1 www.alcor.org Cryonics/Third Quarter 2007 In The Beginning: Vitrification or Freezing? In The Prospect of Immortality, the book which launched cryonics, Robert C.W. Ettinger suggests that glycerol might be used as the cryoprotectant for human cryopreser- vation (1) based largely on the fact that it was the dominant cryoprotective agent (CPA) at that time (1962-64) and most of the positive results with sperm and tissues had been achieved with glycerol. Due largely to the flare and flamboyance of “the Father of Dimethyl Sulfoxide (DMSO*),” Dr. Stanley W. Jacob, who was Assistant Professor of Surgery at the University of Oregon Health Sciences Center Medical School, DMSO entered the public consciousness in a big way in the mid-to-late 1960s (2-4). DMSO’s anti-inflammatory, and seemingly incredible skin-penetrating proper- ties, were much talked about. Sometime between 1966 and 1967, Ettinger asked Dr. Dante Brunol, an Italian national living in the US, to produce a formal, written protocol for cryopreserving cryonics patients. Brunol was a biophysicist (Ph.D.), M.D. and surgeon with experience in car- diopulmonary bypass. Brunol, writing under the nom de plume of Mario Satini, M.D., pro- duced a complicated protocol which, while it has a number of deficiencies, is really quite remarkable, and even visionary in several respects (5). Brunol opens his protocol with the following remarks: “The writer has always favored supercooling rather than the freezing of humans. Supercool- ing does not lead to the formation of ice crystals. It should be possible to find methods to store humans at temperatures warmer than -30 degrees C, for five years, the time necessary to protect humans from freezing. When Professor Ettinger, author of The Pr ospect of Immor tality , asked me to devise a method to freeze humans, at first I declined the offer. In my opinion, only a chemical inducing vitrification could save the cells from (ice) crystal damage.” The First Human Cryopreservation Protocol Brunol then goes on to explain how vit- rification is achieved through ultra-rapid cool- ing and, without naming it, introduces the idea of the glass transition point of water (Tg), a temperature below which water has become a glass and cannot organize into crys- tals. He further explains why such ultra-rapid cooling cannot be applied, even to tissues, let alone whole humans. Brunol details his proto- col which consists of the following core ele- ments: 1) Immediate commencement of CPR at the time medico-legal death is pro- nounced, preferably augmented with an artificial airway and high FiO2 (fraction of inspired oxygen in a gas) oxygen administration (15 liters per minute). He recommends at least 30 compressions per minute, with one ventilation every four minutes. 2) Placement of a thermistor in the rectum to monitor body core temperature. The ther- mistor is affixed to a 10" wooden dowel with straps to hold it deeply in the rectum. 3) Immersion of the patient in a special tub filled with ice and 10% DMSO in water while mechanical CPR continues. The tub supports the patient so that his head remains above the water level allowing manual ventilation to continue. 4) Use of the Westinghouse Iron Heart (a mechanical chest compressor) as soon as possible to continue CPR during cooling. CPR with the Iron Heart is to continue until the patient reaches a core tempera- ture of 15 degrees C, or until extracorpo- real cooling using closed-circuit CPB can be commenced via femoral-femoral bypass using a heat exchanger. 5) Inject 2 liters of ice-cold 5% Dextran in an isotonic solution via both internal carotid arteries to hemodilute and cool the brain. 6) Femoral-femoral cannulation followed by open circuit perfusion (blood washout) of ~20 gallons (80 liters) of heparinized 20% DMSO, 20% glycerol in saline or other isotonic solution at a pressure of 120 mm Hg, and a temperature of between 1 degree and 4 degrees C. 7) Using a fairly complex circuit Brunol demonstrates a good knowledge of phys- iology, and proposes perfusing the pul- monary circulation by turning on the Iron Heart and pressurizing the venous circulation (via retrograde flow through the femoral venous cannulae) to 20 mmHg at very low flow, while opening the arterial cannulae to allow effluent to exhaust retrograde into a discard-reser- voir. Perfusion of the pulmonary circuit is to commence when the patient's tem- perature reaches 10 degrees C and is to continue for 15 minutes. 8) Preferably, perfusion with the CPA mix- ture should terminate when the patient's core temperature is -4 degrees C. 9) Brunol was very concerned about inter- stitial and intracellular ice crystal damage and he proposed vitrifying the cells by initiating ice crystal formation in the vas- culature. He proposed doing this by fol- History of DMSO and Glycerol in Cryonics By Mike Darwin * The correct abbreviation for DMSO is Me2SO, as per the International Union of Biochemistry and Molecular Biology (IUBMB) – International Union of Pure and Applied Chemistry (IUPAC) Joint Commission on Biochemical Nomenclature. Figure 1. Robert Ettinger (foreground) demonstrates use of the Iron Heart, a mechanical chest compressor. Photo by Ted Kraver