- Email: [email protected]
12. Non-ideal thermodynamics in cryobiology
Abstracts / Cryobiology 55 (2007) 324–378 solution and pure EG with 0.01%, 0.1%, 1% different kinds of nanoparticles were measured, and the results show that even 0.01% nanoparticles can lower the devitrification temperatures by 1 3°. These findings can be used to improve the efficiency of cell cryopreservation procedures and open the door to the application of nanoparticles in cryogenics. (Conflicts of interest: None declared. Source of funding: None declared). doi:10.1016/j.cryobiol.2007.10.013
11. Thermodynamics and beyond: connecting modeling with outcome in cryobiology. John C. Bischof, Departments of Mechanical and Biomedical, Engineering and Urology, University of Minnesota, MN, USA Thermodynamics and transport (kinetics) play a crucial role in many biomedical applications in cryobiology including biopreservation and cryosurgery. In these applications a variety of thermodynamic excursions (i.e. thermal, chemical, mechanical, etc.) are used to selectively preserve or destroy cells and tissues. This talk will focus on the use of transport models to predict cryobiological outcome at the cell and tissue level after a thermodynamic excursion. Since models are dependent on input conditions, a review of available and needed thermodynamic and kinetic properties at the molecular (i.e. lipid, protein and water), cellular and tissue (engineered and native) level will be given. (Conflicts of interest: None declared. Source of funding: None declared). doi:10.1016/j.cryobiol.2007.10.014
12. Non-ideal thermodynamics in cryobiology. Janet A.W. Elliott a, Richelle C. Prickett a,b, Heidi Y. Elmoazzen b, Lisa U. Ross-Rodriguez b, Alireza Abazari a, Locksley E. McGann b, a Departments of Chemical and Materials Engineering, University of Alberta, Edmonton, AB, Canada; b Departments of Laboratory Medicine and Pathology, University of Alberta, Edmonton, AB, Canada Cryopreservation is a core enabling technology for the transplantation of natural and engineered cells and tissues. A variety of multipotent stem cells are being heralded for innovative treatments in regenerative medicine and cellular therapies. Blood, heart valves, bone, articular cartilage and corneas may be transplanted with success, and transplantable bioengineered varieties are on the horizon. Cryopreservation is currently the only method to preserve long-term viability and function of mammalian cells and tissue. However, despite decades of research, some cells and the vast majority of tissues have eluded optimal, or even successful, cryopreservation. The removal of pure water upon freezing of the extracellular solution changes the extracellular concentration. As a result, water is passively transported across the plasma membrane at a rate proportional to the osmolality difference. Physical processes determined by the rates of cooling and osmotic transport determine both the formation of intracellular ice (rapid-cool injury) and the temperature and time of exposure of non-physiological extra- and intra-cellular solute concentrations (slow-cool injury). There is rarely an optimal constant cooling rate that provides sufficient cell viability so permeating and/or nonpermeating cryoprotectants are added to increase cell survival by altering the freezing points and avoiding harmful solute concentrations. For the cryopreservation of tissues, there is a further relationship between ice formation in the extracellular matrix and tissue viability and function. The current approach is to use high concentrations of cryoprotectants with the aim of vitrifying the tissue. Thus, in addition to cellular osmotics, cryoprotectant membrane transport and extra- and intra-cellular freezing points that are critical to cellular cryopreservation, heat and mass transfer across the tissue dimensions are also important. While many researchers have used computer modeling to direct and interpret cryobiology experiments, they have used rudimentary solution thermodynamics models often with ideal, dilute solution assumptions. Even though specific advances have been made, solution models that are accurate to the fullest extent of current thermodynamics knowledge have not been used previously. It is well known that the relationship between
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osmolality (and thus freezing point) and solution composition is critical to cryobiological calculations. We will review our use of the osmotic virial equation to improve the mathematical description of the osmolality-concentration relationship and the impact upon cryobiological calculations. We have used the osmotic virial equation to more accurately describe extraand intra-cellular multi-solute solutions. We have derived a non-ideal replacement for the Boyle van’t Hoff relation and non-ideal osmotic membrane transport equations. The improvements in the solution thermodynamics descriptions are important for cellular cryopreservation and may be critical in tissue cryopreservation where highly concentrated solutions with many solutes are being used. (Conflicts of interest: None declared. Source of funding: NSERC and CIHR. J. A. W. Elliott holds a Canada Research Chair in Interfacial Thermodynamics.) doi:10.1016/j.cryobiol.2007.10.015
13. Intracellular ice formation in mouse oocytes subjected to interrupted rapid cooling. Peter Mazur a, Irina L. Pinn a, F.W. Kleinhans a,b, a University of Tennessee, Knoxville, TN, USA; b Indiana U.-Purdue University at Indianapolis, Indianapolis, IN, USA The formation of ice crystals within cells (IIF) is lethal. The classical approach to avoiding it is to cool cells slowly enough so that nearly all their supercooled freezable water leaves the cell osmotically before they have cooled to a temperature that permits IIF. An alternative approach is to cool the cell rapidly to just above its ice nucleation temperature, and hold it there long enough to permit dehydration. Then, the cell is cooled rapidly to 70 °C or below. This approach, often called interrupted rapid cooling, is the subject of this presentation. Mouse oocytes were suspended in 1.5 M ethylene glycol (EG)/PBS, rapidly cooled (50 °C/min) to 25 °C and held for 5, 10, 20, 30, or 40 min before being rapidly cooled (50 °C/min) to 70 °C. In cells held for 5 min, IIF (flashing) occurred abruptly during the second rapid cool. As the holding period was increased to 10 and 20 min, fewer cells flashed during the cooling and more turned black during warming. Finally, when the oocytes were held 30 or 40 min, relatively few flashed during either cooling or warming. Immediately upon thawing, these oocytes were highly shrunken and crenated. However, upon warming to 20 °C, they regained most of their normal volume, shape, and appearance. These oocytes have intact cell membranes, and we refer to them as survivors. We conclude that 30 min at 25 °C removes nearly all intracellular freezable water, the consequence of which is that IIF occurs neither during the subsequent rapid cooling to 70 °C nor during warming. (Conflicts of interest: None declared. Source of funding: NIH Grant R01-RR18470) doi:10.1016/j.cryobiol.2007.10.016
14. The temperature of intracellular ice formation in mouse oocytes vs. the unfrozen fraction at that temperature. Peter Mazur a, Irina L. Pinn a, F.W. Kleinhans a,b, a University of Tennessee, Knoxville, TN, USA; b Indiana U.-Purdue U. at Indianapolis, Indianapolis, IN, USA We have previously reported [Mazur, et al. Cryobiology 51 (2005) 2953] that intracellular ice formation (IIF) in mouse oocytes suspended in various concentrations of glycerol and ethylene glycol (EG) occurs at temperatures where the percentage of unfrozen water is about 6% and 12% respectively even though the IIF temperatures varied from 14° to 41 °C. However, because of the way the solutions were prepared, the concentrations of salt and glycerol or EG in that unfrozen fraction at IIF were also rather tightly grouped. The experiments reported here were designed to separate the effects of the unfrozen fraction at IIF from that of the solute concentration in the unfrozen fraction. This separation makes use of two facts. One is that the concentration of solutes in the residual liquid at a given subzero temperature is fixed regardless of their concentration in the initial unfrozen solution. However, second, the fraction unfrozen at a given temperature is dependent on the initial solute concentration. Experimentally, oocytes were suspended in solutions of glycerol/buffered
11. Thermodynamics and beyond: connecting modeling with outcome in cryobiology. John C. Bischof, Departments of Mechanical and Biomedical, Engineering and Urology, University of Minnesota, MN, USA Thermodynamics and transport (kinetics) play a crucial role in many biomedical applications in cryobiology including biopreservation and cryosurgery. In these applications a variety of thermodynamic excursions (i.e. thermal, chemical, mechanical, etc.) are used to selectively preserve or destroy cells and tissues. This talk will focus on the use of transport models to predict cryobiological outcome at the cell and tissue level after a thermodynamic excursion. Since models are dependent on input conditions, a review of available and needed thermodynamic and kinetic properties at the molecular (i.e. lipid, protein and water), cellular and tissue (engineered and native) level will be given. (Conflicts of interest: None declared. Source of funding: None declared). doi:10.1016/j.cryobiol.2007.10.014
12. Non-ideal thermodynamics in cryobiology. Janet A.W. Elliott a, Richelle C. Prickett a,b, Heidi Y. Elmoazzen b, Lisa U. Ross-Rodriguez b, Alireza Abazari a, Locksley E. McGann b, a Departments of Chemical and Materials Engineering, University of Alberta, Edmonton, AB, Canada; b Departments of Laboratory Medicine and Pathology, University of Alberta, Edmonton, AB, Canada Cryopreservation is a core enabling technology for the transplantation of natural and engineered cells and tissues. A variety of multipotent stem cells are being heralded for innovative treatments in regenerative medicine and cellular therapies. Blood, heart valves, bone, articular cartilage and corneas may be transplanted with success, and transplantable bioengineered varieties are on the horizon. Cryopreservation is currently the only method to preserve long-term viability and function of mammalian cells and tissue. However, despite decades of research, some cells and the vast majority of tissues have eluded optimal, or even successful, cryopreservation. The removal of pure water upon freezing of the extracellular solution changes the extracellular concentration. As a result, water is passively transported across the plasma membrane at a rate proportional to the osmolality difference. Physical processes determined by the rates of cooling and osmotic transport determine both the formation of intracellular ice (rapid-cool injury) and the temperature and time of exposure of non-physiological extra- and intra-cellular solute concentrations (slow-cool injury). There is rarely an optimal constant cooling rate that provides sufficient cell viability so permeating and/or nonpermeating cryoprotectants are added to increase cell survival by altering the freezing points and avoiding harmful solute concentrations. For the cryopreservation of tissues, there is a further relationship between ice formation in the extracellular matrix and tissue viability and function. The current approach is to use high concentrations of cryoprotectants with the aim of vitrifying the tissue. Thus, in addition to cellular osmotics, cryoprotectant membrane transport and extra- and intra-cellular freezing points that are critical to cellular cryopreservation, heat and mass transfer across the tissue dimensions are also important. While many researchers have used computer modeling to direct and interpret cryobiology experiments, they have used rudimentary solution thermodynamics models often with ideal, dilute solution assumptions. Even though specific advances have been made, solution models that are accurate to the fullest extent of current thermodynamics knowledge have not been used previously. It is well known that the relationship between
327
osmolality (and thus freezing point) and solution composition is critical to cryobiological calculations. We will review our use of the osmotic virial equation to improve the mathematical description of the osmolality-concentration relationship and the impact upon cryobiological calculations. We have used the osmotic virial equation to more accurately describe extraand intra-cellular multi-solute solutions. We have derived a non-ideal replacement for the Boyle van’t Hoff relation and non-ideal osmotic membrane transport equations. The improvements in the solution thermodynamics descriptions are important for cellular cryopreservation and may be critical in tissue cryopreservation where highly concentrated solutions with many solutes are being used. (Conflicts of interest: None declared. Source of funding: NSERC and CIHR. J. A. W. Elliott holds a Canada Research Chair in Interfacial Thermodynamics.) doi:10.1016/j.cryobiol.2007.10.015
13. Intracellular ice formation in mouse oocytes subjected to interrupted rapid cooling. Peter Mazur a, Irina L. Pinn a, F.W. Kleinhans a,b, a University of Tennessee, Knoxville, TN, USA; b Indiana U.-Purdue University at Indianapolis, Indianapolis, IN, USA The formation of ice crystals within cells (IIF) is lethal. The classical approach to avoiding it is to cool cells slowly enough so that nearly all their supercooled freezable water leaves the cell osmotically before they have cooled to a temperature that permits IIF. An alternative approach is to cool the cell rapidly to just above its ice nucleation temperature, and hold it there long enough to permit dehydration. Then, the cell is cooled rapidly to 70 °C or below. This approach, often called interrupted rapid cooling, is the subject of this presentation. Mouse oocytes were suspended in 1.5 M ethylene glycol (EG)/PBS, rapidly cooled (50 °C/min) to 25 °C and held for 5, 10, 20, 30, or 40 min before being rapidly cooled (50 °C/min) to 70 °C. In cells held for 5 min, IIF (flashing) occurred abruptly during the second rapid cool. As the holding period was increased to 10 and 20 min, fewer cells flashed during the cooling and more turned black during warming. Finally, when the oocytes were held 30 or 40 min, relatively few flashed during either cooling or warming. Immediately upon thawing, these oocytes were highly shrunken and crenated. However, upon warming to 20 °C, they regained most of their normal volume, shape, and appearance. These oocytes have intact cell membranes, and we refer to them as survivors. We conclude that 30 min at 25 °C removes nearly all intracellular freezable water, the consequence of which is that IIF occurs neither during the subsequent rapid cooling to 70 °C nor during warming. (Conflicts of interest: None declared. Source of funding: NIH Grant R01-RR18470) doi:10.1016/j.cryobiol.2007.10.016
14. The temperature of intracellular ice formation in mouse oocytes vs. the unfrozen fraction at that temperature. Peter Mazur a, Irina L. Pinn a, F.W. Kleinhans a,b, a University of Tennessee, Knoxville, TN, USA; b Indiana U.-Purdue U. at Indianapolis, Indianapolis, IN, USA We have previously reported [Mazur, et al. Cryobiology 51 (2005) 2953] that intracellular ice formation (IIF) in mouse oocytes suspended in various concentrations of glycerol and ethylene glycol (EG) occurs at temperatures where the percentage of unfrozen water is about 6% and 12% respectively even though the IIF temperatures varied from 14° to 41 °C. However, because of the way the solutions were prepared, the concentrations of salt and glycerol or EG in that unfrozen fraction at IIF were also rather tightly grouped. The experiments reported here were designed to separate the effects of the unfrozen fraction at IIF from that of the solute concentration in the unfrozen fraction. This separation makes use of two facts. One is that the concentration of solutes in the residual liquid at a given subzero temperature is fixed regardless of their concentration in the initial unfrozen solution. However, second, the fraction unfrozen at a given temperature is dependent on the initial solute concentration. Experimentally, oocytes were suspended in solutions of glycerol/buffered