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Microfluidic experiments with ice binding proteins: Evidence for irreversible binding
Abstracts / Cryobiology 63 (2011) 306–342 4. Dry state preservation of nucleated cells: Progress and challenge. Ann E. Oliver, Department of Biomedical Engineering, University of California, Davis, CA 94616, USA Effective stabilization of nucleated cells for dry storage would be a transformative development in the field of cell-based biosensors and biotechnologic devices, as well as regenerative medicine and other areas in which stem cells have clinical utility. Ultimately, the tremendous promise of cell-based products will only be fully realized when stable long-term storage becomes available without the use of liquid nitrogen and bulky, energetically expensive freezers. Significant progress has been made over the last 10 years toward this goal, but obstacles still remain. Loading cells with the protective disaccharide trehalose has been achieved by several different techniques and has been shown to increase cell survival at low water contents. Likewise, the protective effect of heat shock proteins and other compounds have also been explored alone and in combination with trehalose. In some cases, the benefit of these molecules is seen not initially upon rehydration, but over time during cellular recovery. Other considerations, such as inhibiting apoptosis and utilizing isotonic buffer conditions have also provided stepwise increases in cell viability and function following drying and rehydration. In all these cases, however, a low level of residual water is required to achieve viability after rehydration. The most significant remaining challenge is to protect nucleated cells such that this residual water can be safely removed, thus allowing vitrification of intra- and extracellular trehalose and stable dry state storage at room temperature. Conflicts of interest: None declared. Source of funding: None declared. doi:10.1016/j.cryobiol.2011.09.007
5. Bulk lyophilization tech transfer. James Brown, Process and Product Development Engineer – ASP, Oregon Freeze Dry, Inc., Albany, OR, USA Several key parameters need to be looked at when transferring lyophilization cycles from one lyophilizer to another in order to (a) eliminate differences of materials of construction between drying units and (b) provide scientific rationale for regulated submission of a repeatable process. These key parameters include but are not limited to chamber pressure, shelf temperature, product temperature, product depth, and condenser temperature. The key parameters developed in the lab or pilot scale are required for producing acceptable product and reproducible lyophilization cycles when transferred to the bulk scale lyophilizer. Taking into account these critical parameters will increase your success by preventing unacceptable product and costly product reformulation. The capabilities of the lyophilizer that will be used for bulk production should be considered during the initial formulation and cycle development to avoid getting unacceptable product that might require reformulation for bulk drying. Conflicts of interest: None declared. Source of funding: None declared. doi:10.1016/j.cryobiol.2011.09.008
6. The case for irreversible binding of ice-binding proteins to ice. Ido Braslavsky * 1,2, Yeliz Celik 1, Ran Drori 2, Maya Bar 2, Aysun Altan 1, Peter L. Davies 3, 1 Department of Physics and Astronomy, Ohio University, Athens, 45701 OH, USA, 2 Institute of Biochemistry, Food Science and Nutrition, Faculty of Agricultural, Food and Environmental Quality Sciences, the Hebrew University of Jerusalem, Rehovot 76100, Israel, 3 Department of Biochemistry, Queen’s University, Kingston, Ontario, Canada K7L 3N6 Ice-binding proteins (IBPs) include proteins that have the ability to stop ice crystal growth and inhibit ice recrystallization. IBPs do this by adsorbing to the surface of the ice and making the addition of water to the crystal unfavourable. Their surface adsorption causes a thermal hysteresis (TH) between the lowered freezing temperature and slightly elevated melting temperature. It has been argued that IBP binding must be irreversible because the ice crystals do not grow in the TH gap. However, this seems inconsistent with the observation that TH values are influenced by IBP concentration. We have made three recent experimental demonstrations that IBP binding to ice is indeed irreversible: (i) photo-bleaching of GFP-tagged IBP residing on the surface of an ice crystal held in the TH gap shows that there is neither exchange nor overgrowth of the bleached IBP; (ii) ice crystals bound by IBPs show a measurable resistance to melting (melting hysteresis) demonstrating that the IBPs remain surface-bound at temperatures above the equilibrium melting point; (iii) using a temperature controlled microfluidics apparatus it is possible to entirely replace the IBP solution surrounding an IBP-bound ice crystal in the TH gap with buffer, without causing the crystal to suddenly grow. Recent experiments also show that the exposure time of a crystal to an IBP solution at a given concentration can change the TH activity up to 10-fold. It appears that IBPs have a slow on-rate for crystal binding. According to the anchored clathrate water hypothesis for the mechanism of IBP binding to ice,
307
the ice-binding site of the IBP forms its ligand before merging with it. A slow on-rate might reflect the infrequency of having sufficient ice-like waters on the ice-binding site of the IBP for it to bind to ice. These results imply that IBP adsorption to the ice surface is irreversible and that TH is a function of the absorbed proteins on the surface and only indirectly a function of the concentration of IBPs in the solution. Conflicts of interest: None declared. Supported by the National Science Foundation (NSF) under Grant No. CHE0848081, the Israel Science Foundation (Grant Nos. 1279/10 and 1281/10), the Marie Curie International Reintegration Grant (Grant No. 256364), the Canadian Institutes for Health Research (CIHR), and the Condensed Matter and Surface Science program at Ohio University (CMSS). doi:10.1016/j.cryobiol.2011.09.009
Ice formation
7. Microfluidic experiments with ice binding proteins: Evidence for irreversible binding. Yeliz Celik * 1, Ran Drori 2, Natalya Pertaya 1, Aysun Altan 1, Maya Bar 2, Alex Groisman 3, Peter L. Davies 4, Ido Braslavsky 1,2, 1 Department of Physics and Astronomy, Ohio University, Athens, OH 45701, USA, 2 Institute of Biochemistry, Food Science and Nutrition, Faculty of Agricultural, Food and Environmental Quality Sciences, the Hebrew University of Jerusalem, Rehovot 76100, Israel, 3 Department of Physics, University of California San Diego, La Jolla, CA 92093, USA, 4 Department of Biochemistry, Queen’s University, Kingston, Ontario, Canada K7L 3N6 Ice binding proteins (IBPs) evolved in cold-adapted organisms, protecting them against freezing conditions by arresting ice crystal growth and inhibiting ice recrystallization. IBPs reduce the freezing temperature of ice below the melting point, a phenomenon defined as thermal hysteresis. The mechanism of action of IBPs is not well understood. In particular, it is not clear why thermal hysteresis activity depends on IBP concentration and whether the binding of IBP to ice is reversible or irreversible. Using fluorescently tagged IBP visualized with fluorescence microscopy in novel temperature controlled microfluidic devices; we found that hyperactive IBPs accumulate on the basal plane of ice. This finding supports the view that hyperactive IBPs adhere to the basal plane and this trait can be a factor contributing to their hyperactivity. Using the temperature-controlled microfluidic devices, we demonstrated that growth of small ice crystals (30–50 lm) covered with IBPs remain arrested for hours and the thermal hysteresis activity was maintained even after the IBP solution surrounding the crystal was replaced by plain buffer. In these experiments, ice crystals were incubated in IBP solution and then the solution was exchanged with a plain buffer. At this point, the reduction of the freezing point (thermal hysteresis activity) was measured. Repeated experiments indicate that thermal hysteresis activity is a function of IBP molecules that accumulated on the surface of the crystal at the start of the experiment and not a function of the concentration of the surrounding IBP molecules in solution after solution exchange with plain buffer. These experimental results provide strong evidence that the binding of IBPs to ice surfaces is irreversible. Conflicts of interest: None declared. Supported by the National Science Foundation (NSF) under Grant No. CHE0848081, the Israel Science Foundation (Grant Nos. 1279/10 and 1281/10), by Marie Curie International Reintegration Grant (Grant No. 256364), the Canadian Institutes for Health Research (CIHR), and the Condensed Matter and Surface Science program at Ohio University (CMSS). doi:10.1016/j.cryobiol.2011.09.010
8. Supercooling–promoting (anti-ice nucleating) flavonoids and tannins. Keita Endoh *, Seizo Fujikawa, Keita Arakawa, Research Faculty and Graduate School of Agriculture, Hokkaido University, Japan Freezing of water is controlled by the presence of ice nucleation substances such as ice nucleation bacterium (INB) which raises freezing temperatures, antifreeze substances such as antifreeze proteins which inhibit ice crystal growth after ice nucleation, and supercooling–promoting (anti-ice nucleation) substances which depress freezing temperatures. These freezing-control substances have potential in applications including low temperature preservation and cryopreservation of biological materials. With regard to these substances, however, studies on the supercooling– promoting substances are very few. As supercooling–promoting substances, only 17 kinds of compounds seem to have been reported so far. From our recent studies examining mechanisms of cold adaptation of xylem parenchyma cells (XPCs) in trees by deep supercooling, however, 4 kinds of novel supercooling–promoting flavonol glycosides and 4 kinds of novel supercooling–promoting hydrolysable tannins were identified. On the basis of the chemical structures of supercooling–promoting substances from XPCs, the present study shows a further 16 kinds of flavonoids and 9 kinds of tannins, although the effects are different depending on solutions. The results by emulsion freezing assay show that all flavonoids and tannins so far examined do not have ability
5. Bulk lyophilization tech transfer. James Brown, Process and Product Development Engineer – ASP, Oregon Freeze Dry, Inc., Albany, OR, USA Several key parameters need to be looked at when transferring lyophilization cycles from one lyophilizer to another in order to (a) eliminate differences of materials of construction between drying units and (b) provide scientific rationale for regulated submission of a repeatable process. These key parameters include but are not limited to chamber pressure, shelf temperature, product temperature, product depth, and condenser temperature. The key parameters developed in the lab or pilot scale are required for producing acceptable product and reproducible lyophilization cycles when transferred to the bulk scale lyophilizer. Taking into account these critical parameters will increase your success by preventing unacceptable product and costly product reformulation. The capabilities of the lyophilizer that will be used for bulk production should be considered during the initial formulation and cycle development to avoid getting unacceptable product that might require reformulation for bulk drying. Conflicts of interest: None declared. Source of funding: None declared. doi:10.1016/j.cryobiol.2011.09.008
6. The case for irreversible binding of ice-binding proteins to ice. Ido Braslavsky * 1,2, Yeliz Celik 1, Ran Drori 2, Maya Bar 2, Aysun Altan 1, Peter L. Davies 3, 1 Department of Physics and Astronomy, Ohio University, Athens, 45701 OH, USA, 2 Institute of Biochemistry, Food Science and Nutrition, Faculty of Agricultural, Food and Environmental Quality Sciences, the Hebrew University of Jerusalem, Rehovot 76100, Israel, 3 Department of Biochemistry, Queen’s University, Kingston, Ontario, Canada K7L 3N6 Ice-binding proteins (IBPs) include proteins that have the ability to stop ice crystal growth and inhibit ice recrystallization. IBPs do this by adsorbing to the surface of the ice and making the addition of water to the crystal unfavourable. Their surface adsorption causes a thermal hysteresis (TH) between the lowered freezing temperature and slightly elevated melting temperature. It has been argued that IBP binding must be irreversible because the ice crystals do not grow in the TH gap. However, this seems inconsistent with the observation that TH values are influenced by IBP concentration. We have made three recent experimental demonstrations that IBP binding to ice is indeed irreversible: (i) photo-bleaching of GFP-tagged IBP residing on the surface of an ice crystal held in the TH gap shows that there is neither exchange nor overgrowth of the bleached IBP; (ii) ice crystals bound by IBPs show a measurable resistance to melting (melting hysteresis) demonstrating that the IBPs remain surface-bound at temperatures above the equilibrium melting point; (iii) using a temperature controlled microfluidics apparatus it is possible to entirely replace the IBP solution surrounding an IBP-bound ice crystal in the TH gap with buffer, without causing the crystal to suddenly grow. Recent experiments also show that the exposure time of a crystal to an IBP solution at a given concentration can change the TH activity up to 10-fold. It appears that IBPs have a slow on-rate for crystal binding. According to the anchored clathrate water hypothesis for the mechanism of IBP binding to ice,
307
the ice-binding site of the IBP forms its ligand before merging with it. A slow on-rate might reflect the infrequency of having sufficient ice-like waters on the ice-binding site of the IBP for it to bind to ice. These results imply that IBP adsorption to the ice surface is irreversible and that TH is a function of the absorbed proteins on the surface and only indirectly a function of the concentration of IBPs in the solution. Conflicts of interest: None declared. Supported by the National Science Foundation (NSF) under Grant No. CHE0848081, the Israel Science Foundation (Grant Nos. 1279/10 and 1281/10), the Marie Curie International Reintegration Grant (Grant No. 256364), the Canadian Institutes for Health Research (CIHR), and the Condensed Matter and Surface Science program at Ohio University (CMSS). doi:10.1016/j.cryobiol.2011.09.009
Ice formation
7. Microfluidic experiments with ice binding proteins: Evidence for irreversible binding. Yeliz Celik * 1, Ran Drori 2, Natalya Pertaya 1, Aysun Altan 1, Maya Bar 2, Alex Groisman 3, Peter L. Davies 4, Ido Braslavsky 1,2, 1 Department of Physics and Astronomy, Ohio University, Athens, OH 45701, USA, 2 Institute of Biochemistry, Food Science and Nutrition, Faculty of Agricultural, Food and Environmental Quality Sciences, the Hebrew University of Jerusalem, Rehovot 76100, Israel, 3 Department of Physics, University of California San Diego, La Jolla, CA 92093, USA, 4 Department of Biochemistry, Queen’s University, Kingston, Ontario, Canada K7L 3N6 Ice binding proteins (IBPs) evolved in cold-adapted organisms, protecting them against freezing conditions by arresting ice crystal growth and inhibiting ice recrystallization. IBPs reduce the freezing temperature of ice below the melting point, a phenomenon defined as thermal hysteresis. The mechanism of action of IBPs is not well understood. In particular, it is not clear why thermal hysteresis activity depends on IBP concentration and whether the binding of IBP to ice is reversible or irreversible. Using fluorescently tagged IBP visualized with fluorescence microscopy in novel temperature controlled microfluidic devices; we found that hyperactive IBPs accumulate on the basal plane of ice. This finding supports the view that hyperactive IBPs adhere to the basal plane and this trait can be a factor contributing to their hyperactivity. Using the temperature-controlled microfluidic devices, we demonstrated that growth of small ice crystals (30–50 lm) covered with IBPs remain arrested for hours and the thermal hysteresis activity was maintained even after the IBP solution surrounding the crystal was replaced by plain buffer. In these experiments, ice crystals were incubated in IBP solution and then the solution was exchanged with a plain buffer. At this point, the reduction of the freezing point (thermal hysteresis activity) was measured. Repeated experiments indicate that thermal hysteresis activity is a function of IBP molecules that accumulated on the surface of the crystal at the start of the experiment and not a function of the concentration of the surrounding IBP molecules in solution after solution exchange with plain buffer. These experimental results provide strong evidence that the binding of IBPs to ice surfaces is irreversible. Conflicts of interest: None declared. Supported by the National Science Foundation (NSF) under Grant No. CHE0848081, the Israel Science Foundation (Grant Nos. 1279/10 and 1281/10), by Marie Curie International Reintegration Grant (Grant No. 256364), the Canadian Institutes for Health Research (CIHR), and the Condensed Matter and Surface Science program at Ohio University (CMSS). doi:10.1016/j.cryobiol.2011.09.010
8. Supercooling–promoting (anti-ice nucleating) flavonoids and tannins. Keita Endoh *, Seizo Fujikawa, Keita Arakawa, Research Faculty and Graduate School of Agriculture, Hokkaido University, Japan Freezing of water is controlled by the presence of ice nucleation substances such as ice nucleation bacterium (INB) which raises freezing temperatures, antifreeze substances such as antifreeze proteins which inhibit ice crystal growth after ice nucleation, and supercooling–promoting (anti-ice nucleation) substances which depress freezing temperatures. These freezing-control substances have potential in applications including low temperature preservation and cryopreservation of biological materials. With regard to these substances, however, studies on the supercooling– promoting substances are very few. As supercooling–promoting substances, only 17 kinds of compounds seem to have been reported so far. From our recent studies examining mechanisms of cold adaptation of xylem parenchyma cells (XPCs) in trees by deep supercooling, however, 4 kinds of novel supercooling–promoting flavonol glycosides and 4 kinds of novel supercooling–promoting hydrolysable tannins were identified. On the basis of the chemical structures of supercooling–promoting substances from XPCs, the present study shows a further 16 kinds of flavonoids and 9 kinds of tannins, although the effects are different depending on solutions. The results by emulsion freezing assay show that all flavonoids and tannins so far examined do not have ability