X-Message-Number: 7678 Date: Tue, 11 Feb 1997 23:50:56 +0100 (MET) From: Eugene Leitl <> Subject: FYI: (looong) Cryobiology now online (at last!) It's pretty bare, but it's a start... http://www.apnet.com/www/journal/cy.htm Things which can be found there, e.g.: The Effect of Cooling Rate and Temperature on the Toxicity of Ethylene Glycol in the Rabbit Internal Carotid Artery Monica Wusteman, Serena Boylan, David E. Pegg The smooth muscle and vascular endothelium of small elastic arteries (the rabbit common carotid artery) are injured by exposure to 40% ethylene glycol (EG) at 4=B0C, and additional damage occurs when the arteries are cooled without freezing to -20=B0C. This paper reports attempts to reduce this injury by altering the cooling rate and temperature of exposure to the cryoprotectant. Very slow cooling (0.1=B0C/min) removed all residual smooth muscle and endothelial function when assessed in vitro after rewarming and removal of the cryoprotectant. Very rapid cooling to -20=B0C also increased the injury, both to the endothelium and to the smooth muscle. Reducing the temperature of exposure to 40% EG from +4=B0C to -20=B0C had no beneficial effect on the smooth muscle but enabled the vascular endothelium to retain some functional activity. These data suggest that the mechanism responsible may be related to the physical properties of ethylene glycol rather than to a biochemical interaction with metabolic processes, and that it is a mechanism which is highly specific for the cell types involved. It also underlines the difficulties involved in the successful cryopreservation of complex tissues and organs. Cryobiology, v 33, n 4, August 1996, p423-429 (ID CY960042) Copyright 1996 Academic Press, Inc. ...or... An Experimental Study of the Mechanical Response of Frozen Biological Tissues at Cryogenic Temperatures YOED RABIN, Paul S. Steif, Michael J. Taylor, Thomas B. Julian, Norman Wolmark An experimental study of the mechanical response of frozen soft biological tissues to applied compressive stresses is presented. This study is related to the mechanical stresses that develop due to the contraction of frozen tissues in cryopreservation as well as in cryosurgical procedures. The main concept in this study is that the stresses associated with the constrained contraction of the frozen tissue, i.e., due to temperature variations within the frozen tissue, can be simulated by an external mechanical load which is applied to the frozen tissue while the tissue is maintained at a uniform temperature. A new apparatus for measuring compressive stresses and strains of frozen biological tissues in cryogenic temperature range is presented. A new technique for processing the fresh biological tissue into a cylindrical frozen sample for mechanical testing is introduced. Results of compression tests on rabbit liver, kidney, and brain are presented and are compared with available data from the literature on sea ice and single ice crystals. An unusual response of frozen biological tissues to compressive stress was observed: after the initial, roughly linear elastic portion there was a series of sudden stress drops at constant strain, each followed by a linear increase of stress with strain to the next drop. This phenomenon, which is attributed to the accumulation of microcracks, broadly resembles plastic deformation, and thus provides some support for simple mechanical models invoked in theoretical studies. Cryobiology, v 33, n 4, August 1996, p472-482 (ID CY960048) Copyright 1996 Academic Press, Inc. ...or... SHINICHI HOCHI, MEGUMI KOZAWA, TAKENORI FUJIMOTO, EIICHI HONDO, JUNZO YAMADA, NORIHIKO OGURI This study was designed to examine the suitability of immature horse oocytes for vitrification. Immature oocytes derived from slaughtered horse ovaries were transferred to a vitrification solution (EFS; 40% ethylene glycol, 18% Ficoll, and 0.3 M sucrose in modified phosphate-buffered saline) directly (Groups 1 and 4) or were first exposed to 20% ethylene glycol solution for 10 min (Groups 2 and 5) or 20 min (Groups 3 and 6). Oocytes were handled at 20=B0C (Groups 1, 2, and 3) or 30=B0C (Groups 4, 5, and 6). After vitrification and warming, their viability was assessed by maturation culture for 32 h. The percentages of oocytes reaching the metaphase II stage after the in vitro maturation in Groups 2, 3, 5, and 6 (16.0, 16.7, 10.0, and 8.2%, respectively) were higher than those in Groups 1 and 4 (2.2 and 1.9%, respectively). In untreated control oocytes, 55.6% completed meiosis in vitro. Transmission electron microscopy was used to compare the fine structure of vitrified oocytes (treated as Group 2) with those of untreated control oocytes and EFS-exposed, nonvitrified oocytes (n =3D 10 each). The viability of EFS-exposed oocytes, assessed by in vitro maturation, was 27.7%. Vitrification induced some ultrastructural changes, such as the swelling of mitochondria together with reduced matrix density, the destruction of communication between oocytes and their surrounding cumulus cells, and the presence of vacuoles located in the periphery of the ooplasm. However, these changes were not always observed. Exposure of the oocytes to EFS solution induced similar ultrastructural changes in mitochondria and cell-cell communication but to a lesser extent. However, the exposure to EFS induced vacuoles in the periphery of the ooplasm to the same extent as did the vitrification. Thus, immature horse oocytes can be cryopreserved by vitrification with EFS solution. Reduced viability of EFS-exposed and/or vitrified horse oocytes may relate to morphological changes such as destruction of the intercellular communications between cumulus cells and oocytes. Cryobiology, v 33, n 3, June 1996, p300-310 (ID CY960030) Copyright 1996 Academic Press, Inc. ...or... M. A. J. ZIEGER, E. E. TREDGET, L. E. MCGANN Successful cryopreservation of tissues will ultimately require a more detailed understanding of how the in situ environment modifies cell responses during cooling and warming. Low-temperature responses of porcine split-thickness skin and isolated basal keratinocytes were compared after various cooling protocols and in the presence and absence of cryoprotectants. Recovery was assessed by measuring oxygen consumption kinetics in skin and tetrazolium reduction in isolated cells. Freeze substitution was used to visualize ice nucleation and growth in skin. The results indicated that the time required for diffusion of water in split-thickness skin delayed osmotic responses in the basal keratinocytes and resulted in increased intracellular and intercellular ice formation. Rapid cooling (-200=B0C/min) in the presence of cryoprotectants resulted in a reduction in the number of cells containing ice and the size of the intercellular ice crystals and an increase in tissue recovery. These observations support other reports which suggest that cell-to-cell and cell-to-substrate interactions are sensitive sites for cryoinjury. A practical recommendation from this study is that high recovery of split-thickness skin may be achieved with protocols using high cooling rates. Cryobiology, v 33, n 3, June 1996, p376-389 (ID CY960038) Copyright 1996 Academic Press, Inc. ...or... A. BAUDOT, J. F. PEYRIDIEU, P. BOUTRON, J. MAZUER, J. ODIN The effect of sugars or reduced saccharides trehalose, sucrose, sorbitol, or mannitol on the glass-forming tendency during cooling and the stability of the wholly amorphous state during warming has been studied with 2,3-butanediol, 1,2-propanediol, or 1,3-butanediol in three different carrier solutions. The 2,3-butanediol contained 96.7% (w/w) racemic mixture of the levo and dextro isomers and 3.1% (w/w) of the meso isomer (called 2,3-butanediol 97% dl). The carrier solutions were water, a phosphate-buffered saline, and two organ preservation solutions (Euro-Collins and Saint Thomas). The latter two were chosen because they are often used for kidney and heart preservation, respectively. The concentrations of 2,3-butanediol, 1,2-propanediol, and 1,3-butanediol varied respectively from 25 to 34, 30 to 35, and 30% (w/w). The concentrations of saccharides were 4 or 5% (w/w). In the absence of saccharides, for a given 2,3-butanediol concentration, the glass-forming tendency increased in the following order: water, Saint Thomas, the phosphate buffer, Euro-Collins. Addition of 4 or 5% (w/w) saccharide resulted in a large increase in the glass-forming ability of the solution during cooling and increased the stability of the glass during warming; but replacement of 4 or 5% diol by an equivalent weight (percentage) of a saccharide decreased, though to a lesser extent, these properties. Cryobiology, v 33, n 3, June 1996, p363-375 (ID CY960037) Copyright 1996 Academic Press, Inc. ...or... Crystallization of Ice in Aqueous Solutions of Glycerol and Dimethyl Sulfoxide. 1. A Comparison of Mechanisms J. M. HEY, D. R. MACFARLANE The crystallization of ice from aqueous solutions of glycerol and dimethyl sulfoxide (Me2SO) has been studied using differential scanning calorimetry. In particular, the ice crystallization behavior of glycerol and Me2SO solutions containing approximately the same mole percent solute concentration (i.e., approximately 16 mol%) has been compared. These solutions (45 w/w% Me2SO (15.9 mol%) and 50 w/w% glycerol (16.4 mol%)) were shown to exhibit markedly different ice crystallization properties. For example, the peak homogeneous nucleation temperature of the Me2SO solution was observed to be 3=B0C above Tg, whereas the peak homogeneous nucleation temperature of the glycerol solution was shown to be 20=B0C above Tg. Further, the 50 w/w% glycerol solution was shown to devitrify at temperatures close to those of the peak nucleation rate, whereas the Me2SO solution was found to devitrify at temperatures much higher than the peak nucleation temperature. This, along with evidence from emulsion-based calorimetry experiments, indicates that the nucleation leading to devitrification in 45 w/w% Me2SO solutions is largely heterogeneous in nature. Cryobiology, v 33, n 2, April 1996, p205-216 (ID CY960021) Copyright 1996 Academic Press, Inc. ...or... Analysis of Thermal Stresses around a Cryosurgical Probe YOED RABIN, PAUL SSTEIF Large thermal stresses easily exceeding the tissue yield strength may develop in the frozen region around a cryosurgical probe. A new integrodifferential solution for the heat transfer problem of biological tissues freezing around a cryosurgical probe is presented in this article. This solution is suitable for cases of high Stephan numbers and for a temperature-dependent forcing function at the cryoprobe. A new solution for the thermal stresses around a cryosurgical probe is also presented, based on an elastic-perfectly plastic model. It is proposed that thermal stresses beyond the elastic limit of the frozen region may sharply increase the mechanical damage to the cell membranes due to plastic deformation. It was found that plastic deformation always starts at the cryoprobe surface; however, plastic deformation may also be formed near the freezing front at high cooling rates and large cryoprobes. It is demonstrated that under some conditions plastic deformation may occur in the entire frozen region. A parametric study to identify the best cooling protocol for maximal plastic deformation is presented. Cryobiology, v 33, n 2, April 1996, p276-290 (ID CY960028) Copyright 1996 Academic Press, Inc. ...or... A New Cryosurgical Device for Controlled Freezing YOED RABIN, AVRAHAM SHITZER A new cryosurgical device utilizing liquid nitrogen, which is a modification of an existing commercial system, was developed. In the new computer-controlled cryodevice the temperature of the cryoprobe is controlled by means of an electrical heating element. The desired temperature-forcing function is calculated to ensure a specified constant cooling rate at the freezing front. The new device facilitates real-time data processing, and, in particular, simulation of the heat transfer processes. A series of tests was performed to study the characteristics of the cryodevice and to validate the underlying assumptions. These tests were performed using organic tissue, i.e., potatoes, as an in vivo simulating medium of biological tissue. The differences between experimental data and computed results were found to be within =B10.5=B0C, which falls within the uncertainty range of the experimental temperature measurements. A typical control error of the new device is within =B10.3=B0C, prior to the formation of the freezing front, and =B10.6=B0C thereafter, which is of the same order of magnitude as the uncertainty range of the temperature measurements. The new device is capable of producing maximal cooling rates of 50=B0C/min down to temperatures of -165=B0C and a maximal heating rate of 300=B0C/min. The maximal cooling power of the cryoprobe, due to LN2 boiling, is 80 W; the maximal electrical heating power of the cryoprobe is 160 W. Precooling of the device requires about 30 min, and it can be operated continuously for about 3 h. Initial results of experimental in vivo cryosurgery performed on rabbit hindlimbs, including histological observations and thermal analysis, are presented in the second part of this study. Cryobiology, v 33, n 1, February 1996, p82-92 (ID CY960009) Copyright 1996 Academic Press, Inc. Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=7678