaper brands like ice-milks which have minimal concentrations of fat and sucrose (sucrose is a good glass former and inhibitor of recrystalization) and are very susceptible to recrystalization. Mike Darwin Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=5096