- Email: [email protected]
ethylene glycol revisited
336
Abstracts / Cryobiology 57 (2008) 315–340
100. The effect of salt on macromolecule solutions: Implications for modeling. Richelle C. Prickett, Janet A.W. Elliott, Locksley E. McGann, University of Alberta, Edmonton, AB, Canada Macromolecules and salts are ubiquitous in biology in both intra- and extracellular solutions. As both of these solutes exhibit non-ideal behaviour at very low concentrations, they have a marked effect on the solution thermodynamics, especially when the solutes are concentrated during freezing by the removal of water as ice. We have previously shown that salts alone or salts in combination with certain small molecules have osmotic behaviour that is well predicted by our osmotic virial equation. However, it has been shown that the solution behaviour of macromolecules is altered when the salt concentration is varied, but equations have not been developed to predict the osmolality of these solutions accurately. The objective of this study was to determine the osmotic virial coefficients for macromolecules in various salt solutions and investigate the changes in these coefficients as a function of the salt concentration. The phase diagrams for hydroxyethyl starch (HES) in varying concentrations of NaCl were obtained from the literature for mass ratios (R values) ranging from 0.5 to 20. Our multisolute osmotic virial equation was fit to each HES + NaCl phase diagram, using the osmotic virial coefficients for NaCl that were previously determined, to obtain the osmotic virial coefficients for HES at each R value. The osmotic virial coefficients were plotted as a function of R value and analyzed in order to obtain equations to determine the osmotic virial coefficient dependence on salt concentration. The effect of salt on macromolecules should be taken into account when modeling these solutions. The osmotic virial coefficients show a strong dependence on the R value, which may lead to the development of equations to predict osmotic virial coefficients as a function of the salt concentration. (Conflicts of interest: None declared. Source of funding: R.C. Prickett, received support from NSERC, the Alberta Ingenuity Fund and CIHR; Janet A.W. Elliott holds the Canada Research Chair in Interfacial Thermodynamics.) Note: This presentation was the winner of the Society’s Poster Award.
ments using a new differential scanning calorimeter (DSC) and analyzed the resulting data. For the new study, melting points for five R values (R = 5, 10, 15, 30, and 45) each measured at five levels of concentration within each R were determined. These melting points were averaged from 3 measurements and plotted as a function of total solute concentration for each R value studied. The new measurements differed from our original values, however they did agree with predicted values using both theoretical models. The data were also fit simultaneously utilizing a method similar to our previous report and the resulting equation was obtained: T m ¼ 0:565x þ ð3:749 103 Þx2 ð1:708 104 Þx3 þ ð2:104 103 ÞxR ð3:007 105 Þx2 R ð1:934 108 Þx3 R: Where x = the total solute concentration. This new equation provided good fits to the experimental data as well as published values and relates the determined polynomial constants to the R value of the corresponding isopleths of the three dimensional phase diagram, allowing the liquidus curve for any R value to be obtained. (Conflicts of interest: E.J. Woods is a General Biotechnology, LLC Shareholder. Source of funding: None declared.)
doi:10.1016/j.cryobiol.2008.10.103
Poster presentations 2. Mammalian systems 103. Towards cryopreservation of peromyscus embryos. Amanda R. Duselis a, Paul B. Vrana a, Monika Veres b, Xiaoming He c, Gabor Szalai b, a University of California at Irvine, Department of Biological Chemistry, Irvine, CA, b University of South Carolina, Peromyscus Genetic Stock Center, Columbia, SC, c University of South Carolina, Department of Mechanical Engineering and Biomedical Engineering Program, Columbia, SC
doi:10.1016/j.cryobiol.2008.10.101
101. Optimizing desiccation buffers utilizing disaccharides. Justin Reis, Ranjan Sitaula, Sankha Bhowmick, University of Masachusetts Dartmouth, North Dartmouth, MA, USA Sugars, particularly disaccharides, have proven to be attractive excipients for desiccation preservation of biologics. Our research aimed towards replacement of water in mammalian cells with sugars to sustain life. The goal is to optimize the in vitro environment, a ternary mix of buffer salts, stabilizing sugar, and water. The buffers we have chosen are TRIS and citrate due to their usage as bovine sperm extenders. Trehalose and sucrose where chosen for their glass transitions as well as proven effectiveness in previous desiccation studies. Glass transition temperature (Tg) is a key thermophysical parameter governing stability of mammalian cells in the desiccated state. Sugar/buffer concentrations were equilibrated to different end moisture contents and Tg was determined using Differential Scanning Calorimetry (DSC). Solutions void of moisture showed that at low concentrations of the sugar Tg’s assimilated themselves with the Tg of the buffer while yielding a Tg slightly below that of the sugar at lower buffer concentrations. Once moisture was added to the sugar/salt solution we saw the expected decrease of glass transition as moisture content increased. Modeling experimental Tg’s was accomplished through the usage of the Gordon Taylor equation for samples with and without moisture and the Pochan-Beatty-Hinman equation for samples void of moisture. These models are able to predict Tg both for intermediary solutions as well as extrapolate Tg for larger residual moisture content sample. (Conflicts of interest: None declared. Source of funding: None declared.) doi:10.1016/j.cryobiol.2008.10.102
102. Melting point equations for the ternary system water/sodium chloride/ethylene glycol revisited. Erik J. Woods a,b, Aniruddha Bagchi a, James D. Benson c, Xu Han c, John K. Critser a,c, a General BioTechnology, LLC, Indianapolis, IN, USA, b Indiana University School of Medicine, Indianapolis, IN, USA, c University of Missouri, Columbia, MO, USA Previously, our group measured melting points (TM) of four different ratios (R) of ethylene glycol to NaCl and developed an equation to fit the experimental measurements [Woods et al., 1999]. This equation was meant to characterize the liquidus surface above the eutectic for any value of R and thus facilitate modeling of cryopreservation using the ternary system. More recently, however, theoretical methods to predict ternary solution properties have been developed which seem to provide consistent and accurate predictions for several different ternary aqueous solutions [Kleinhans and Mazur, 2007; Elliott et al., 2007]. Comparison of our previous report with these new models indicated a discrepancy and a potential problem with our formerly measured values. To test this, we revisited our previous experi-
Peromyscus, of the Rodent genus, is the most prevalent North American mammal. The Perormyscus Genetic Stock Center houses 7 species and several mutant lines of these animals. While Peromyscus provide an attractive model to study different areas of research they are still lagging in the methods of mammalian reproduction. This information is necessary to achieve successful embryo cryopreservation. We have made a significant progress in producing 2-cell stage P. maniculatus embryos. To achieve this, we had to deviate from the protocols published for Mus musculus in several ways. We have found that females that respond to our hormonal priming must be at least 3 months old and receive 10 IU PMS twice (24 h intervals) and 10 IU hCG after 48 h. An overnight mating with a male is also necessary prior to artificial insemination (AI). On average half of the females will produce about 7–10 embryos per animal. The recovered 2 cell stage embryos underwent several divisions in culture in KSOM. We have performed cryopreservation studies on the embryos and obtained promising morphological survival. Currently we are optimizing the freezing and thawing conditions with the goal to achieve successful late-stage development (i.e., 4-cell, morula, and hatch) of the cryopreserved embryo both in culture and in animal model. (Conflicts of interest: None declared. Source of funding: None declared.)
doi:10.1016/j.cryobiol.2008.10.104
104. The coupled effect of freezing and cisplatin on human ovarian cancer COC1 cells. Xiaofeng Li a, Baolin Liu a, Xin Wang a, Tse-Chao Hua a, Kaidong Ma b, a Institute of Cryoengineering, University of Shanghai for Science and Technology, Shanghai, China, b Obstetrics and Gynecology Hospital, Fudan University, Shanghai, China Cisplatin is often used in the treatment of human ovarian cancer cells (COC1). The cells usually have a high drug-resisitance against cisplatin. The effect of cryosurgery on human ovarian cancer cells (COC1) is rarely studied. The aim of this study is to evaluate the effects of some factors on the ovarian cancer cell (COC1) in the freezing process. The cells will be treated with cisplatin before freezing to verify if the drug can enhance the cell damage during freezing process. COC1 cultures were exposed to a range of sub-zero temperatures (5 to 40 °C), and cells were thawed at 37 °C. The Trypan Blue method was used for cell viability assay. Freeze duration was investigated using single and double freezing cycles (5, 10, and 20 min). COC1 cells treated with Cisplatin were more susceptible to freezing than cells without Cisplatin. At 20 °C, cells without Cisplatin yielded 58.9% viability versus 30.4% viability for cells with Cisplatin at the cooling rate of 1 °C/min. Double freezing cycles were found to be more than twice as destructive than a single freeze-thaw cycle,especially for the cells treated with Cisplatin. At the same final temperatures and cooling rates, slower warming at 5 °C/min yielded greater cell death than faster warming rate at 10 °C/min. A 10% and 15% difference in viability between slow and fast warming was obtained for cells treated with and without cisplatin respectively. Finally, COC1 cultures were exposed to 40 °C using a range of cooling rates (1 to 20 °C/min). The results demonstrate that while COC1 cells without
Abstracts / Cryobiology 57 (2008) 315–340
100. The effect of salt on macromolecule solutions: Implications for modeling. Richelle C. Prickett, Janet A.W. Elliott, Locksley E. McGann, University of Alberta, Edmonton, AB, Canada Macromolecules and salts are ubiquitous in biology in both intra- and extracellular solutions. As both of these solutes exhibit non-ideal behaviour at very low concentrations, they have a marked effect on the solution thermodynamics, especially when the solutes are concentrated during freezing by the removal of water as ice. We have previously shown that salts alone or salts in combination with certain small molecules have osmotic behaviour that is well predicted by our osmotic virial equation. However, it has been shown that the solution behaviour of macromolecules is altered when the salt concentration is varied, but equations have not been developed to predict the osmolality of these solutions accurately. The objective of this study was to determine the osmotic virial coefficients for macromolecules in various salt solutions and investigate the changes in these coefficients as a function of the salt concentration. The phase diagrams for hydroxyethyl starch (HES) in varying concentrations of NaCl were obtained from the literature for mass ratios (R values) ranging from 0.5 to 20. Our multisolute osmotic virial equation was fit to each HES + NaCl phase diagram, using the osmotic virial coefficients for NaCl that were previously determined, to obtain the osmotic virial coefficients for HES at each R value. The osmotic virial coefficients were plotted as a function of R value and analyzed in order to obtain equations to determine the osmotic virial coefficient dependence on salt concentration. The effect of salt on macromolecules should be taken into account when modeling these solutions. The osmotic virial coefficients show a strong dependence on the R value, which may lead to the development of equations to predict osmotic virial coefficients as a function of the salt concentration. (Conflicts of interest: None declared. Source of funding: R.C. Prickett, received support from NSERC, the Alberta Ingenuity Fund and CIHR; Janet A.W. Elliott holds the Canada Research Chair in Interfacial Thermodynamics.) Note: This presentation was the winner of the Society’s Poster Award.
ments using a new differential scanning calorimeter (DSC) and analyzed the resulting data. For the new study, melting points for five R values (R = 5, 10, 15, 30, and 45) each measured at five levels of concentration within each R were determined. These melting points were averaged from 3 measurements and plotted as a function of total solute concentration for each R value studied. The new measurements differed from our original values, however they did agree with predicted values using both theoretical models. The data were also fit simultaneously utilizing a method similar to our previous report and the resulting equation was obtained: T m ¼ 0:565x þ ð3:749 103 Þx2 ð1:708 104 Þx3 þ ð2:104 103 ÞxR ð3:007 105 Þx2 R ð1:934 108 Þx3 R: Where x = the total solute concentration. This new equation provided good fits to the experimental data as well as published values and relates the determined polynomial constants to the R value of the corresponding isopleths of the three dimensional phase diagram, allowing the liquidus curve for any R value to be obtained. (Conflicts of interest: E.J. Woods is a General Biotechnology, LLC Shareholder. Source of funding: None declared.)
doi:10.1016/j.cryobiol.2008.10.103
Poster presentations 2. Mammalian systems 103. Towards cryopreservation of peromyscus embryos. Amanda R. Duselis a, Paul B. Vrana a, Monika Veres b, Xiaoming He c, Gabor Szalai b, a University of California at Irvine, Department of Biological Chemistry, Irvine, CA, b University of South Carolina, Peromyscus Genetic Stock Center, Columbia, SC, c University of South Carolina, Department of Mechanical Engineering and Biomedical Engineering Program, Columbia, SC
doi:10.1016/j.cryobiol.2008.10.101
101. Optimizing desiccation buffers utilizing disaccharides. Justin Reis, Ranjan Sitaula, Sankha Bhowmick, University of Masachusetts Dartmouth, North Dartmouth, MA, USA Sugars, particularly disaccharides, have proven to be attractive excipients for desiccation preservation of biologics. Our research aimed towards replacement of water in mammalian cells with sugars to sustain life. The goal is to optimize the in vitro environment, a ternary mix of buffer salts, stabilizing sugar, and water. The buffers we have chosen are TRIS and citrate due to their usage as bovine sperm extenders. Trehalose and sucrose where chosen for their glass transitions as well as proven effectiveness in previous desiccation studies. Glass transition temperature (Tg) is a key thermophysical parameter governing stability of mammalian cells in the desiccated state. Sugar/buffer concentrations were equilibrated to different end moisture contents and Tg was determined using Differential Scanning Calorimetry (DSC). Solutions void of moisture showed that at low concentrations of the sugar Tg’s assimilated themselves with the Tg of the buffer while yielding a Tg slightly below that of the sugar at lower buffer concentrations. Once moisture was added to the sugar/salt solution we saw the expected decrease of glass transition as moisture content increased. Modeling experimental Tg’s was accomplished through the usage of the Gordon Taylor equation for samples with and without moisture and the Pochan-Beatty-Hinman equation for samples void of moisture. These models are able to predict Tg both for intermediary solutions as well as extrapolate Tg for larger residual moisture content sample. (Conflicts of interest: None declared. Source of funding: None declared.) doi:10.1016/j.cryobiol.2008.10.102
102. Melting point equations for the ternary system water/sodium chloride/ethylene glycol revisited. Erik J. Woods a,b, Aniruddha Bagchi a, James D. Benson c, Xu Han c, John K. Critser a,c, a General BioTechnology, LLC, Indianapolis, IN, USA, b Indiana University School of Medicine, Indianapolis, IN, USA, c University of Missouri, Columbia, MO, USA Previously, our group measured melting points (TM) of four different ratios (R) of ethylene glycol to NaCl and developed an equation to fit the experimental measurements [Woods et al., 1999]. This equation was meant to characterize the liquidus surface above the eutectic for any value of R and thus facilitate modeling of cryopreservation using the ternary system. More recently, however, theoretical methods to predict ternary solution properties have been developed which seem to provide consistent and accurate predictions for several different ternary aqueous solutions [Kleinhans and Mazur, 2007; Elliott et al., 2007]. Comparison of our previous report with these new models indicated a discrepancy and a potential problem with our formerly measured values. To test this, we revisited our previous experi-
Peromyscus, of the Rodent genus, is the most prevalent North American mammal. The Perormyscus Genetic Stock Center houses 7 species and several mutant lines of these animals. While Peromyscus provide an attractive model to study different areas of research they are still lagging in the methods of mammalian reproduction. This information is necessary to achieve successful embryo cryopreservation. We have made a significant progress in producing 2-cell stage P. maniculatus embryos. To achieve this, we had to deviate from the protocols published for Mus musculus in several ways. We have found that females that respond to our hormonal priming must be at least 3 months old and receive 10 IU PMS twice (24 h intervals) and 10 IU hCG after 48 h. An overnight mating with a male is also necessary prior to artificial insemination (AI). On average half of the females will produce about 7–10 embryos per animal. The recovered 2 cell stage embryos underwent several divisions in culture in KSOM. We have performed cryopreservation studies on the embryos and obtained promising morphological survival. Currently we are optimizing the freezing and thawing conditions with the goal to achieve successful late-stage development (i.e., 4-cell, morula, and hatch) of the cryopreserved embryo both in culture and in animal model. (Conflicts of interest: None declared. Source of funding: None declared.)
doi:10.1016/j.cryobiol.2008.10.104
104. The coupled effect of freezing and cisplatin on human ovarian cancer COC1 cells. Xiaofeng Li a, Baolin Liu a, Xin Wang a, Tse-Chao Hua a, Kaidong Ma b, a Institute of Cryoengineering, University of Shanghai for Science and Technology, Shanghai, China, b Obstetrics and Gynecology Hospital, Fudan University, Shanghai, China Cisplatin is often used in the treatment of human ovarian cancer cells (COC1). The cells usually have a high drug-resisitance against cisplatin. The effect of cryosurgery on human ovarian cancer cells (COC1) is rarely studied. The aim of this study is to evaluate the effects of some factors on the ovarian cancer cell (COC1) in the freezing process. The cells will be treated with cisplatin before freezing to verify if the drug can enhance the cell damage during freezing process. COC1 cultures were exposed to a range of sub-zero temperatures (5 to 40 °C), and cells were thawed at 37 °C. The Trypan Blue method was used for cell viability assay. Freeze duration was investigated using single and double freezing cycles (5, 10, and 20 min). COC1 cells treated with Cisplatin were more susceptible to freezing than cells without Cisplatin. At 20 °C, cells without Cisplatin yielded 58.9% viability versus 30.4% viability for cells with Cisplatin at the cooling rate of 1 °C/min. Double freezing cycles were found to be more than twice as destructive than a single freeze-thaw cycle,especially for the cells treated with Cisplatin. At the same final temperatures and cooling rates, slower warming at 5 °C/min yielded greater cell death than faster warming rate at 10 °C/min. A 10% and 15% difference in viability between slow and fast warming was obtained for cells treated with and without cisplatin respectively. Finally, COC1 cultures were exposed to 40 °C using a range of cooling rates (1 to 20 °C/min). The results demonstrate that while COC1 cells without