- Email: [email protected]
71. Oxygenated hypothermic machine perfusion improves liver function
Abstracts / Cryobiology 63 (2011) 306–342 206105; 98 (2007) 035703]. In addition to this, several mechanisms have been incorporated to form new grains at the perimeter of growing crystals, a process we term as ‘‘growth front nucleation” [Nat. Mater. 3 (2004) 635]. The equations of motion (three stochastic partial differential equations in 2D, and six in 3D) are solved numerically in a massively parallel environment. The model requires a detailed information on the system incorporating the free energy of the phases as a function of composition and temperature, the magnitude and anisotropy of the solid–liquid interface free energy and of the grain boundary energy, and the mobilities for the phase-, orientation and concentration fields, which are related to the translational, rotational and inter-diffusion coefficients. Advantages, problems and possible solutions for quantitative simulations will also be outlined. Numerous examples will be presented to demonstrate that the phase-field model supplied with orientation field (s) can successfully describe the formation of complex polycrystalline structures, such as impinging symmetric dendrites, disordered (‘‘dizzy”) dendrites emerging as a result of dendrite-particle interaction, fractallike morphologies, and a broad variety of spherulitic patterns. Finally, possible adaptation of the phase-field model to morphology evolution of ice in undercooled water will be outlined. Conflict of interest: None declared. Source of funding: None declared.
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practical, subject. For cryobiology applications, an understanding of the Gibbsian thermodynamics of multi-component composite systems is required. Solution thermodynamics, i.e. equations for chemical potentials of solvent and solutes, are necessary to predict solution freezing points and driving forces for osmotic transport across cell membranes and through tissue extra-cellular matrix. A number of equations have been proposed and used to describe solution thermodynamics in cryobiology. Our group has done much research with a broadly applicable osmotic virial equation. This equation allows solution thermodynamics of multi-solute solutions to be developed using only single-solute knowledge. In addition to solution chemical potentials, thermodynamics predicts the role of curvature of the ice-solution interface which is important in cryobiology since ice growing in confined spaces, or growing quickly through free space, can be highly curved. Through the Gibbs–Thompson equation, curvature affects the freezing point of a solution in a way that is additive to the effect of composition. I will share with the audience my love of thermodynamics while reviewing our current understanding of ice-solution thermodynamics in cryobiology. Conflict of interest: None declared. Source of funding: None declared.
doi:10.1016/j.cryobiol.2011.09.073
doi:10.1016/j.cryobiol.2011.09.071 Cell preservation protocols I 69. Molecular dynamics of water below freezing. Masakazu Matsumoto, Graduate School of Natural Science and Technology, Okayama University, Japan Molecular dynamics simulation gives us total information on the structure and motion of the molecules in liquids. Recent simulation studies on water even elucidate the microscopic aspects of various phase transitions between solid and liquid phases. In every transition, one finds that the specific form of amorphous ice, called low-density amorphous (LDA) ice, plays the key role. In the freezing and melting processes of ice, for example, LDA-like structure ‘‘wets” the surface between water and ice and reduces the surface energy. When liquid water is supercooled, various thermodynamic properties behave divergently toward the characteristic temperature, Ts = 228 K. Polyamorphism, i.e. the hypothesis on the co-existence of two distinct metastable amorphous (or liquid) phase below the melting point and the existence of metastable critical point between them, seems to explain consistently the anomalous behavior of water below freezing. Many experimental and simulation results also support the idea. Polyamorphism is commonly found in the tetravalent network-forming materials such as silicon, germanium, and silica. LDA is supposed to bring about first-order phase transition with another metastable form of water, i.e. high-density amorphous ice, below the critical temperature. LDA is more ordered than HDA. Sharp transition to HDA is also observed when an electron beam is radiated to LDA ice at very low temperature, which causes serious image degradation in electron microscopy. It has been advocated that there are two different local structures in liquid water at room temperature. We casually use the term ‘‘structured water” for the surface water in the confined geometry, water near the solute molecule, and vicinal water near biomolecules as the counterpart of the ‘‘normal water”. In the framework of polyamorphism, two-state-like behavior of water is explained by the critical fluctuation above the metastable critical point hidden at around 228 K. If so, it is natural that the structure of liquid water is influenced by the weak external field such as walls and solutes even at room temperature. Then a question arises. If two amorphous phases coexist in an appropriate condition, they must form the interface between them (otherwise they will mix). However, it is hard to imagine that the two amorphous ices of the same spatial symmetry and similar density (differs only 15%) can form the interface where its local structure is incompatible to the both amorphous structures. How can they selforganize themselves to maintain the interface? Why are their structures different so sharply? We carried out a thorough investigation of the local network structure of supercooled water and gave a conjecture on LDA structure in terms of locally stable amorphous ‘‘fragments”. It gives a consistent explanation to the self-organizability of the LDA ice and provides the way to distinguish the local order of it directly. Conflict of interest: None declared. Source of funding: None declared. doi:10.1016/j.cryobiol.2011.09.072
70. Ice-solution thermodynamics in cryobiology. Janet A.W. Elliott, Department of Chemical and Materials Engineering, University of Alberta, Canada Cryobiology is an inherently interdisciplinary research area – from the physical effects of cold on solutions including ice-solution phase change and osmotic transport, to the biological effects of cold on membranes, cytoskeleton, organelles, proteins, reaction cascades, gene expression and ultimately life. Over the past 14 years, my research group has contributed to the subject of thermodynamics in cryobiology. Thermodynamics is the study of mathematical relationships arising from physical laws governing energy and entropy. Thermodynamics is a rich, mathematical, yet profoundly
71. Oxygenated hypothermic machine perfusion improves liver function. Kelvin G.M. Brockbank * 1,2,3, Charles Y. Lee 4, Barry J. Fuller 5, Elizabeth D. Greene 1, Zhenzhen Chen 1, Lindsay K. Freeman 1, Hans R. Kershaw 1, David Kravitz 6, Lia H. Campbell 1, 1 Cell & Tissue Systems, Inc. North Charleston, SC, USA, 2 Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA, USA, 3 Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, SC, USA, 4 University of North Carolina, Charlotte, NC, USA, 5 Cell, Tissue and Organ Preservation Unit, Department of Surgery & Liver Transplant Unit, UCL Medical School, Royal Free Hospital Campus, London, UK, 6 Organ Recovery Systems, Inc., Itasca, IL, USA The long-term goal of our liver research program is to increase the number and quality of donor livers available for transplantation by developing a clinically usable, portable, hypothermic perfusion device and methods for liver preservation. The shortage of organs for transplantation continues to be a major impediment to providing optimal treatment for patients with end stage liver failure. There is no dialysis-equivalent therapy for these patients and the prospect of death while waiting for a transplantable organ is a realistic probability. In this report we have compared 4–6 °C hypothermic machine perfusion (HMP) with and without oxygenation employing a prototype hypothermic liver perfusion device and Belzer’s machine perfusion solution (KPS-1) for preservation of porcine heart beating donor liver functions for 12 h. Adult domestic Yorkshire cross farm pigs (25–30 kg) were used as donors. All livers were flushed with Belzer’s solution and placed on ice for 2 h during transport and preparation for studies. Experimental HMP livers +/ O2 were compared with 2 h static cold stored control livers using a battery of cell/tissue damage and function assays by sampling during 3 h normothermic ex vivo perfusions with washed erythrocytes in Krebs Henseleit solution. The HMP oxygenated livers exhibited no statistically significant differences in liver lactate dehydrogenase, hyaluronic acid uptake, total bile production, albumin, glucose concentrations and lactate, alanine aminotransferase, interleukin-8 or b-galactosidase on the normothermic test circuit relative to control livers. Statistically significant increases in alanine aminotransferase were observed during normothermic blood perfusion after HMP without oxygen (200 U/L) compared with <100 U/L in all other groups (p < 0.05). In addition, significant changes (p < 0.05) were observed after HMP without oxygenation in bile production, lactate dehydrogenase, interleukin-8, b-galactosidase, and albumin versus either control livers and/or HMP livers with oxygen. Tumor necrosis factor-a was significantly decreased in both HMP groups compared with 2 h SCS controls (p < 0.05). These results provide strong support for continuous oxygenation during HMP of livers. Future research will focus on exploring novel alternatives to tanked oxygen to improve machine preservation for practical field use and evaluation of the potential benefits of inhibiting mediators of ischemic reperfusion injury during HMP prior to preclinical liver transplant studies. Conflict of interest: None declared. Source of funding: Supported by NIH Grant #1R43DK082063–01.
doi:10.1016/j.cryobiol.2011.09.074
72. Development of a cryopreservation protocol for pancreatic cells using plant proteins. Mélanie Grondin 1,2,3, Isabelle Robinson * 1,2,3, Sonia DoCarmo 1,2, François Ouellet 1, Catherine Mounier 1,2, Fathey Sarhan 1, Diana Averill-Bates 1,2,3, 1 Département des Sciences biologiques, Université du Québec à Montréal, Canada, 2 Centre de recherche BioMed, Université du Québec à Montréal, Canada, 3 Centre de recherche en toxicologie de l’environnement (TOXEN), Université du Québec à Montréal, Canada
325
practical, subject. For cryobiology applications, an understanding of the Gibbsian thermodynamics of multi-component composite systems is required. Solution thermodynamics, i.e. equations for chemical potentials of solvent and solutes, are necessary to predict solution freezing points and driving forces for osmotic transport across cell membranes and through tissue extra-cellular matrix. A number of equations have been proposed and used to describe solution thermodynamics in cryobiology. Our group has done much research with a broadly applicable osmotic virial equation. This equation allows solution thermodynamics of multi-solute solutions to be developed using only single-solute knowledge. In addition to solution chemical potentials, thermodynamics predicts the role of curvature of the ice-solution interface which is important in cryobiology since ice growing in confined spaces, or growing quickly through free space, can be highly curved. Through the Gibbs–Thompson equation, curvature affects the freezing point of a solution in a way that is additive to the effect of composition. I will share with the audience my love of thermodynamics while reviewing our current understanding of ice-solution thermodynamics in cryobiology. Conflict of interest: None declared. Source of funding: None declared.
doi:10.1016/j.cryobiol.2011.09.073
doi:10.1016/j.cryobiol.2011.09.071 Cell preservation protocols I 69. Molecular dynamics of water below freezing. Masakazu Matsumoto, Graduate School of Natural Science and Technology, Okayama University, Japan Molecular dynamics simulation gives us total information on the structure and motion of the molecules in liquids. Recent simulation studies on water even elucidate the microscopic aspects of various phase transitions between solid and liquid phases. In every transition, one finds that the specific form of amorphous ice, called low-density amorphous (LDA) ice, plays the key role. In the freezing and melting processes of ice, for example, LDA-like structure ‘‘wets” the surface between water and ice and reduces the surface energy. When liquid water is supercooled, various thermodynamic properties behave divergently toward the characteristic temperature, Ts = 228 K. Polyamorphism, i.e. the hypothesis on the co-existence of two distinct metastable amorphous (or liquid) phase below the melting point and the existence of metastable critical point between them, seems to explain consistently the anomalous behavior of water below freezing. Many experimental and simulation results also support the idea. Polyamorphism is commonly found in the tetravalent network-forming materials such as silicon, germanium, and silica. LDA is supposed to bring about first-order phase transition with another metastable form of water, i.e. high-density amorphous ice, below the critical temperature. LDA is more ordered than HDA. Sharp transition to HDA is also observed when an electron beam is radiated to LDA ice at very low temperature, which causes serious image degradation in electron microscopy. It has been advocated that there are two different local structures in liquid water at room temperature. We casually use the term ‘‘structured water” for the surface water in the confined geometry, water near the solute molecule, and vicinal water near biomolecules as the counterpart of the ‘‘normal water”. In the framework of polyamorphism, two-state-like behavior of water is explained by the critical fluctuation above the metastable critical point hidden at around 228 K. If so, it is natural that the structure of liquid water is influenced by the weak external field such as walls and solutes even at room temperature. Then a question arises. If two amorphous phases coexist in an appropriate condition, they must form the interface between them (otherwise they will mix). However, it is hard to imagine that the two amorphous ices of the same spatial symmetry and similar density (differs only 15%) can form the interface where its local structure is incompatible to the both amorphous structures. How can they selforganize themselves to maintain the interface? Why are their structures different so sharply? We carried out a thorough investigation of the local network structure of supercooled water and gave a conjecture on LDA structure in terms of locally stable amorphous ‘‘fragments”. It gives a consistent explanation to the self-organizability of the LDA ice and provides the way to distinguish the local order of it directly. Conflict of interest: None declared. Source of funding: None declared. doi:10.1016/j.cryobiol.2011.09.072
70. Ice-solution thermodynamics in cryobiology. Janet A.W. Elliott, Department of Chemical and Materials Engineering, University of Alberta, Canada Cryobiology is an inherently interdisciplinary research area – from the physical effects of cold on solutions including ice-solution phase change and osmotic transport, to the biological effects of cold on membranes, cytoskeleton, organelles, proteins, reaction cascades, gene expression and ultimately life. Over the past 14 years, my research group has contributed to the subject of thermodynamics in cryobiology. Thermodynamics is the study of mathematical relationships arising from physical laws governing energy and entropy. Thermodynamics is a rich, mathematical, yet profoundly
71. Oxygenated hypothermic machine perfusion improves liver function. Kelvin G.M. Brockbank * 1,2,3, Charles Y. Lee 4, Barry J. Fuller 5, Elizabeth D. Greene 1, Zhenzhen Chen 1, Lindsay K. Freeman 1, Hans R. Kershaw 1, David Kravitz 6, Lia H. Campbell 1, 1 Cell & Tissue Systems, Inc. North Charleston, SC, USA, 2 Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA, USA, 3 Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, SC, USA, 4 University of North Carolina, Charlotte, NC, USA, 5 Cell, Tissue and Organ Preservation Unit, Department of Surgery & Liver Transplant Unit, UCL Medical School, Royal Free Hospital Campus, London, UK, 6 Organ Recovery Systems, Inc., Itasca, IL, USA The long-term goal of our liver research program is to increase the number and quality of donor livers available for transplantation by developing a clinically usable, portable, hypothermic perfusion device and methods for liver preservation. The shortage of organs for transplantation continues to be a major impediment to providing optimal treatment for patients with end stage liver failure. There is no dialysis-equivalent therapy for these patients and the prospect of death while waiting for a transplantable organ is a realistic probability. In this report we have compared 4–6 °C hypothermic machine perfusion (HMP) with and without oxygenation employing a prototype hypothermic liver perfusion device and Belzer’s machine perfusion solution (KPS-1) for preservation of porcine heart beating donor liver functions for 12 h. Adult domestic Yorkshire cross farm pigs (25–30 kg) were used as donors. All livers were flushed with Belzer’s solution and placed on ice for 2 h during transport and preparation for studies. Experimental HMP livers +/ O2 were compared with 2 h static cold stored control livers using a battery of cell/tissue damage and function assays by sampling during 3 h normothermic ex vivo perfusions with washed erythrocytes in Krebs Henseleit solution. The HMP oxygenated livers exhibited no statistically significant differences in liver lactate dehydrogenase, hyaluronic acid uptake, total bile production, albumin, glucose concentrations and lactate, alanine aminotransferase, interleukin-8 or b-galactosidase on the normothermic test circuit relative to control livers. Statistically significant increases in alanine aminotransferase were observed during normothermic blood perfusion after HMP without oxygen (200 U/L) compared with <100 U/L in all other groups (p < 0.05). In addition, significant changes (p < 0.05) were observed after HMP without oxygenation in bile production, lactate dehydrogenase, interleukin-8, b-galactosidase, and albumin versus either control livers and/or HMP livers with oxygen. Tumor necrosis factor-a was significantly decreased in both HMP groups compared with 2 h SCS controls (p < 0.05). These results provide strong support for continuous oxygenation during HMP of livers. Future research will focus on exploring novel alternatives to tanked oxygen to improve machine preservation for practical field use and evaluation of the potential benefits of inhibiting mediators of ischemic reperfusion injury during HMP prior to preclinical liver transplant studies. Conflict of interest: None declared. Source of funding: Supported by NIH Grant #1R43DK082063–01.
doi:10.1016/j.cryobiol.2011.09.074
72. Development of a cryopreservation protocol for pancreatic cells using plant proteins. Mélanie Grondin 1,2,3, Isabelle Robinson * 1,2,3, Sonia DoCarmo 1,2, François Ouellet 1, Catherine Mounier 1,2, Fathey Sarhan 1, Diana Averill-Bates 1,2,3, 1 Département des Sciences biologiques, Université du Québec à Montréal, Canada, 2 Centre de recherche BioMed, Université du Québec à Montréal, Canada, 3 Centre de recherche en toxicologie de l’environnement (TOXEN), Université du Québec à Montréal, Canada