- Email: [email protected]
30. Roles of ice-active agents in organ cryopreservation
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 activity. Low molecular weight PVA and PGL are backbones of the advanced M22 and VM3 vitrification solutions. There are other INIs in the literature, such as flavonol glycosides, and new families of synthetic IRI inhibitors that may also be useful for cryopreservation. Even in circumstances in which ice nucleation cannot be avoided during vitrification, injury might be prevented if IRIs can keep ice crystals sufficiently small during warming. There is, however, a deficiency of small synthetic molecules with the same broad anti-ice activity as PVA. Small molecules are preferred because of greater mobility and decreased contribution to solution viscosity (low viscosity being important for perfusion cryoprotection of organs). Even though syndiotactic PVA oligomers have stable conformations with excellent alignment of hydroxyls for bonding to the basal plane of ice, a custom synthesized four ‘‘mer” PVA oligomer (1,3,5,7heptanetetrol) was found to be inactive as an ice blocker in our laboratory. Subsequent structure-activity studies by Gibson’s group showed that PVA IRI activity ceases somewhere between 10 and 19 mers (400–800 MW). Evidently if a molecule is small, molecular models showing structural matching to an ice crystal surface, such as is seen with cyclohexanediols and triols, are not sufficient to establish identity as an ice blocker. Nor are empirical results at concentrations high enough (>1 mg/g) to enable bulk cryoprotective effects. Until good models and understanding of how known synthetic ice blockers or IRIs work are developed, prospects for finding or making new ones are limited. Protein-based compounds are understood better, but have obstacles of cost, stability in cryoprotectant solutions, and possible antigenicity. http://dx.doi.org/10.1016/j.cryobiol.2015.05.036
31. Cryoprotectant toxicity: Biochemical mechanisms and functional genomics. Joao Pedro Magalhaes, Leader of the Integrative Genomics of Aging Group at the Institute of Integrative Biology, University of Liverpool, UK The constraints placed by delicate biological structures set many challenges for the science of cryopreservation. The cryoprotective agents (CPAs) which block ice formation and remove intracellular water are also toxic, to varying degrees. The complexity of this toxicity effect has been a major barrier to clinical application of vitrification in human tissues and organs. In this talk, I will discuss biochemical mechanisms of CPA toxicity. Moreover, we are employing high-throughput gene expression profiling to study CPA toxicity and cryopreservation. Our goal is to identify genes and pathways at work in CPA toxicity; this may give us direct targets for drug discovery. Ultimately we aim to improve cryopreservation protocols to make long-term storage of stem cells, engineered tissues and organs more efficient. Results will be available online (http://cryopreservation.org.uk/) as our project proceeds.
173
in this resistance. When we expose mutated ESC to the toxin, we find some cells forming colonies and these cells grow and divide under conditions that kill all unmutated cells. These ESC seem to maintain pluripotency. The sites of insertion are many and we have found many (some very unusual) targets for drug development. We hope to expand our studies to a variety of other stressful cryogenic agents and conditions. We also want to initiate drug screening and clinical chemistry, allowing us to move these methods to the organ transplantation community. http://dx.doi.org/10.1016/j.cryobiol.2015.05.038
33. Protecting cells and proteins in multiple organ systems. Kenneth Storey Canada Research Chair in Molecular Physiology and Professor in Biochemistry at Carleton University Molecular studies of cold and frozen systems – be they naturally cold-hardy animals, cryopreserved cells/tissues/organs, or tissues ablated by cryosurgery – all have one thing in common. This is a need to identify and quantify biochemical markers of stress, injury and survival. Physical assessment methods (cell rupture, enzyme leakage, etc.) have long been used as have ‘‘bulk” protective strategies (e.g. cryoprotectant infusion). However, in-depth knowledge of the key biochemical parameters of cell systems under freezing stress, the negative versus positive responses that can constitute molecular markers of stress versus endurance, and the features that differentiate natural versus man-made freezing survival still leave much to be determined. Assessing complex cell functions has traditionally been difficult and time consuming – absorbing many grad student careers for energy-intensive methods like gene screening, PCR, immunoblotting and enzyme assay. However, new technologies have recently emerged that are re-focusing research efforts and allowing huge amounts of data to be gathered from very small cell/tissue samples. Among these, accelerated rates of genome sequencing are giving us access to genomic tools to find inherent differences in genes between species and tools for screening a stress-responsive transcriptome (e.g. RNA seq.; the RT2 profiler array technology) or proteome are becoming easier and cheaper. This is providing ways to identify signatures of cells/ organisms under stress. A relatively new analytical tool, multiplex suspension array technology (Luminex), is also radically changing biomedical analytical capabilities for both research and diagnostic purposes by providing a way to quantify panels of multiple analytes simultaneously in single small samples in a very short time. For more information visit: www.carleton.ca/kbstorey. http://dx.doi.org/10.1016/j.cryobiol.2015.05.039
http://dx.doi.org/10.1016/j.cryobiol.2015.05.037
32. Eliminating toxicity during long-term cryogenic storage of human organs. Tom Johnson, Professor at the Institute for Behavioral Genetics, University of Colorado; Professor of Integrative Physiology and Fellow of Biofrontiers Program a, W.E. Chick b, G. Fahy c, a Institute for Behavioral Genetics, University of Colorado Boulder, United States, b Cell and Developmental Biology, University of Colorado Denver, United States, c 21st Century Medicine, Inc., Fontana, CA, United States We have developed a novel genetic platform enabling high-throughput identification of genetic targets in embryonic stem cells (ESC) of the mouse (Mamm Genome 20: 11, 2009). We propose to utilize this platform to identify genes causing cryotoxicity. The method consists of mass screening of mutated populations of ESC. Mutated cells are then exposed to a near-lethal concentration of a cryogenic agent or a stressful condition. Mutant ESC surviving exposure to the agent or stress grow and form colonies, which are picked and verified to be resistant to the stress. Hundreds of thousands of mutations can be screened simultaneously to identify those rare mutants causing resistance resulting from a mutational event. In our platform, mutations are generated by a transposable element called ‘‘PiggyBack” (PB). When activated by an appropriate transposase, the PB vectors move throughout the genome inserting themselves into the genome almost randomly and disrupting correct gene expression in a variety of ways. The sites of insertion and the genes disrupted by the PB element are identified by PCR off the ends of the PB element into the flanking DNA. The ability of the ESC to develop into mice (called pluripotency) is maintained by using a replica plating strategy so that a so-called ‘‘Master Plate” of the ESC are never exposed to a stressor. Thus, these mutant ESC can be used to generate intact, completely normal, adult mice, which can be used for organ harvest. These organs are composed entirely of genetically modified ESC, which can then be tested in organ transplants. For use in human (and perhaps in xenograft) transplants, drugs that block cytotoxicity will be developed. Genes and their protein products that lead to resistance become targets for developing drugs that should enhance organ viability during cryogenic storage. In preliminary results, it seems that we can establish conditions, using M22, that will completely kill the parental unmutated ESC. We have demonstrated that we can identify genes mediating resistance to cryotoxins and verify their role
34. Designing next generation protectants. Gloria Elliot, Director of the Biostability Lab and Professor, Lindong Weng. Department of Mechanical Engineering and Engineering Science, University of North Carolina at Charlotte, 9201 University City Blvd., Charlotte, NC 28223, United States In order to advance towards the vitrification of complex organs, further advances in cryoprotectant compositions will be necessary. Systematic solution optimization has enabled the identification of superior combinations of cryoprotectants for various cell and tissue types, yet the range of chemicals used as protectants is still quite narrow and it is oftentimes not well understood why some compositions work better than others. The White House recently launched a ‘‘Materials Genome Initiative” to catalyze an integrated design process that uses feedback from computational studies to improve the efficiency of advanced materials development. This approach is very suitable to the design of Next Generation Protectants. For vitrification applications the main materials optimization parameters are cryoprotectant toxicity and glassy state properties. While advances have been made in understanding and predicting CPA toxicity, it is much harder to predict the glassy state characteristics of complex mixtures. Additive mixing rules provide little insight, as superior compositions often exhibit strong deviations from ideal solution behavior. In recent work we have combined advanced thermal analysis techniques, specifically Dynamic Mechanical Analysis (DMA) and Differential Scanning Calorimetry (DSC), together with molecular modelling to explore the underling molecular mechanisms that give rise to Tg enhancements in protectant compositions in an effort to better understand the design rules that lead to improved compositions. Using molecular dynamics (MD) simulation, we have recently shown that divalent cations can strengthen the interacting network in electrolyte/glycerol mixtures via strong cation-dipole attractions, leading to an increase in T 1g . In contrast, monovalent cations were observed to have little effect on Tg as the cation-dipole attraction was only slightly stronger than the original hydrogen-bonding network amongst glycerol molecules. The precursor of salt crystallization was also observed in these monovalent ion compositions, potentially contributing to weak Tg-enhancing ability. Using both thermal analysis and MD simulation we were also able to better understand the effects of phosphate anions on the glassy state properties of trehalose2. We observed that the addition of NaH2PO4 decreased both the glass transition temperature and the a-relaxation
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 activity. Low molecular weight PVA and PGL are backbones of the advanced M22 and VM3 vitrification solutions. There are other INIs in the literature, such as flavonol glycosides, and new families of synthetic IRI inhibitors that may also be useful for cryopreservation. Even in circumstances in which ice nucleation cannot be avoided during vitrification, injury might be prevented if IRIs can keep ice crystals sufficiently small during warming. There is, however, a deficiency of small synthetic molecules with the same broad anti-ice activity as PVA. Small molecules are preferred because of greater mobility and decreased contribution to solution viscosity (low viscosity being important for perfusion cryoprotection of organs). Even though syndiotactic PVA oligomers have stable conformations with excellent alignment of hydroxyls for bonding to the basal plane of ice, a custom synthesized four ‘‘mer” PVA oligomer (1,3,5,7heptanetetrol) was found to be inactive as an ice blocker in our laboratory. Subsequent structure-activity studies by Gibson’s group showed that PVA IRI activity ceases somewhere between 10 and 19 mers (400–800 MW). Evidently if a molecule is small, molecular models showing structural matching to an ice crystal surface, such as is seen with cyclohexanediols and triols, are not sufficient to establish identity as an ice blocker. Nor are empirical results at concentrations high enough (>1 mg/g) to enable bulk cryoprotective effects. Until good models and understanding of how known synthetic ice blockers or IRIs work are developed, prospects for finding or making new ones are limited. Protein-based compounds are understood better, but have obstacles of cost, stability in cryoprotectant solutions, and possible antigenicity. http://dx.doi.org/10.1016/j.cryobiol.2015.05.036
31. Cryoprotectant toxicity: Biochemical mechanisms and functional genomics. Joao Pedro Magalhaes, Leader of the Integrative Genomics of Aging Group at the Institute of Integrative Biology, University of Liverpool, UK The constraints placed by delicate biological structures set many challenges for the science of cryopreservation. The cryoprotective agents (CPAs) which block ice formation and remove intracellular water are also toxic, to varying degrees. The complexity of this toxicity effect has been a major barrier to clinical application of vitrification in human tissues and organs. In this talk, I will discuss biochemical mechanisms of CPA toxicity. Moreover, we are employing high-throughput gene expression profiling to study CPA toxicity and cryopreservation. Our goal is to identify genes and pathways at work in CPA toxicity; this may give us direct targets for drug discovery. Ultimately we aim to improve cryopreservation protocols to make long-term storage of stem cells, engineered tissues and organs more efficient. Results will be available online (http://cryopreservation.org.uk/) as our project proceeds.
173
in this resistance. When we expose mutated ESC to the toxin, we find some cells forming colonies and these cells grow and divide under conditions that kill all unmutated cells. These ESC seem to maintain pluripotency. The sites of insertion are many and we have found many (some very unusual) targets for drug development. We hope to expand our studies to a variety of other stressful cryogenic agents and conditions. We also want to initiate drug screening and clinical chemistry, allowing us to move these methods to the organ transplantation community. http://dx.doi.org/10.1016/j.cryobiol.2015.05.038
33. Protecting cells and proteins in multiple organ systems. Kenneth Storey Canada Research Chair in Molecular Physiology and Professor in Biochemistry at Carleton University Molecular studies of cold and frozen systems – be they naturally cold-hardy animals, cryopreserved cells/tissues/organs, or tissues ablated by cryosurgery – all have one thing in common. This is a need to identify and quantify biochemical markers of stress, injury and survival. Physical assessment methods (cell rupture, enzyme leakage, etc.) have long been used as have ‘‘bulk” protective strategies (e.g. cryoprotectant infusion). However, in-depth knowledge of the key biochemical parameters of cell systems under freezing stress, the negative versus positive responses that can constitute molecular markers of stress versus endurance, and the features that differentiate natural versus man-made freezing survival still leave much to be determined. Assessing complex cell functions has traditionally been difficult and time consuming – absorbing many grad student careers for energy-intensive methods like gene screening, PCR, immunoblotting and enzyme assay. However, new technologies have recently emerged that are re-focusing research efforts and allowing huge amounts of data to be gathered from very small cell/tissue samples. Among these, accelerated rates of genome sequencing are giving us access to genomic tools to find inherent differences in genes between species and tools for screening a stress-responsive transcriptome (e.g. RNA seq.; the RT2 profiler array technology) or proteome are becoming easier and cheaper. This is providing ways to identify signatures of cells/ organisms under stress. A relatively new analytical tool, multiplex suspension array technology (Luminex), is also radically changing biomedical analytical capabilities for both research and diagnostic purposes by providing a way to quantify panels of multiple analytes simultaneously in single small samples in a very short time. For more information visit: www.carleton.ca/kbstorey. http://dx.doi.org/10.1016/j.cryobiol.2015.05.039
http://dx.doi.org/10.1016/j.cryobiol.2015.05.037
32. Eliminating toxicity during long-term cryogenic storage of human organs. Tom Johnson, Professor at the Institute for Behavioral Genetics, University of Colorado; Professor of Integrative Physiology and Fellow of Biofrontiers Program a, W.E. Chick b, G. Fahy c, a Institute for Behavioral Genetics, University of Colorado Boulder, United States, b Cell and Developmental Biology, University of Colorado Denver, United States, c 21st Century Medicine, Inc., Fontana, CA, United States We have developed a novel genetic platform enabling high-throughput identification of genetic targets in embryonic stem cells (ESC) of the mouse (Mamm Genome 20: 11, 2009). We propose to utilize this platform to identify genes causing cryotoxicity. The method consists of mass screening of mutated populations of ESC. Mutated cells are then exposed to a near-lethal concentration of a cryogenic agent or a stressful condition. Mutant ESC surviving exposure to the agent or stress grow and form colonies, which are picked and verified to be resistant to the stress. Hundreds of thousands of mutations can be screened simultaneously to identify those rare mutants causing resistance resulting from a mutational event. In our platform, mutations are generated by a transposable element called ‘‘PiggyBack” (PB). When activated by an appropriate transposase, the PB vectors move throughout the genome inserting themselves into the genome almost randomly and disrupting correct gene expression in a variety of ways. The sites of insertion and the genes disrupted by the PB element are identified by PCR off the ends of the PB element into the flanking DNA. The ability of the ESC to develop into mice (called pluripotency) is maintained by using a replica plating strategy so that a so-called ‘‘Master Plate” of the ESC are never exposed to a stressor. Thus, these mutant ESC can be used to generate intact, completely normal, adult mice, which can be used for organ harvest. These organs are composed entirely of genetically modified ESC, which can then be tested in organ transplants. For use in human (and perhaps in xenograft) transplants, drugs that block cytotoxicity will be developed. Genes and their protein products that lead to resistance become targets for developing drugs that should enhance organ viability during cryogenic storage. In preliminary results, it seems that we can establish conditions, using M22, that will completely kill the parental unmutated ESC. We have demonstrated that we can identify genes mediating resistance to cryotoxins and verify their role
34. Designing next generation protectants. Gloria Elliot, Director of the Biostability Lab and Professor, Lindong Weng. Department of Mechanical Engineering and Engineering Science, University of North Carolina at Charlotte, 9201 University City Blvd., Charlotte, NC 28223, United States In order to advance towards the vitrification of complex organs, further advances in cryoprotectant compositions will be necessary. Systematic solution optimization has enabled the identification of superior combinations of cryoprotectants for various cell and tissue types, yet the range of chemicals used as protectants is still quite narrow and it is oftentimes not well understood why some compositions work better than others. The White House recently launched a ‘‘Materials Genome Initiative” to catalyze an integrated design process that uses feedback from computational studies to improve the efficiency of advanced materials development. This approach is very suitable to the design of Next Generation Protectants. For vitrification applications the main materials optimization parameters are cryoprotectant toxicity and glassy state properties. While advances have been made in understanding and predicting CPA toxicity, it is much harder to predict the glassy state characteristics of complex mixtures. Additive mixing rules provide little insight, as superior compositions often exhibit strong deviations from ideal solution behavior. In recent work we have combined advanced thermal analysis techniques, specifically Dynamic Mechanical Analysis (DMA) and Differential Scanning Calorimetry (DSC), together with molecular modelling to explore the underling molecular mechanisms that give rise to Tg enhancements in protectant compositions in an effort to better understand the design rules that lead to improved compositions. Using molecular dynamics (MD) simulation, we have recently shown that divalent cations can strengthen the interacting network in electrolyte/glycerol mixtures via strong cation-dipole attractions, leading to an increase in T 1g . In contrast, monovalent cations were observed to have little effect on Tg as the cation-dipole attraction was only slightly stronger than the original hydrogen-bonding network amongst glycerol molecules. The precursor of salt crystallization was also observed in these monovalent ion compositions, potentially contributing to weak Tg-enhancing ability. Using both thermal analysis and MD simulation we were also able to better understand the effects of phosphate anions on the glassy state properties of trehalose2. We observed that the addition of NaH2PO4 decreased both the glass transition temperature and the a-relaxation