- Email: [email protected]
28. Thermodynamics in cryopreservation: Understanding ice formation
172
Abstracts / Cryobiology 71 (2015) 164–180
pressure can be rapidly and equally distributed throughout the entire volume of an organ, creating unique conditions for cryopreservation, which cannot be realized at ambient pressure. The effect of pressure and a low concentration of dimethyl sulfoxide (Me2SO) or glycerol, on hemolysis of human red blood cells after freezing and thawing were investigated. Pressure was applied during cooling and freezing the red blood cells and a minimum in hemolysis was reached at approximately 120 MPa. Either 5% v/v Me2SO or 8% v/v glycerol concentration in combination with 120 MPa pressure was sufficient to obtain 8% or less hemolysis of red blood cells after cooling at a 35 °C/ min or a 160 °C/min rate. The preliminary results suggest that the method may help to solve the cryoprotectant toxicity problem. Since cryoprotectants in high concentrations are harmful to tissues and organs, the development of the method for freezing under pressure with a reduced cryoprotectant concentration may be another step towards successful cryopreservation and recovery of viable organs. More research is needed to optimize the method and determine if it is clinically applicable. http://dx.doi.org/10.1016/j.cryobiol.2015.05.032
27. Biological matter in isochoric systems. Boris Rubinsky Professor at UC Berkeley, Discoverer of fish antifreeze proteins for cryopreservation solutions, and innovative isochoric cryopreservation approach, Department of Mechanical Engineering, University of California, Berkeley, United States Most of the research in biology deals with biological systems under thermodynamic conditions of variable volume and constant pressure; either atmospheric pressure or hyperbaric pressure or hypobaric pressure. These are the conditions that exist on the planet Earth. Interest in astrobiology and the possibility of life on other systems, such as the ice moons of Jupiter and Saturn, was the original motive behind our study of the thermodynamics of life in a constant volume environment, isochoric; as in the fluid layer between the ground and the thick layer of ice surrounding moons of Jupiter and Saturn. Fundamental and later applied studies on isochoric systems of ice and water, have revealed that at the interface between ice and water, the isochoric systems behave differently from isobaric systems, with possible applications in preservation of biological systems at below atmospheric freezing temperatures. This presentation will review the thermodynamics of isochoric systems with physiological relevant compositions, the effects of isochoric cooling on nucleation and vitrification, the interaction between antifreeze proteins and ice under isochoric conditions and previously unpublished data on isochoric preservation of cells and organs (heart). http://dx.doi.org/10.1016/j.cryobiol.2015.05.033
28. Thermodynamics in cryopreservation: Understanding ice formation. Janet Elliott, Canada Research Chair in Thermodynamics and Professor at University of Alberta, Department of Chemical and Materials Engineering & Department of Laboratory Medicine and Pathology, University of Alberta, Canada Email address: [email protected] Thermodynamics is the study of mathematical relationships arising from physical laws governing energy and entropy. Thermodynamic equilibrium includes thermal equilibrium, mechanical equilibrium and chemical equilibrium. If one of these equilibria is not satisfied in a system, there will be a change towards equilibrium: heat will be transferred, mass will be transferred or change phase (ice will form or melt), or acceleration will occur due to a mechanical force imbalance. As such, thermodynamics (including both equilibrium and nonequilibrium thermodynamics) is the overarching physical science of cryobiology. Thermodynamics describes the freezing point of intra and extracellular solutions and how much ice is formed at a given temperature. Thermodynamics describes the flux of water and cryoprotectants into and out of cells and across tissues. Thermodynamics describes the heat transfer that occurs during cooling and rewarming. Though vitrification is not strictly speaking a process of thermodynamic equilibrium, since vitrifiability is governed by how far the system is from its thermodynamic freezing point (vitrification is out-running thermodynamic equilibrium) and since the process of ice recrystallization of a vitrified solution is a thermodynamic one, thermodynamics plays a key role here too. Any approach to cryopreservation must be well-based in sound thermodynamic understanding. For more than 15 years, we have worked to improve thermodynamic modelling in cryobiology. We have introduced a multi-solute osmotic virial equation to make the most accurate predictions of multi-component extra- and intracellular solution freezing points and driving forces for osmotic transport. We have described the transport of water and non-dilute components across cell membranes and across complex tissues. Our modelling has introduced an understanding of mechanisms of injury such as Mazur’s rapid-cool and slow-cool injury for cells or the mechanical stress of spatially uneven tissue dehydration during cryoprotectant loading in tissues. We have explored curvature-induced freezing point depression and its implications for the growth of ice through cell membrane pores and tissue porosity. We have
investigated physical conditions for intracellular ice formation. We have coupled thermodynamics to fluid mechanics to describe complicated phenomena that occur in the freezing of colloidal suspensions. We have described vitrifiability with empirical mathematical models. Even though we have made many improvements in thermodynamic modelling in cryobiology, there exists a great deal of other welldeveloped thermodynamics that has yet to be applied to cryopreservation challenges. http://dx.doi.org/10.1016/j.cryobiol.2015.05.034
29. Ice-binding proteins and their interactions with ice crystals. Ido Braslavsky Director of the Food-Biophysics and Cryobiology Laboratory and Professor at The Hebrew University of Jerusalem a,b, Ran Drori a, Yeliz Celik b, Maya Bar Dolev a, Peter L. Davies c, a The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 7610001, Israel, b Department of Physics and Astronomy, Ohio University, Athens, OH 45701, USA, c Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, Ontario K7L 3N6, Canada Email address: [email protected] Ice-binding proteins (IBPs) depress the freezing point of the body fluids below the melting point, resulting in a thermal hysteresis (TH) that prevents freezing of the organism in supercooled conditions and inhibits ice re-crystallization in frozen tissues. The potential of these proteins in the medical sector, in cryopreservation, in the frozen food industry, and in agriculture is enormous. The mechanisms by which IBPs interact with ice surfaces are still not completely understood and the potential of IBPs as cryoprotecting agents has not yet been realized. At the molecular level it was found that the IBPs coordinate and stabilize water molecules on their binding surface to form an ice-like water film. While this ice-like film is too small to nucleate supper-cooled water it serves as a mechanism of tight binding to ice. Still, the way these IBPs influence ice growth and the activity differences between difference types of IBPs is the subject of current research. We are investigating the interactions of IBPs with ice surfaces. In particular we are interested in the difference between hyperactive antifreeze proteins and moderately active ones, and the dynamic nature of the protein:ice interaction. We have developed novel methods to study these issues, including fluorescence microscopy techniques combined with temperaturecontrolled microfluidic devices (Celik et al., PNAS 2013, Drori et al., J.R. Soc. Interface 2014, Drori et al., RSC Adv. 2015). These techniques enable the replacement of the IBP solution surrounding an IBP-bound ice crystal by other solutions, without perturbing the system, which enables us to investigate the dynamic nature of the interactions between IBP and ice. The results show that binding of IBP to ice is irreversible, and that the TH-gap is sensitive to the time allowed for the proteins to accumulate on ice surfaces. This sensitivity changes dramatically between different types of IBPs. In a study of ice shaping during growth and melting we have demonstrated a correlation between ice crystal shapes, the shaping process, and the affinity of IBPs for the basal plane (Bar-Dolev et al., J.R. Soc. Interface 2012). Our results point to a connection between the dynamics and level of activity of different types of IBP to their ability to bind to specific ice orientations, in particularly to the basal plane of the ice. These results contribute to an understanding of the mechanisms by which diverse IBPs act that will be critical for the successful use of IBP in cryobiological applications. Supported by ERC, NSF, ISF, and CIHR. http://dx.doi.org/10.1016/j.cryobiol.2015.05.035
30. Roles of ice-active agents in organ cryopreservation. Brian Wowk, Cryobiologist and Senior Physicist at 21st Century Medicine Inc., Fontana, CA 92336, USA Email address: [email protected] The components of cryopreservation solutions can be classified into three general classes: (1) carrier solutes, which are the non-penetrating osmolytes, pH buffers, and nutritive ingredients that support viability of cells at hypothermic temperatures; (2) bulk cryoprotectants, which are ingredients (either membrane-penetrating or nonpenetrating) typically added at multi-percent concentrations that reduce availability of bulk liquid water for ice formation by hydrogen bonding and dilution; (3) solutes that specifically interact with ice nucleating particles or ice crystals to inhibit or modify the growth of ice, and which are typically effective at very low concentrations. Class 3 solutes may exhibit ice nucleation inhibition (INI), ice growth inhibition (IGI), and/or ice recrystallization inhibition (IRI). Such solutes are valuable additives in vitrification solutions because they can replace much larger concentrations of more toxic bulk cryoprotectants while achieving similar suppression of ice formation. Antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs) are the prototypical examples of class 3 solutes. Fahy proposed in 1995 that synthetic analogs of AFPs would be useful additives for vitrification solutions, especially if lower molecular weights conveyed higher mobility in the high viscosity of vitrification solutions at low temperature. Subsequently, low molecular weight versions of the polymers polyvinyl alcohol (PVA) and polyglycerol (PGL) were found to show efficacy for numerous applications as ‘‘ice blockers” in vitrification solutions. PGL is apparently a specific INI against ice-nucleating protein contaminants, while PVA exhibits general INI, IGI, and IRI
Abstracts / Cryobiology 71 (2015) 164–180
pressure can be rapidly and equally distributed throughout the entire volume of an organ, creating unique conditions for cryopreservation, which cannot be realized at ambient pressure. The effect of pressure and a low concentration of dimethyl sulfoxide (Me2SO) or glycerol, on hemolysis of human red blood cells after freezing and thawing were investigated. Pressure was applied during cooling and freezing the red blood cells and a minimum in hemolysis was reached at approximately 120 MPa. Either 5% v/v Me2SO or 8% v/v glycerol concentration in combination with 120 MPa pressure was sufficient to obtain 8% or less hemolysis of red blood cells after cooling at a 35 °C/ min or a 160 °C/min rate. The preliminary results suggest that the method may help to solve the cryoprotectant toxicity problem. Since cryoprotectants in high concentrations are harmful to tissues and organs, the development of the method for freezing under pressure with a reduced cryoprotectant concentration may be another step towards successful cryopreservation and recovery of viable organs. More research is needed to optimize the method and determine if it is clinically applicable. http://dx.doi.org/10.1016/j.cryobiol.2015.05.032
27. Biological matter in isochoric systems. Boris Rubinsky Professor at UC Berkeley, Discoverer of fish antifreeze proteins for cryopreservation solutions, and innovative isochoric cryopreservation approach, Department of Mechanical Engineering, University of California, Berkeley, United States Most of the research in biology deals with biological systems under thermodynamic conditions of variable volume and constant pressure; either atmospheric pressure or hyperbaric pressure or hypobaric pressure. These are the conditions that exist on the planet Earth. Interest in astrobiology and the possibility of life on other systems, such as the ice moons of Jupiter and Saturn, was the original motive behind our study of the thermodynamics of life in a constant volume environment, isochoric; as in the fluid layer between the ground and the thick layer of ice surrounding moons of Jupiter and Saturn. Fundamental and later applied studies on isochoric systems of ice and water, have revealed that at the interface between ice and water, the isochoric systems behave differently from isobaric systems, with possible applications in preservation of biological systems at below atmospheric freezing temperatures. This presentation will review the thermodynamics of isochoric systems with physiological relevant compositions, the effects of isochoric cooling on nucleation and vitrification, the interaction between antifreeze proteins and ice under isochoric conditions and previously unpublished data on isochoric preservation of cells and organs (heart). http://dx.doi.org/10.1016/j.cryobiol.2015.05.033
28. Thermodynamics in cryopreservation: Understanding ice formation. Janet Elliott, Canada Research Chair in Thermodynamics and Professor at University of Alberta, Department of Chemical and Materials Engineering & Department of Laboratory Medicine and Pathology, University of Alberta, Canada Email address: [email protected] Thermodynamics is the study of mathematical relationships arising from physical laws governing energy and entropy. Thermodynamic equilibrium includes thermal equilibrium, mechanical equilibrium and chemical equilibrium. If one of these equilibria is not satisfied in a system, there will be a change towards equilibrium: heat will be transferred, mass will be transferred or change phase (ice will form or melt), or acceleration will occur due to a mechanical force imbalance. As such, thermodynamics (including both equilibrium and nonequilibrium thermodynamics) is the overarching physical science of cryobiology. Thermodynamics describes the freezing point of intra and extracellular solutions and how much ice is formed at a given temperature. Thermodynamics describes the flux of water and cryoprotectants into and out of cells and across tissues. Thermodynamics describes the heat transfer that occurs during cooling and rewarming. Though vitrification is not strictly speaking a process of thermodynamic equilibrium, since vitrifiability is governed by how far the system is from its thermodynamic freezing point (vitrification is out-running thermodynamic equilibrium) and since the process of ice recrystallization of a vitrified solution is a thermodynamic one, thermodynamics plays a key role here too. Any approach to cryopreservation must be well-based in sound thermodynamic understanding. For more than 15 years, we have worked to improve thermodynamic modelling in cryobiology. We have introduced a multi-solute osmotic virial equation to make the most accurate predictions of multi-component extra- and intracellular solution freezing points and driving forces for osmotic transport. We have described the transport of water and non-dilute components across cell membranes and across complex tissues. Our modelling has introduced an understanding of mechanisms of injury such as Mazur’s rapid-cool and slow-cool injury for cells or the mechanical stress of spatially uneven tissue dehydration during cryoprotectant loading in tissues. We have explored curvature-induced freezing point depression and its implications for the growth of ice through cell membrane pores and tissue porosity. We have
investigated physical conditions for intracellular ice formation. We have coupled thermodynamics to fluid mechanics to describe complicated phenomena that occur in the freezing of colloidal suspensions. We have described vitrifiability with empirical mathematical models. Even though we have made many improvements in thermodynamic modelling in cryobiology, there exists a great deal of other welldeveloped thermodynamics that has yet to be applied to cryopreservation challenges. http://dx.doi.org/10.1016/j.cryobiol.2015.05.034
29. Ice-binding proteins and their interactions with ice crystals. Ido Braslavsky Director of the Food-Biophysics and Cryobiology Laboratory and Professor at The Hebrew University of Jerusalem a,b, Ran Drori a, Yeliz Celik b, Maya Bar Dolev a, Peter L. Davies c, a The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 7610001, Israel, b Department of Physics and Astronomy, Ohio University, Athens, OH 45701, USA, c Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, Ontario K7L 3N6, Canada Email address: [email protected] Ice-binding proteins (IBPs) depress the freezing point of the body fluids below the melting point, resulting in a thermal hysteresis (TH) that prevents freezing of the organism in supercooled conditions and inhibits ice re-crystallization in frozen tissues. The potential of these proteins in the medical sector, in cryopreservation, in the frozen food industry, and in agriculture is enormous. The mechanisms by which IBPs interact with ice surfaces are still not completely understood and the potential of IBPs as cryoprotecting agents has not yet been realized. At the molecular level it was found that the IBPs coordinate and stabilize water molecules on their binding surface to form an ice-like water film. While this ice-like film is too small to nucleate supper-cooled water it serves as a mechanism of tight binding to ice. Still, the way these IBPs influence ice growth and the activity differences between difference types of IBPs is the subject of current research. We are investigating the interactions of IBPs with ice surfaces. In particular we are interested in the difference between hyperactive antifreeze proteins and moderately active ones, and the dynamic nature of the protein:ice interaction. We have developed novel methods to study these issues, including fluorescence microscopy techniques combined with temperaturecontrolled microfluidic devices (Celik et al., PNAS 2013, Drori et al., J.R. Soc. Interface 2014, Drori et al., RSC Adv. 2015). These techniques enable the replacement of the IBP solution surrounding an IBP-bound ice crystal by other solutions, without perturbing the system, which enables us to investigate the dynamic nature of the interactions between IBP and ice. The results show that binding of IBP to ice is irreversible, and that the TH-gap is sensitive to the time allowed for the proteins to accumulate on ice surfaces. This sensitivity changes dramatically between different types of IBPs. In a study of ice shaping during growth and melting we have demonstrated a correlation between ice crystal shapes, the shaping process, and the affinity of IBPs for the basal plane (Bar-Dolev et al., J.R. Soc. Interface 2012). Our results point to a connection between the dynamics and level of activity of different types of IBP to their ability to bind to specific ice orientations, in particularly to the basal plane of the ice. These results contribute to an understanding of the mechanisms by which diverse IBPs act that will be critical for the successful use of IBP in cryobiological applications. Supported by ERC, NSF, ISF, and CIHR. http://dx.doi.org/10.1016/j.cryobiol.2015.05.035
30. Roles of ice-active agents in organ cryopreservation. Brian Wowk, Cryobiologist and Senior Physicist at 21st Century Medicine Inc., Fontana, CA 92336, USA Email address: [email protected] The components of cryopreservation solutions can be classified into three general classes: (1) carrier solutes, which are the non-penetrating osmolytes, pH buffers, and nutritive ingredients that support viability of cells at hypothermic temperatures; (2) bulk cryoprotectants, which are ingredients (either membrane-penetrating or nonpenetrating) typically added at multi-percent concentrations that reduce availability of bulk liquid water for ice formation by hydrogen bonding and dilution; (3) solutes that specifically interact with ice nucleating particles or ice crystals to inhibit or modify the growth of ice, and which are typically effective at very low concentrations. Class 3 solutes may exhibit ice nucleation inhibition (INI), ice growth inhibition (IGI), and/or ice recrystallization inhibition (IRI). Such solutes are valuable additives in vitrification solutions because they can replace much larger concentrations of more toxic bulk cryoprotectants while achieving similar suppression of ice formation. Antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs) are the prototypical examples of class 3 solutes. Fahy proposed in 1995 that synthetic analogs of AFPs would be useful additives for vitrification solutions, especially if lower molecular weights conveyed higher mobility in the high viscosity of vitrification solutions at low temperature. Subsequently, low molecular weight versions of the polymers polyvinyl alcohol (PVA) and polyglycerol (PGL) were found to show efficacy for numerous applications as ‘‘ice blockers” in vitrification solutions. PGL is apparently a specific INI against ice-nucleating protein contaminants, while PVA exhibits general INI, IGI, and IRI