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concentrations on vitrification of mouse germinal vesicle (GV)—and metaphase II (MII)—oocytes
Abstracts / Cryobiology 59 (2009) 370–418 to freeze, but it does increase the average time between that IIF and IIF in an adjoining blastomere from 0.6 s to 3.9 s. (Conflicts of interest: None. Source of funding: NIH Grant R01 RR018470.) doi:10.1016/j.cryobiol.2009.10.108
95. Effect of the expression of aquaporins 1 and 3 in mouse MII oocytes on the nucleation temperature for intracellular ice formation. *Shinsuke Seki a, Keisuke Edashige b, Peter Mazur a, a Fundamental and Applied Cryobiology Group, Department of Biochemistry and Cellular and Molecular Biology, The University of Tennessee, Knoxville, TN 37996, USA, b Laboratory of Animal Science College of Agriculture, Kochi University, Kochi, Japan When cells are cooled too rapidly to keep the chemical potential of their intracellular water in chemical potential equilibrium with external water and ice by osmotic dehydration, they become increasingly supercooled; and at some subzero temperature, they undergo intracellular ice formation (IIF). In some cases, IIF occurs at or very near the minimum temperature at which the supercooled state can be maintained, the so-called temperature for homogeneous nucleation (Th). In cells the size of mouse oocytes that temperature is 40 °C. But in many other cases, IIF occurs at temperatures well above Th and is said to occur by heterogeneous nucleation. There is increasing circumstantial evidence that the heterogeneous nucleator is external ice itself. What is not clear is the mechanism or route by which an external ice crystal can traverse the plasma membrane and cause the nucleation of the supercooled solution within the cell. When we began this series of studies on IIF in mouse oocytes some 5 years ago, we hypothesized that one route by which an external ice crystal might traverse the lipid bilayer membrane is through pores in preexisting transmembrane proteins. One family of structures that are constitutively present in the membranes of many cell types and contain central pores are the aquaporins. In one way, their pores are not good candidates for our hypothetical pores, for there is a constriction in the pore that will not allow the passage of more than one molecule of water at a time, much less allow the passage of the assembly of ice molecules that constitute even a tiny ice crystal. But we considered it possible that the force generated by ice crystal growth might be sufficient to enlarge the pore. The plasma membrane of mature MII mouse oocytes does not express aquaporins, but Edashige et al. showed that it could be induced to express aquaporin 3 by the injection of the appropriate cRNA [Biol. Reprod. 77 (2007) 365]. We decided, therefore, to compare the ice nucleation temperature of native oocytes with that of oocytes expressing AQP 3 and 1. The oocytes were suspended in 1.0 M ethylene glycol in PBS for 15 minutes, cooled in a Linkam cryostage to 7.0 °C, induced to freeze externally, and finally cooled at 20 °C/min to 70 °C. IIF occurred during the 20 °C/min cooling as manifested by abrupt black flashing. The temperature of that flashing was recorded. The mean IIF temperatures for native oocytes, for oocytes sham injected with water, for oocytes expressing AQP 1, and for those expressing AQP-3 were 35 °C, 40 °C, 35 °C, and 27 °C, respectively. The fact that the ice nucleation temperature of oocytes expressing AQP-3 was higher than the others is consistent with our hypothesis. However, it does not prove it because the apparent higher temperature may have been due to other, indirect consequences of the expression of the aquaporin entity in the membrane. (Conflicts of interest: None declared. Source of funding: Grant R01 RR018470.) doi:10.1016/j.cryobiol.2009.10.109
96. Effects of using slush nitrogen (SN2) and various cryoprotectants (CPA) compositions/ concentrations on vitrification of mouse germinal vesicle (GV)—and metaphase II (MII)— oocytes. S.K. Cha, B.Y. Kim, Y.Y. Lee, T.H. Kim, D.R. Lee, M.K. Kim, H.J. Won, J.E. Han, W.S. Lee, T.K. Yoon, Fertility Center of CHA General Hospital, CHA Research Institute, CHA University College of Medicine, Seoul 135-081, Republic of Korea Recent studies suggest that vitrification may be more effective than slow freezing, resulting in improved oocyte survival and pregnancy rates. However, a major concern of vitrification relates to the use of higher concentrations of membrane-permeable cryoprotectants (CPA) in the equilibrating solution. In order to optimize an oocyte vitrification method, we have recently introduced new methodology, super-rapid cooling by using slush nitrogen (SN2). Boiling of liquid nitrogen (LN2) occurs when a sample is immersed and results in gas bubbles around the specimen, which, in turn, result in poor heat transfer. By applying negative pressure with a vacuum, LN2 will freeze and converts into a slush state. SN2 has a lower internal temperature of 210 °C without vaporization. Since it may offer high-speed cooling rates, 135,000 °C/min, it may be possible to increase the survival rate as well as other characteristics. The objectives of this study were to improve the efficacy of mouse GV and MII oocytes after vitrifying and warming by applying various compositions and concentrations of CPA and/or super-rapid cooling using SN2. Female mice (3–4 weeks old ICR) were injected with PMSG for GV stage oocytes and injected hCG after 48 h for
397
MII stage oocytes. Experimental groups were divided into 4 groups. Group 1: preequilibrated with 1.5 M ethylene glycol (EG) for 2.5 min and then equilibrated with 5.5 M EG and 1.0 M sucrose for 20 s. Group 2–4: pre-equilibrated with 7.5% EG + 7.5 dimethylsulfoxide (Me2 SO) for 2.5 min. Then, Group 2 (4.78 M): equilibrated 15% EG + 15%Me2SO + 1M sucrose for 20 s. Group 3 (3.19 M): equilibrated with 10%EG + 10%Me2SO + 1 M sucrose for 20 s. Group 4 (2.39 M): equilibrated with 7.5% EG + 7.5%Me2SO + 1 M sucrose for 20 s. The oocytes were loaded onto electron microscopic grids and plunged into SN2 or LN2 and then stored in LN2. Stored oocytes were warmed by a five-step method, and then their survival, maturation, fertilization and development rates were observed. Survival, maturation, fertilization and development rates after thawing of GV oocytes vitrified using SN2 showed no significant difference in all concentrations compared with those of oocytes using conventional LN2. However, survival and blastocyst rates after thawing of MII oocytes vitrified using SN2 were increased in group 2 (79.0%, 33.9% vs. 60.0%, 16.3%) and 3 (30.0%, 12.5% vs. 5.0%, 0%) compared with those of oocytes using conventional LN2. The higher Me2SO and EG concentration group showed an increased developmental rate than the group with EG only. However, there was a decrease of survival rate as the CPA concentration decreased. We conclude that SN2 may improve the efficiency by reducing cryoinjury and high-speed cooling rate during vitrification. Combinations of CPA could be used as an alternative to reduce the relative concentration of CPA and improve survival and further development rates. The difference of survival and fertilization rates by cooling speed differs according to oocyte stages. Therefore, the further study is required to know the precise mechanism of these differences in oocyte cryopreservation. (Conflicts of interest: None declared. Source of funding: A grant of the Korea Healthcare Technology R& D Project, Ministry of Health, Welfare & Family affairs, Republic of Korea (A084923).) doi:10.1016/j.cryobiol.2009.10.110
97. Biomolecules for the improvement of cryopreservation of human endothelial cells. H. Sun, N. Hofmann, *B. Glasmacher, Institute of Multiphase Processes/Leibniz Universitaet Hannover, Hannover, Germany Cryopreservation plays an important role in the long-term storage of cells and tissues and a high survival rate of cryopreserved cells requires an optimal cooling rate and the presence of a cryoprotective agent (CPA) in a sufficiently high concentration. The most widely used CPAs, dimethyl sulfoxide (Me2SO) and glycerol, however are toxic at high concentrations and have detrimental effects on the biological functioning of a cell. Therefore, it is of great interest to develop new cryoprotective strategies to replace the currently used CPAs or to reduce their concentration. For this purpose we investigated the amino acid proline and the heat shock proteins (HSP) as potential CPAs, since these biomolecules are natural protectants in cells that undergo stress. Human pulmonary microvascular endothelial cells (HPMEC) were used in the freezing experiments. Cells were frozen in 1.8 ml NalgeneÒ cryovials (1.5 ml, 4.5 105 cells/ml) using the optimal cooling rate gained from a previous study. For the investigation of proline, cells were frozen either directly with freezing medium containing proline with a 10 min equilibration period (method A) or after incubation for 48 h in a praline-containing culture medium (method B). The concentrations of proline were 5, 10, 20, 50 and 100 mM both in the freezing medium and in the culture medium, the concentrations of Me2SO in freezing medium were 0, 1, 2.5, 7.5 and 10% (v/v). To study the HSPs, cells were exposed to hyper- (41 °C) or hypothermal (4 °C) stress for 30 min followed by 4 h or 10 h cultivation for cell recovery and HSPs expression. The survival rate of the cells after thawing was determined after 24 h recultivation. Cells that were attached to the surface of the culture flask were considered as viable cells, and the survival rate is the percentage of viable cells to the total cell number in recultivation. With methods A & B a cell survival rate of 90% was achieved with 20 mM proline and 2.5% Me2SO, while without proline cell survival rate reaches up to 90% with 10% Me2SO. Using method A, a reduction to 5 mM as well as an increase to 100 mM did not give better results, while by method B, 5 mM proline and 2.5% Me2 SO also gave a 90% cell survival rate, and a cell survival rate of 80% could be achieved even with 1% Me2SO and 20 mM proline. First experiments with cells activated by thermal stresses showed only a small improvement in cell survival rates without Me2SO but sufficient cell survival rates were also achieved with 2.5% Me2SO. Our results show that proline can be used as an additional CPA to reduce the Me2SO concentration if a short equilibration time before freezing is applied and a further reduction of Me2 SO is possible with praline-containing culture medium. HSPs exhibit a higher influence in respond to stronger stresses. If high Me2SO concentrations are necessary, HSPs could be used to increase the survival rates. (Conflicts of interest: None declared. Source of funding: The Cluster of Excellence ‘‘REBIRTH” (DFG).) doi:10.1016/j.cryobiol.2009.10.111
95. Effect of the expression of aquaporins 1 and 3 in mouse MII oocytes on the nucleation temperature for intracellular ice formation. *Shinsuke Seki a, Keisuke Edashige b, Peter Mazur a, a Fundamental and Applied Cryobiology Group, Department of Biochemistry and Cellular and Molecular Biology, The University of Tennessee, Knoxville, TN 37996, USA, b Laboratory of Animal Science College of Agriculture, Kochi University, Kochi, Japan When cells are cooled too rapidly to keep the chemical potential of their intracellular water in chemical potential equilibrium with external water and ice by osmotic dehydration, they become increasingly supercooled; and at some subzero temperature, they undergo intracellular ice formation (IIF). In some cases, IIF occurs at or very near the minimum temperature at which the supercooled state can be maintained, the so-called temperature for homogeneous nucleation (Th). In cells the size of mouse oocytes that temperature is 40 °C. But in many other cases, IIF occurs at temperatures well above Th and is said to occur by heterogeneous nucleation. There is increasing circumstantial evidence that the heterogeneous nucleator is external ice itself. What is not clear is the mechanism or route by which an external ice crystal can traverse the plasma membrane and cause the nucleation of the supercooled solution within the cell. When we began this series of studies on IIF in mouse oocytes some 5 years ago, we hypothesized that one route by which an external ice crystal might traverse the lipid bilayer membrane is through pores in preexisting transmembrane proteins. One family of structures that are constitutively present in the membranes of many cell types and contain central pores are the aquaporins. In one way, their pores are not good candidates for our hypothetical pores, for there is a constriction in the pore that will not allow the passage of more than one molecule of water at a time, much less allow the passage of the assembly of ice molecules that constitute even a tiny ice crystal. But we considered it possible that the force generated by ice crystal growth might be sufficient to enlarge the pore. The plasma membrane of mature MII mouse oocytes does not express aquaporins, but Edashige et al. showed that it could be induced to express aquaporin 3 by the injection of the appropriate cRNA [Biol. Reprod. 77 (2007) 365]. We decided, therefore, to compare the ice nucleation temperature of native oocytes with that of oocytes expressing AQP 3 and 1. The oocytes were suspended in 1.0 M ethylene glycol in PBS for 15 minutes, cooled in a Linkam cryostage to 7.0 °C, induced to freeze externally, and finally cooled at 20 °C/min to 70 °C. IIF occurred during the 20 °C/min cooling as manifested by abrupt black flashing. The temperature of that flashing was recorded. The mean IIF temperatures for native oocytes, for oocytes sham injected with water, for oocytes expressing AQP 1, and for those expressing AQP-3 were 35 °C, 40 °C, 35 °C, and 27 °C, respectively. The fact that the ice nucleation temperature of oocytes expressing AQP-3 was higher than the others is consistent with our hypothesis. However, it does not prove it because the apparent higher temperature may have been due to other, indirect consequences of the expression of the aquaporin entity in the membrane. (Conflicts of interest: None declared. Source of funding: Grant R01 RR018470.) doi:10.1016/j.cryobiol.2009.10.109
96. Effects of using slush nitrogen (SN2) and various cryoprotectants (CPA) compositions/ concentrations on vitrification of mouse germinal vesicle (GV)—and metaphase II (MII)— oocytes. S.K. Cha, B.Y. Kim, Y.Y. Lee, T.H. Kim, D.R. Lee, M.K. Kim, H.J. Won, J.E. Han, W.S. Lee, T.K. Yoon, Fertility Center of CHA General Hospital, CHA Research Institute, CHA University College of Medicine, Seoul 135-081, Republic of Korea Recent studies suggest that vitrification may be more effective than slow freezing, resulting in improved oocyte survival and pregnancy rates. However, a major concern of vitrification relates to the use of higher concentrations of membrane-permeable cryoprotectants (CPA) in the equilibrating solution. In order to optimize an oocyte vitrification method, we have recently introduced new methodology, super-rapid cooling by using slush nitrogen (SN2). Boiling of liquid nitrogen (LN2) occurs when a sample is immersed and results in gas bubbles around the specimen, which, in turn, result in poor heat transfer. By applying negative pressure with a vacuum, LN2 will freeze and converts into a slush state. SN2 has a lower internal temperature of 210 °C without vaporization. Since it may offer high-speed cooling rates, 135,000 °C/min, it may be possible to increase the survival rate as well as other characteristics. The objectives of this study were to improve the efficacy of mouse GV and MII oocytes after vitrifying and warming by applying various compositions and concentrations of CPA and/or super-rapid cooling using SN2. Female mice (3–4 weeks old ICR) were injected with PMSG for GV stage oocytes and injected hCG after 48 h for
397
MII stage oocytes. Experimental groups were divided into 4 groups. Group 1: preequilibrated with 1.5 M ethylene glycol (EG) for 2.5 min and then equilibrated with 5.5 M EG and 1.0 M sucrose for 20 s. Group 2–4: pre-equilibrated with 7.5% EG + 7.5 dimethylsulfoxide (Me2 SO) for 2.5 min. Then, Group 2 (4.78 M): equilibrated 15% EG + 15%Me2SO + 1M sucrose for 20 s. Group 3 (3.19 M): equilibrated with 10%EG + 10%Me2SO + 1 M sucrose for 20 s. Group 4 (2.39 M): equilibrated with 7.5% EG + 7.5%Me2SO + 1 M sucrose for 20 s. The oocytes were loaded onto electron microscopic grids and plunged into SN2 or LN2 and then stored in LN2. Stored oocytes were warmed by a five-step method, and then their survival, maturation, fertilization and development rates were observed. Survival, maturation, fertilization and development rates after thawing of GV oocytes vitrified using SN2 showed no significant difference in all concentrations compared with those of oocytes using conventional LN2. However, survival and blastocyst rates after thawing of MII oocytes vitrified using SN2 were increased in group 2 (79.0%, 33.9% vs. 60.0%, 16.3%) and 3 (30.0%, 12.5% vs. 5.0%, 0%) compared with those of oocytes using conventional LN2. The higher Me2SO and EG concentration group showed an increased developmental rate than the group with EG only. However, there was a decrease of survival rate as the CPA concentration decreased. We conclude that SN2 may improve the efficiency by reducing cryoinjury and high-speed cooling rate during vitrification. Combinations of CPA could be used as an alternative to reduce the relative concentration of CPA and improve survival and further development rates. The difference of survival and fertilization rates by cooling speed differs according to oocyte stages. Therefore, the further study is required to know the precise mechanism of these differences in oocyte cryopreservation. (Conflicts of interest: None declared. Source of funding: A grant of the Korea Healthcare Technology R& D Project, Ministry of Health, Welfare & Family affairs, Republic of Korea (A084923).) doi:10.1016/j.cryobiol.2009.10.110
97. Biomolecules for the improvement of cryopreservation of human endothelial cells. H. Sun, N. Hofmann, *B. Glasmacher, Institute of Multiphase Processes/Leibniz Universitaet Hannover, Hannover, Germany Cryopreservation plays an important role in the long-term storage of cells and tissues and a high survival rate of cryopreserved cells requires an optimal cooling rate and the presence of a cryoprotective agent (CPA) in a sufficiently high concentration. The most widely used CPAs, dimethyl sulfoxide (Me2SO) and glycerol, however are toxic at high concentrations and have detrimental effects on the biological functioning of a cell. Therefore, it is of great interest to develop new cryoprotective strategies to replace the currently used CPAs or to reduce their concentration. For this purpose we investigated the amino acid proline and the heat shock proteins (HSP) as potential CPAs, since these biomolecules are natural protectants in cells that undergo stress. Human pulmonary microvascular endothelial cells (HPMEC) were used in the freezing experiments. Cells were frozen in 1.8 ml NalgeneÒ cryovials (1.5 ml, 4.5 105 cells/ml) using the optimal cooling rate gained from a previous study. For the investigation of proline, cells were frozen either directly with freezing medium containing proline with a 10 min equilibration period (method A) or after incubation for 48 h in a praline-containing culture medium (method B). The concentrations of proline were 5, 10, 20, 50 and 100 mM both in the freezing medium and in the culture medium, the concentrations of Me2SO in freezing medium were 0, 1, 2.5, 7.5 and 10% (v/v). To study the HSPs, cells were exposed to hyper- (41 °C) or hypothermal (4 °C) stress for 30 min followed by 4 h or 10 h cultivation for cell recovery and HSPs expression. The survival rate of the cells after thawing was determined after 24 h recultivation. Cells that were attached to the surface of the culture flask were considered as viable cells, and the survival rate is the percentage of viable cells to the total cell number in recultivation. With methods A & B a cell survival rate of 90% was achieved with 20 mM proline and 2.5% Me2SO, while without proline cell survival rate reaches up to 90% with 10% Me2SO. Using method A, a reduction to 5 mM as well as an increase to 100 mM did not give better results, while by method B, 5 mM proline and 2.5% Me2 SO also gave a 90% cell survival rate, and a cell survival rate of 80% could be achieved even with 1% Me2SO and 20 mM proline. First experiments with cells activated by thermal stresses showed only a small improvement in cell survival rates without Me2SO but sufficient cell survival rates were also achieved with 2.5% Me2SO. Our results show that proline can be used as an additional CPA to reduce the Me2SO concentration if a short equilibration time before freezing is applied and a further reduction of Me2 SO is possible with praline-containing culture medium. HSPs exhibit a higher influence in respond to stronger stresses. If high Me2SO concentrations are necessary, HSPs could be used to increase the survival rates. (Conflicts of interest: None declared. Source of funding: The Cluster of Excellence ‘‘REBIRTH” (DFG).) doi:10.1016/j.cryobiol.2009.10.111