X-Message-Number: 4797 Newsgroups: sci.cryonics Date: Mon, 21 Aug 1995 17:18:56 +0200 (MET DST) From: Eugene Leitl <> Subject: Fahy (II) Message-Id: <> essential Fahy cot'd... do not fold, mutilate or bit-mutate -- Eugene =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= [5] Practical Aspects of Ice-Free Cryopreservation (G. M. Fahy, T. Takahashi, H. T. Meryman), <unknown source> (<unknown year>). ABSTRACT Low temperatures are used by biologists primarily because they slow or prevent unwanted physical and chemical events. Unfortunately, the utility of low temperatures is usually compromised by the inconvenient fact that cooling also leads to the crystallization of water and thereby creates new and unwanted physical and chemical event which may injure the system the biologist wishes to preserve. Although the penalties imposed by freezing are in many cases acceptable, ice formation renders biological preservation generally imperfect and sometimes inconvenient. Here we will consider an alternative to this situation. It is now possible, after after suitable pretreatment, to prevent ice formation in biological systems during cooling to liquid nitrogen temperature in such a way that when these systems are warmed rapidly, little or no injury is observed. Instead of forming ice, these systems become glassy rather than crystalline, and event known as vitrification. CONCLUSION Vitrification has become a successful method for cryopreserving human monocytes and erythrocytes, murine embryos, and guinea pig smooth muscle and is likely to be useful for other cell types and tissues relevant to blood banking. It can be a simple, quick, inexpensive and reliable process when carried out properly, but it must be executed carefully. Only the future will decide the limits of applicability of this relavely new approach to cryopreservation. =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= [6] Simplified Calculation on Cell Water Content during Freezing and Thawing in Nonideal Solutions of Cryoprotective Agents and its Possible Application to the Study of "Solution Effects" Injury, (Gregory M. Fahy) CRYOBIOLOGY 18, 473-482 (1981). ABSTRACT In 1964 Mazur (10) introduced a second-order differential equation, hereinafter referred to as the Mazur equation, which permitted the calculation of cell water content as a function of temperature, cooling rate, and cellular properties (i.e., volume, surface area, solute content, hydraulic conductivity). However, this formidable equation can only be solved using an involved approximation method (the Runge-Kutta method) with iteration 0.001 K. To the average cryobiologist, the difficulty of solving the Mazur equation is sufficient to preclude its use; certainly this was the experience of the present author. A simplified method for calculating the extent of cellular dehydration during freezing at different rates was therefore sought. In addition, it was desired to improve upon the Mazur equation by incorporating the nonideal behaviour of glycerol and dimethyl sulfoxide (Me_2SO) described elsewhere (2). Both of these goals have been achieved. The derivation of the desired simplified equation for nonideal cells is presented here. In addition, posible application of the new equation to the study of "solutions effect" injury is discussed. CONCLUSION A simplified equation has been derived which reduces the time and complexity of calculating subzero cell water content during freezing and thawing as compared to caculation by means of the Mazur equation. The simplified equation also allows inclusion of the effects of nonideality of glycerol and dimethyl sulfoxide aqueous electrolyte solutions. Furthermore, a very simple, iterative method of solving the simplified equation has been shown to give results which are equivalent to those obtained using the far more difficult and involved Runge-Kutta technique. It is hoped that these simplifications will make calculation of cell water content accessible to more cryobiologists. In addition, possible applications of such calculations to mechanistic issues in the area of "solution effeects" injure are discussed. =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= [7] Microwave Hypothermia III; An Approach for Rapidly Rewarming Cryogenically Preserved Organs, (Paul S. Ruggera, Gregory M. Fahy), Proc. Ann. Int. Conf. of the IEEE Engineering in Medicine & Biology Soc., 10: 884-885, (1988). ABSTRACT Wire-length resonant radio frequence (RF) coils [1], which are based on equating a wavelength of RF with the wire length used in winding a coil, were first developed at CDRH for potential use in hypothermia treatment for cancer therapy. Design modifications, coupled with a series of experiments [2], established their potential use for rapidly and uniformly rewarming canines following hypothermic cardiac surgery. This paper details a new design which incorporates a single-end coaxial input and RF shielding, allowing it to be used outside of screen room environment. Data from early experiments to investigate this system's usefulness in rewarming cryogenically preserved donor organs are presented. INTRODUCTION Vitrification has been under development by the American Reed Cross as a means for organ cryopreservation. By infusing a rabbit kidney with a cryoprotective fluid and cooling it under extreme pressure, vitrification, i.e. solidification without ice crystal formation has been demonstrated [3]. However, ice crystal formation during warming from the vitreous state can only be avoided by using rapid and uniform heating. The indications are that the conventional heating techniques do not produce heating which is sufficiently rapid, uniform, efficient, and readily applicable inside a high-pressure vessel. Given the demonstrated deep, uniform heating capability of wire-length resonant coils, and the desire to investigate potential use of the longer wavelengths 10-100 MHz region of the spectrum in this application, two coils were constructed and tested at CDRH. Details of the construction and results of testing thus far are presented here. CONCLUSION The results of the heating peak's shift is of little consequence in this application. Prior to vitrification, the organ can be positioned in the container to align it with the area of maximum heating. The available heating volume, however, is of concern. The 12.5-cm coil's volume is too small for a rabbit kidney, the first organ which will be investigated. The 16.5-cm coil's volume is adequate. However, as can be seen from Figs. 2 and 3, the longer coild requires 33 percent more power to attain a given temperature in given volume. The target heating rate for vitrified organs is 1000 deg C/min. The data indicated that this would require 4.2 kW for the coil of Fig, and 5.6 kW for the coil of figure 3. It is interesting to note that for this design the power increase required to achieve peak temperatures may well be related to the ration of the coils' lengths (i.e. 16.5/12.5 = 1.32). If this holds, it indicates that for a coil length approaching the limit of the pressure vessel's length, which will maximize the available heating volume, RF power in the region of 10 kW will be required. These results also suggest that the exposure system appears to work well and, therefore, may be of use to other researchers who ues this frequency range. It offers a high, uniform field intensity with virtually no external emission and with relatively low power requirements. =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= [8] The Relevance of Cryoprotectant "Toxicity" to Cryobiology (Gregory M. Fahy), CRYOBIOLOGY 23, 1-13 (1986) ABSTRACT Cryoprotective agents are essential for the cryopreservation of almost all biological systems. These additives, however, do not usually permit 100 percent survival after freezing and thawing, though from a theoretical point of view they should be able to fully suppress all known types of freezing injury. In view of the known biological and physicochemical effects of cryoprotectants, it is suggested that the toxicity of these agents is a key limiting factor in cryobiology. Not only does this toxicity prevent the use of fully protective levels of additive, but it may also be manifested in the form of cryoinjury over and beyond the cryoinjury due to classical causes. Evidence for this extra injury ("cryoprotectant-associated freezing injury") is reviewed. It is suggested that better supression of toxicity is possible and will lead to advances in cryopreservation. CONCLUSION Nevertheless, I submit that the detrimental effects of cryoprotectants are almost as relevant to cryobiology as are their cryoprotective effects, and that our understanding of cryobiology will remain incomplete until we have finished examining both sides of the cryoprotectant coin. We very much neeed the services of talented biochemists and biophysicists who can work out the mechanisms and the sites of injury so that we might be permitted to protect cells againstt the "side effects" of cryoprotectants. Of particular interest would be more studies which explore those effects of cryoprotectants which have been shown to be irreversible. We have substantial clues with regards to both where and how to look for answers. We also have evidence that deeper insight can lead to practical benefits (3, 13). =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= .. to be continued -- Eugene Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=4797