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181. Evaluation of glass transition and frozen water in plant tissues by temperature modulated differential scanning calorimetry
Abstracts / Cryobiology 53 (2006) 367–446 than in neotrehalose, (3) the free volume formed in the glassy matrix is relatively smaller in trehalose than in neotrehalose, and (4) in the glassy matrix, the glycosidic bond of trehalose takes a single conformation but that of neotrehalose has more than two conformations. These results suggested that the higher Tg and higher stability of trehalose glass is due to the increasing packing density of the glassy matrix, which originates from the conformational simplicity of this sugar, a characteristic feature of a,a-1,1linkage. (Source of funding: None declared. Conflict of interest: None declared.) doi:10.1016/j.cryobiol.2006.10.179
179. Spectrophotometric measurement of intra-liposomal trehalose. Jelena L. Holovati a,b, Jason P. Acker a,b, a Department of Laboratory Medicine and Pathology, University of Alberta, T6G 2R8 Edmonton, Canada; b Research and Development, Canadian Blood Services, T6G 2R8 Edmonton, Canada Trehalose, a non-reducing glucose disaccharide found at high concentrations in many species of anhydrobiotic organisms, shows significant promise in protecting cellular viability and structural integrity during freezing and desiccation. There has been extensive evidence that the maximum protection efficiency occurs when trehalose is present on both sides of cell membrane. Another important determinant of stabilization is the amount of trehalose that is accumulated intracellularly. As mammalian cell membranes are impermeable to trehalose, we are investigating the use of liposomes for the intracellular delivery of trehalose. Our previous study described methods for the synthesis and characterization of trehalose-containing liposomes. The purpose of this study is to measure the trehalose content of the liposomal aqueous core using the commercially available Megazyme spectrophotometric method, and therefore, to evaluate the encapsulation efficiency of the liposome synthesis process. Liposomes were synthesized by hydrating a phospholipid/cholesterol (70:30 mol%) lipid bilayer with 200 mM trehalose buffer and repeatedly extruding the lipid suspension to form unilamellar vesicles 400 nm in size. Non-encapsulated trehalose was removed with a series of washings, and trehalose-containing liposomes were resuspended in NaCl–HEPES buffer. Liposome preparations were then lysed with 1% (wt/vol) Triton X-100, and the trehalose concentration of the liposomal extract was measured either spectrophotometrically or using a normal-phase partition HPLC with an evaporative light scattering detector. The sensitivity (7 lM), linearity (7 lM–3 mM), specificity (no interference from L-glucose), and cost of the Megazyme spectrophotometric technique ($4.05/test) make it a great alternative to other trehalose measurement methods. The trehalose content of the 1 mM liposomal lysate was spectrophotometrically determined to be 364 ± 38 lM, and was confirmed with HPLC measurements (397 ± 53 lM, n = 3). The number of liposomes was calculated from the phosphate content of the liposomal preparation and an estimated number of lipid molecules in a 401 ± 18 nm liposome. Assuming a spherical liposomal shape and a lipid bilayer thickness of 5 nm, the volume of the liposomal aqueous core was calculated to be 0.03 fL, which translated to an intra-liposomal trehalose content of 183.9 ± 14.3 mM (n = 6). Based on these values, the liposomal encapsulation efficiency was of 92 ± 0.7%. Characterizing the entrapment of trehalose inside liposomes is essential for the controlled, reproducible and effective liposomal delivery of trehalose
443
into mammalian cells for applications in biopreservation. (Source of funding: The Bayer/Canadian Blood Services/Hema-Quebec Partnership Fund; Canadian Blood Services; Canada Foundation for Innovation; University of Alberta. Conflict of interest: None declared.) doi:10.1016/j.cryobiol.2006.10.180
180. Quantitative analysis of the effect of sugars on membrane phase transitions and interlamellar hydration forces. Thomas Lenne´ a, Gary Bryant a, Karen L. Koster b, Roland Holcomb b, a Applied Physics, School of Applied Sciences, RMIT University, 3001 Melbourne, Australia; b University of South Dakota, Department of Biology, 57069 Vermillion, SD, USA It is well known that sugars and other small solutes can reduce the temperature at which membranes undergo the fluid-gel phase transition at low hydration. The mechanisms for this are now well understood [Bryant et al. Abstract No. 85]. Naively, one might expect that this ability would be a direct function of sugar concentration, and that the effects should increase as the amount of sugar increases. However, the real situation is more complex. Previous work [K.L. Koster, Y.P. Lei, M. Anderson, S. Martin, G. Bryant, Biophys. J. 78 (2000) 1932–1946.] has shown that there are two distinct mechanisms for reduction in the transition temperature: first, if the sugar concentration is too low to form a glass, then the transition temperature can be reduced to (at best) the full hydration value; and second, if a glass forms, the transition temperature can be depressed to a fixed value, largely independent of sugar concentration. However, to the authors’ knowledge there has been no systematic study of the membrane transition temperature as a function of sugar/lipid ratio and level of hydration. In this paper we present the results of such a study. We show that in the absence of a glass, the reduction in the membrane phase transition temperature reaches a maximum value at a limiting sugar:lipid ratio. Beyond that value, the addition of further sugar no longer alters the membrane phase transition temperature. We explain these results in terms of hydration forces between membranes, and comment on the implications of these results for the prevention of damage to membranes during dehydration. (Source of funding: None declared. Conflict of interest: None declared.) doi:10.1016/j.cryobiol.2006.10.181
181. Evaluation of glass transition and frozen water in plant tissues by temperature modulated differential scanning calorimetry. Jiri Zamecnik a, Alois Bilavcik a, Milos Faltus a, Antonin Sikora b, a Molecular Biology, Research Institute of Crop Production, CZ 161 06 Prague, Czech Republic; b Institute of Macromolecular Chemistry CAS CR, CZ 162 06 Prague, Czech Republic The main problem arising during the cryopreservation of plant tissues is the determination and characterization of water status. Recent cryopreservation methods are mainly based on vitrification as a result of removing the excess of freezable water until a glass is formed. One of the most convenient methods for determination of the state of matter and transition characteristics is differential scanning calorimetry (DSC). The frozen water is
444
Abstracts / Cryobiology 53 (2006) 367–446
characterized by the DSC as an endothermic/exothermic peak contrary to glass transition, which is defined by the change in the heat capacity (DCp). The plant tissues usually used for cryopreservation often show overlapping of these thermal events. The heat flow changes during the glass transition are often smaller than during melting. This is the case with the conventional DSC method which is optimized for the best ratio of sensitivity and resolution at a cooling/heating rate of 10 C min1. In two biological models, twigs and seeds, it was not possible to separate the glass transition from the endothermic peak in many cases using this method. The second DSC method, temperature modulated DSC (TMDSC) is based on the superposition of a periodic modulation (amplitude and frequency) to a constant cooling/ heating rate. It is possible to separate the heat flow of the sample into reversing (thermodynamic) and non-reversing (kinetic) components by the TMDSC method. The amplitude, frequency, and heating rate used were in the range 0.1–1 C, 20–60 s, and 2– 5 C min1, respectively. Even if the Lissajous plot indicating quality of the DSC output was used, it was still not possible to clearly differentiate between the glass transition and the melting peak. The third method, quasi-isothermal temperature modulated DSC (QITMDSC), is based on the temperature modulation at constant modulating temperature until a constant value of the reverse Cp and later on the temperature change by a jump to a new constant modulating temperature. QITMDSC method gave us unambiguous results of melting characterized by a discontinuity of the reverse Cp. It had also a typical change in the reverse Cp characteristic for glass transition. The advantage of the QITMDSC is that the measured Cp is not influenced by cooling/heating rate. The data obtained previously by the other DSC methods can be clarified by the QITMDSC method. The amount of frozen water and the glass transition of chives seeds (Allium schoenoprasum) and apple twigs was evaluated by a combination of these three methods—DSC, TMDSC, and QITMDSC. The exact measurement of endothermic peaks is necessary to assess the dehydration level of plant samples to avoid ice crystal formation during freezing and thawing and to create the glassy state, which is important for long-term cryopreservation of plant samples. (Source of funding: Partially from the Grant Agency of the Czech Republic (522/04/0384) and The Ministry of Agriculture of the Czech Republic (0002700602). Conflict of interest: None declared.)
freezing front in the range 0.8 to 80 lm/s and the temperature gradient in the range from 12 to 30 C min1 were independently and systematically varied. Physiological saline was used as the solution. It was found that the patterns of interaction between the ice crystals and cells could be classified into two types: (1) the cells were swept from the freezing front causing them to accumulate in the unfrozen solution between the ice crystals, (2) the cells were trapped by the ice crystals. It was shown that the probability of being swept at the freezing front increased with increasing cooling rate. The trapped and swept phenomena for biological cells due to freezing was also analyzed by using the model proposed by Aoki et al. in 2003 for a solid particle and a flat freezing front, taking account of the electrical double layer force, van der Waals force, viscous force as the force acting on a cell. The critical velocity of the freezing front is defined as the maximum velocity where the cells were swept out from the freezing front. When the velocity of the freezing front becomes higher than this threshold, water cannot flow into the gap and the cell is trapped in the ice layer. The calculated critical velocity of the freezing front was close to the experimental results in the crystal-tip region. It was also clarified that the critical velocity mainly depends on the velocity of the freezing front. The model was next extended into the ‘mushy’ zone. In the solidification of multicomponent systems, the redistribution of solute causes constitutional supercooling in the liquid adjacent to the interface. This thermodynamically unstable state creates a mushy zone, in which cellular ice-crystals with finger-like shape are equally spaced and grow. Since the advancing velocity of ice-crystal tip is larger than that of side surface, the cells are more easily trapped in the tip region than the side surface. Therefore, we measured the probability of cells encountering the ice-crystal tip as defined by the tip radius and the primary arm spacing. Taking account of the probability of encounter and the probability of being trapped at the crystal tip, the probability of the trapping phenomenon in the mushy zone was calculated. The calculated results was quantitatively agree with the experimental results. The reason for the discrepancy is considered to be due to the approximation of the encounter probability using the tip radius, and the effect of concentrating the solution between the ice crystals. (Source of funding: None declared. Conflict of interest: None declared.) doi:10.1016/j.cryobiol.2006.10.183
doi:10.1016/j.cryobiol.2006.10.182
182. Trapped and swept phenomenon of biological cells during freezing. Yukio Tada a, Akira Takimoto a, Hajime Onishi a, Azusa Oomori b, a Division of Innovative Technology and Science, Graduate School of Natural Science and Technology, Kanazawa University, 920-1192 Kanazawa, Japan; b Canon Inc., 321-3298 Utsunomiya, Japan Mechanical interaction between cells and ice crystals during freezing causes mechanical damage to the cells. The cells swept out from the freezing front would also be damaged by solution effect injury. Therefore, the accurate prediction of the trapping and the sweeping phenomenon during the freezing of cells is an important research issue in fundamental cryobiology. In this study, the microscale behavior of ice crystals and yeast cells during directional solidification of cell suspensions has been experimentally and theoretically investigated. The velocity of the
183. Effect of instrument dynamic range on the estimation of osmotic properties using electronic cell sizing techniques. Adam Z. Higgins, Jens O.M. Karlsson, Institute for Bioengineering and Bioscience, Georgia Institute of Technology, 30332 Atlanta, GA, USA Electronic sizing (Coulter counter) methods are becoming increasingly popular for measurement of cellular osmotic properties. Although it is known that cells of a given type do not have a uniform size, conventional methods typically base analysis on curve fits to population means (or similar measures of central tendency), neglecting size variations. The present study compares such conventional approaches with new techniques that account for the distribution of cell sizes. The validity of conventional methods for determining the osmotically inactive volume fraction (Vb) was investigated by simulating the Boyle–vant Hoff experiment for a cell population comprising a distribution of cell sizes. The conventional method was found to be accurate when most of
179. Spectrophotometric measurement of intra-liposomal trehalose. Jelena L. Holovati a,b, Jason P. Acker a,b, a Department of Laboratory Medicine and Pathology, University of Alberta, T6G 2R8 Edmonton, Canada; b Research and Development, Canadian Blood Services, T6G 2R8 Edmonton, Canada Trehalose, a non-reducing glucose disaccharide found at high concentrations in many species of anhydrobiotic organisms, shows significant promise in protecting cellular viability and structural integrity during freezing and desiccation. There has been extensive evidence that the maximum protection efficiency occurs when trehalose is present on both sides of cell membrane. Another important determinant of stabilization is the amount of trehalose that is accumulated intracellularly. As mammalian cell membranes are impermeable to trehalose, we are investigating the use of liposomes for the intracellular delivery of trehalose. Our previous study described methods for the synthesis and characterization of trehalose-containing liposomes. The purpose of this study is to measure the trehalose content of the liposomal aqueous core using the commercially available Megazyme spectrophotometric method, and therefore, to evaluate the encapsulation efficiency of the liposome synthesis process. Liposomes were synthesized by hydrating a phospholipid/cholesterol (70:30 mol%) lipid bilayer with 200 mM trehalose buffer and repeatedly extruding the lipid suspension to form unilamellar vesicles 400 nm in size. Non-encapsulated trehalose was removed with a series of washings, and trehalose-containing liposomes were resuspended in NaCl–HEPES buffer. Liposome preparations were then lysed with 1% (wt/vol) Triton X-100, and the trehalose concentration of the liposomal extract was measured either spectrophotometrically or using a normal-phase partition HPLC with an evaporative light scattering detector. The sensitivity (7 lM), linearity (7 lM–3 mM), specificity (no interference from L-glucose), and cost of the Megazyme spectrophotometric technique ($4.05/test) make it a great alternative to other trehalose measurement methods. The trehalose content of the 1 mM liposomal lysate was spectrophotometrically determined to be 364 ± 38 lM, and was confirmed with HPLC measurements (397 ± 53 lM, n = 3). The number of liposomes was calculated from the phosphate content of the liposomal preparation and an estimated number of lipid molecules in a 401 ± 18 nm liposome. Assuming a spherical liposomal shape and a lipid bilayer thickness of 5 nm, the volume of the liposomal aqueous core was calculated to be 0.03 fL, which translated to an intra-liposomal trehalose content of 183.9 ± 14.3 mM (n = 6). Based on these values, the liposomal encapsulation efficiency was of 92 ± 0.7%. Characterizing the entrapment of trehalose inside liposomes is essential for the controlled, reproducible and effective liposomal delivery of trehalose
443
into mammalian cells for applications in biopreservation. (Source of funding: The Bayer/Canadian Blood Services/Hema-Quebec Partnership Fund; Canadian Blood Services; Canada Foundation for Innovation; University of Alberta. Conflict of interest: None declared.) doi:10.1016/j.cryobiol.2006.10.180
180. Quantitative analysis of the effect of sugars on membrane phase transitions and interlamellar hydration forces. Thomas Lenne´ a, Gary Bryant a, Karen L. Koster b, Roland Holcomb b, a Applied Physics, School of Applied Sciences, RMIT University, 3001 Melbourne, Australia; b University of South Dakota, Department of Biology, 57069 Vermillion, SD, USA It is well known that sugars and other small solutes can reduce the temperature at which membranes undergo the fluid-gel phase transition at low hydration. The mechanisms for this are now well understood [Bryant et al. Abstract No. 85]. Naively, one might expect that this ability would be a direct function of sugar concentration, and that the effects should increase as the amount of sugar increases. However, the real situation is more complex. Previous work [K.L. Koster, Y.P. Lei, M. Anderson, S. Martin, G. Bryant, Biophys. J. 78 (2000) 1932–1946.] has shown that there are two distinct mechanisms for reduction in the transition temperature: first, if the sugar concentration is too low to form a glass, then the transition temperature can be reduced to (at best) the full hydration value; and second, if a glass forms, the transition temperature can be depressed to a fixed value, largely independent of sugar concentration. However, to the authors’ knowledge there has been no systematic study of the membrane transition temperature as a function of sugar/lipid ratio and level of hydration. In this paper we present the results of such a study. We show that in the absence of a glass, the reduction in the membrane phase transition temperature reaches a maximum value at a limiting sugar:lipid ratio. Beyond that value, the addition of further sugar no longer alters the membrane phase transition temperature. We explain these results in terms of hydration forces between membranes, and comment on the implications of these results for the prevention of damage to membranes during dehydration. (Source of funding: None declared. Conflict of interest: None declared.) doi:10.1016/j.cryobiol.2006.10.181
181. Evaluation of glass transition and frozen water in plant tissues by temperature modulated differential scanning calorimetry. Jiri Zamecnik a, Alois Bilavcik a, Milos Faltus a, Antonin Sikora b, a Molecular Biology, Research Institute of Crop Production, CZ 161 06 Prague, Czech Republic; b Institute of Macromolecular Chemistry CAS CR, CZ 162 06 Prague, Czech Republic The main problem arising during the cryopreservation of plant tissues is the determination and characterization of water status. Recent cryopreservation methods are mainly based on vitrification as a result of removing the excess of freezable water until a glass is formed. One of the most convenient methods for determination of the state of matter and transition characteristics is differential scanning calorimetry (DSC). The frozen water is
444
Abstracts / Cryobiology 53 (2006) 367–446
characterized by the DSC as an endothermic/exothermic peak contrary to glass transition, which is defined by the change in the heat capacity (DCp). The plant tissues usually used for cryopreservation often show overlapping of these thermal events. The heat flow changes during the glass transition are often smaller than during melting. This is the case with the conventional DSC method which is optimized for the best ratio of sensitivity and resolution at a cooling/heating rate of 10 C min1. In two biological models, twigs and seeds, it was not possible to separate the glass transition from the endothermic peak in many cases using this method. The second DSC method, temperature modulated DSC (TMDSC) is based on the superposition of a periodic modulation (amplitude and frequency) to a constant cooling/ heating rate. It is possible to separate the heat flow of the sample into reversing (thermodynamic) and non-reversing (kinetic) components by the TMDSC method. The amplitude, frequency, and heating rate used were in the range 0.1–1 C, 20–60 s, and 2– 5 C min1, respectively. Even if the Lissajous plot indicating quality of the DSC output was used, it was still not possible to clearly differentiate between the glass transition and the melting peak. The third method, quasi-isothermal temperature modulated DSC (QITMDSC), is based on the temperature modulation at constant modulating temperature until a constant value of the reverse Cp and later on the temperature change by a jump to a new constant modulating temperature. QITMDSC method gave us unambiguous results of melting characterized by a discontinuity of the reverse Cp. It had also a typical change in the reverse Cp characteristic for glass transition. The advantage of the QITMDSC is that the measured Cp is not influenced by cooling/heating rate. The data obtained previously by the other DSC methods can be clarified by the QITMDSC method. The amount of frozen water and the glass transition of chives seeds (Allium schoenoprasum) and apple twigs was evaluated by a combination of these three methods—DSC, TMDSC, and QITMDSC. The exact measurement of endothermic peaks is necessary to assess the dehydration level of plant samples to avoid ice crystal formation during freezing and thawing and to create the glassy state, which is important for long-term cryopreservation of plant samples. (Source of funding: Partially from the Grant Agency of the Czech Republic (522/04/0384) and The Ministry of Agriculture of the Czech Republic (0002700602). Conflict of interest: None declared.)
freezing front in the range 0.8 to 80 lm/s and the temperature gradient in the range from 12 to 30 C min1 were independently and systematically varied. Physiological saline was used as the solution. It was found that the patterns of interaction between the ice crystals and cells could be classified into two types: (1) the cells were swept from the freezing front causing them to accumulate in the unfrozen solution between the ice crystals, (2) the cells were trapped by the ice crystals. It was shown that the probability of being swept at the freezing front increased with increasing cooling rate. The trapped and swept phenomena for biological cells due to freezing was also analyzed by using the model proposed by Aoki et al. in 2003 for a solid particle and a flat freezing front, taking account of the electrical double layer force, van der Waals force, viscous force as the force acting on a cell. The critical velocity of the freezing front is defined as the maximum velocity where the cells were swept out from the freezing front. When the velocity of the freezing front becomes higher than this threshold, water cannot flow into the gap and the cell is trapped in the ice layer. The calculated critical velocity of the freezing front was close to the experimental results in the crystal-tip region. It was also clarified that the critical velocity mainly depends on the velocity of the freezing front. The model was next extended into the ‘mushy’ zone. In the solidification of multicomponent systems, the redistribution of solute causes constitutional supercooling in the liquid adjacent to the interface. This thermodynamically unstable state creates a mushy zone, in which cellular ice-crystals with finger-like shape are equally spaced and grow. Since the advancing velocity of ice-crystal tip is larger than that of side surface, the cells are more easily trapped in the tip region than the side surface. Therefore, we measured the probability of cells encountering the ice-crystal tip as defined by the tip radius and the primary arm spacing. Taking account of the probability of encounter and the probability of being trapped at the crystal tip, the probability of the trapping phenomenon in the mushy zone was calculated. The calculated results was quantitatively agree with the experimental results. The reason for the discrepancy is considered to be due to the approximation of the encounter probability using the tip radius, and the effect of concentrating the solution between the ice crystals. (Source of funding: None declared. Conflict of interest: None declared.) doi:10.1016/j.cryobiol.2006.10.183
doi:10.1016/j.cryobiol.2006.10.182
182. Trapped and swept phenomenon of biological cells during freezing. Yukio Tada a, Akira Takimoto a, Hajime Onishi a, Azusa Oomori b, a Division of Innovative Technology and Science, Graduate School of Natural Science and Technology, Kanazawa University, 920-1192 Kanazawa, Japan; b Canon Inc., 321-3298 Utsunomiya, Japan Mechanical interaction between cells and ice crystals during freezing causes mechanical damage to the cells. The cells swept out from the freezing front would also be damaged by solution effect injury. Therefore, the accurate prediction of the trapping and the sweeping phenomenon during the freezing of cells is an important research issue in fundamental cryobiology. In this study, the microscale behavior of ice crystals and yeast cells during directional solidification of cell suspensions has been experimentally and theoretically investigated. The velocity of the
183. Effect of instrument dynamic range on the estimation of osmotic properties using electronic cell sizing techniques. Adam Z. Higgins, Jens O.M. Karlsson, Institute for Bioengineering and Bioscience, Georgia Institute of Technology, 30332 Atlanta, GA, USA Electronic sizing (Coulter counter) methods are becoming increasingly popular for measurement of cellular osmotic properties. Although it is known that cells of a given type do not have a uniform size, conventional methods typically base analysis on curve fits to population means (or similar measures of central tendency), neglecting size variations. The present study compares such conventional approaches with new techniques that account for the distribution of cell sizes. The validity of conventional methods for determining the osmotically inactive volume fraction (Vb) was investigated by simulating the Boyle–vant Hoff experiment for a cell population comprising a distribution of cell sizes. The conventional method was found to be accurate when most of