- Email: [email protected]
036 Cryobiology of the Natural World: Linking the laboratory to the Field
408
Abstracts / Cryobiology 67 (2013) 398–442
between cooling rate and vitrification of the medium (EAFS), and between survival and the vitrification of the medium. The relative concentration of the EAFS vitrification solution (1, 0.75, 0.6, 0.5, 0.4, 0.33, and 0.25) and the cooling rate (522, 1827, or 69,250 °C/min) had a major effect on whether the media vitrified or froze, but when those results were compared with previous morphological survival and membrane integrity data from Seki and Mazur [PLoS One (2012)], the survival showed little or no correlation with the vitrification or freezing of the medium. The procedure consisted of placing a 0.1–0.2 ll droplet of the desired concentration of EAFS medium on a Cryotop and abruptly immersing the naked Cryotop in liquid nitrogen (LN2). This produces a cooling rate of 69,250 °C/min. For slower cooling, the Cryotops were insulated in various ways. Two questions arose. One was how much dehydration of the droplets of EAFS occurs between the time a droplet is placed on the Cryotop and the time cooling is initiated? The second question was what is the effect of droplet size on whether it freezes or vitrifies? The answer to the second question was that the percentage of droplets vitrifying did not differ significantly in drops ranging in volume from 0.1 to 1.0 ll. The answer to the first question is that the droplets dehydrate rather rapidly in room-temperature air; namely, they lose 20–30% of their mass in the first 2 minutes. This dehydration increases the solute concentration of the EAFS which artifactually increases the likelihood of vitrification. Consequently, if it is not minimized, it can cause confounding. Source of funding: E. Paredes was sponsored by a fellowship from the Spanish government (FPU Research Stays). The research was supported by NIH grant 8R01 OD 011201, P. Mazur, P.I. Conflict of interest: None declared. Email address: [email protected] http://dx.doi.org/10.1016/j.cryobiol.2013.09.039
034 Survival of mouse oocytes after cooling in lower cryoprotectant concentrations by ultra rapid warming using an IR laser pulse. Bo Jin 1, F.W. Kleinhans 1,2, Peter Mazur 1, 1 Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, TN, USA, 2 Physics, IUPUI, Indianapolis, IN, USA Seki and Mazur have recently shown [PLoS One. 2012;7(4)] that about 90% of MII mouse oocytes survive vitrification in solutions of EAFS 10/10 that contain only half the usual concentration of solutes, provided that the warming rate is extremely high (117,000 °C/min). They reported that fewer than 10% survived if the concentration of EAFS were further reduced to 33% of normal. We are currently investigating whether high survivals could be obtained in 0.33 and 0.25 EAFS if the warming rates were still higher. For this purpose we have cooled MII oocytes in these media on Cryotops to 196 °C and warmed them 5 times more rapidly at 600,000 °C/min by subjecting them to a 13 joule pulse of 15 msec duration from a Nd:YAG laser. Kleinhans et al in a companion paper at this meeting discuss the physics attributes of the laser and the interactions of the pulse with the sample, as well as the jig he developed to permit the manipulation of the Cryotop before and during its exposure to the laser pulse. The osmotic/morphological survival of 31 samples suspended in 0.25 EAFS was 15.2 ± 22% (standard deviation). The survival of two samples in 0.33 EAFS was 30%. The total molality of these solutions is 1.27 and 1.72, respectively. These are in the range used in standard slow-freeze cryopreservation. For comparison, note that the total molality of full strength 1x EAFS is 7.37 molal, 5.8 and 4.3-fold higher. Although these preliminary results are encouraging, we have two caveats. One is that the variability of the former is high as evidenced by the large standard errors. The other is that the survival of controls subjected to the same cooling procedure but warmed at 117,000 °C/min (no laser) was close to the same (13.3% vs. 15.2%). Source of funding: NIH Grant 8R01 OD 011201, Peter Mazur, PI. Conflict of interest: None declared. Email address: [email protected] http://dx.doi.org/10.1016/j.cryobiol.2013.09.040
035 Novel computer-generated strategies for rapid cryoprotectant loading into mouse and human oocytes. Jens O.M. Karlsson 1, Edyta A. Szurek 2, Sang R. Lee 2, Ali Eroglu 2,3, 1 Department of Mechanical Engineering, and Cellular & Molecular Bioengineering Research Group, Villanova University, Villanova, PA, USA, 2 Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA, USA, 3 Department of Obstetrics and Gynecology, and Cancer Center, Medical College of Georgia, Augusta, GA, USA A physics-based mathematical optimization algorithm was used to devise novel strategies for intracellular loading of penetrating cryoprotectants. By coupling computer simulations of mass transport with a cost function that minimizes cryoprotectant exposure time while limiting volumetric excursions, a simplex algorithm was
used to optimize two-step cryoprotectant loading procedures for mouse and human oocytes. This resulted in the identification of two new strategies to reduce the time required for cryoprotectant loading, which were then tested experimentally. For example, prior approaches to osmotic manipulation during cryoprotectant loading have been limited to the addition of nonpenetrating excipients (e.g., sugars) that help concentrate intracellular cryoprotectant by drawing out water. In contrast, the optimal computer-generated loading protocol prescribed the use of hypotonic buffers during the initial cryoprotectant exposure step. In particular, to load 1.5 M propane-1,2-diol (PROH) into human metaphase II oocytes at room temperature, the optimization algorithm predicted that oocytes should initially be exposed for 5.5 min to a solution containing 1.3 M of the cryoprotectant and 50 mOsmol/L saline, following which oocytes are predicted to reach equilibrium with only 0.5 min additional exposure to the final 1.5 M PROH solution (in isotonic buffer). This protocol results in less than half the 15 min cryoprotectant exposure time required to load the same amount of PROH using a conventional one-step addition method, while also reducing the magnitude of deleterious volume excursions. A second strategy suggested by computer simulations was to perform cryoprotectant loading at elevated temperatures, to increase the rate of molecular transport. Higher temperatures are typically assumed to result in increased cytotoxicity, to wit, the fertilization rate of mouse oocytes after one-step loading 1.5 M Me2SO at 30 °C (8%) was found to be much lower than the fertilization rate for one-step loading at room temperature (34%). However, this assumption has not previously been tested using experimental protocols based on optimal cryoprotectant exposure times. When using a mathematically optimized two-step loading protocol, fertilizability of mouse oocytes after Me2SO addition at 30 °C was 86%, comparable to that of controls (96%). Combining the temperature elevation technique with the hypotonic diluent strategy, an optimized protocol (2.4 min exposure to 1.4 M Me2SO in 50 mOsmol/L phosphate-buffered saline, followed by an 8-s exposure to isotonic saline with 1.5 M Me2SO) resulted in fertilization and blastocyst formation rates of 92% and 88%, respectively, comparable to those of controls. Applying the same approaches to the development of methods for loading human oocytes with penetrating cryoprotectant, the computer algorithm generated an optimal two-step protocol that is predicted to require only 83 seconds to load 1.5 M PROH. The optimal protocol consists of an initial 79-s exposure at 30 °C to a solution containing 1.4 M PROH and 50 mOsm salt, followed by a 4-s equilibration in the 1.5-M cryoprotectant solution. Source of funding: National Institute of Child Health and Human Development Grant No. R01HD049537, awarded to AE. Conflict of interest: None declared. Email address: [email protected] http://dx.doi.org/10.1016/j.cryobiol.2013.09.041
036 Cryobiology of the Natural World: Linking the laboratory to the Field. Andrew Clarke, British Antarctic Survey, Cambridge, UK Life has been found on Earth wherever water is liquid. Whilst considerable attention has been directed at microbes growing in boiling geysers and hydrothermal vents, much of the natural environment is seasonally or permanently cold. The coldest seawater is 1.96 °C, but highly saline aquatic environments may reach 18 °C. On land the interior of Siberia and Alaska may reach 60 °C, a temperature which poses a significant physiological challenge to plants and animals living there. Laboratory physiology has elucidated how temperature affects physiological performance in all organisms. Of particular importance is the temperature dependence in two important processes: ATP synthesis and contractile protein function. These temperature dependencies underpin much of the thermal physiology of cellular performance. They are not, however generally the cause of chilling injury which can lead to impairment or death of many organisms at temperatures above zero. These appear to be membrane events or neurophysiological functions in more complex organisms. Extra factors come into play at the freezing point, as the phase change of water from liquid to solid presents an extra suite of problems. Key challenges for the cell are the freezeconcentration of extracellular solutes, and the need to avoid ice penetration into the cell. Experimental cryobiology in the laboratory has been important in elucidating how organisms cope with these subzero temperatures. Work with model systems has allowed us to define several fundamental aspects, including the effects of low temperature on the structure and function of cell membranes, the circumstances under which intracellular freezing may occur, and the role of vitrification. Work on organisms in the field has revealed the presence of antifreeze molecules, the importance of small molecular weight cryoprotectants, and the importance of dehydration. Bringing laboratory and field cryobiology together is important to allow us to develop a more complete picture of how organisms survive very low temperatures. Source of funding: British Antarctic Survey. Conflict of interest: None declared. Email address: [email protected] http://dx.doi.org/10.1016/j.cryobiol.2013.09.042
Abstracts / Cryobiology 67 (2013) 398–442
between cooling rate and vitrification of the medium (EAFS), and between survival and the vitrification of the medium. The relative concentration of the EAFS vitrification solution (1, 0.75, 0.6, 0.5, 0.4, 0.33, and 0.25) and the cooling rate (522, 1827, or 69,250 °C/min) had a major effect on whether the media vitrified or froze, but when those results were compared with previous morphological survival and membrane integrity data from Seki and Mazur [PLoS One (2012)], the survival showed little or no correlation with the vitrification or freezing of the medium. The procedure consisted of placing a 0.1–0.2 ll droplet of the desired concentration of EAFS medium on a Cryotop and abruptly immersing the naked Cryotop in liquid nitrogen (LN2). This produces a cooling rate of 69,250 °C/min. For slower cooling, the Cryotops were insulated in various ways. Two questions arose. One was how much dehydration of the droplets of EAFS occurs between the time a droplet is placed on the Cryotop and the time cooling is initiated? The second question was what is the effect of droplet size on whether it freezes or vitrifies? The answer to the second question was that the percentage of droplets vitrifying did not differ significantly in drops ranging in volume from 0.1 to 1.0 ll. The answer to the first question is that the droplets dehydrate rather rapidly in room-temperature air; namely, they lose 20–30% of their mass in the first 2 minutes. This dehydration increases the solute concentration of the EAFS which artifactually increases the likelihood of vitrification. Consequently, if it is not minimized, it can cause confounding. Source of funding: E. Paredes was sponsored by a fellowship from the Spanish government (FPU Research Stays). The research was supported by NIH grant 8R01 OD 011201, P. Mazur, P.I. Conflict of interest: None declared. Email address: [email protected] http://dx.doi.org/10.1016/j.cryobiol.2013.09.039
034 Survival of mouse oocytes after cooling in lower cryoprotectant concentrations by ultra rapid warming using an IR laser pulse. Bo Jin 1, F.W. Kleinhans 1,2, Peter Mazur 1, 1 Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, TN, USA, 2 Physics, IUPUI, Indianapolis, IN, USA Seki and Mazur have recently shown [PLoS One. 2012;7(4)] that about 90% of MII mouse oocytes survive vitrification in solutions of EAFS 10/10 that contain only half the usual concentration of solutes, provided that the warming rate is extremely high (117,000 °C/min). They reported that fewer than 10% survived if the concentration of EAFS were further reduced to 33% of normal. We are currently investigating whether high survivals could be obtained in 0.33 and 0.25 EAFS if the warming rates were still higher. For this purpose we have cooled MII oocytes in these media on Cryotops to 196 °C and warmed them 5 times more rapidly at 600,000 °C/min by subjecting them to a 13 joule pulse of 15 msec duration from a Nd:YAG laser. Kleinhans et al in a companion paper at this meeting discuss the physics attributes of the laser and the interactions of the pulse with the sample, as well as the jig he developed to permit the manipulation of the Cryotop before and during its exposure to the laser pulse. The osmotic/morphological survival of 31 samples suspended in 0.25 EAFS was 15.2 ± 22% (standard deviation). The survival of two samples in 0.33 EAFS was 30%. The total molality of these solutions is 1.27 and 1.72, respectively. These are in the range used in standard slow-freeze cryopreservation. For comparison, note that the total molality of full strength 1x EAFS is 7.37 molal, 5.8 and 4.3-fold higher. Although these preliminary results are encouraging, we have two caveats. One is that the variability of the former is high as evidenced by the large standard errors. The other is that the survival of controls subjected to the same cooling procedure but warmed at 117,000 °C/min (no laser) was close to the same (13.3% vs. 15.2%). Source of funding: NIH Grant 8R01 OD 011201, Peter Mazur, PI. Conflict of interest: None declared. Email address: [email protected] http://dx.doi.org/10.1016/j.cryobiol.2013.09.040
035 Novel computer-generated strategies for rapid cryoprotectant loading into mouse and human oocytes. Jens O.M. Karlsson 1, Edyta A. Szurek 2, Sang R. Lee 2, Ali Eroglu 2,3, 1 Department of Mechanical Engineering, and Cellular & Molecular Bioengineering Research Group, Villanova University, Villanova, PA, USA, 2 Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA, USA, 3 Department of Obstetrics and Gynecology, and Cancer Center, Medical College of Georgia, Augusta, GA, USA A physics-based mathematical optimization algorithm was used to devise novel strategies for intracellular loading of penetrating cryoprotectants. By coupling computer simulations of mass transport with a cost function that minimizes cryoprotectant exposure time while limiting volumetric excursions, a simplex algorithm was
used to optimize two-step cryoprotectant loading procedures for mouse and human oocytes. This resulted in the identification of two new strategies to reduce the time required for cryoprotectant loading, which were then tested experimentally. For example, prior approaches to osmotic manipulation during cryoprotectant loading have been limited to the addition of nonpenetrating excipients (e.g., sugars) that help concentrate intracellular cryoprotectant by drawing out water. In contrast, the optimal computer-generated loading protocol prescribed the use of hypotonic buffers during the initial cryoprotectant exposure step. In particular, to load 1.5 M propane-1,2-diol (PROH) into human metaphase II oocytes at room temperature, the optimization algorithm predicted that oocytes should initially be exposed for 5.5 min to a solution containing 1.3 M of the cryoprotectant and 50 mOsmol/L saline, following which oocytes are predicted to reach equilibrium with only 0.5 min additional exposure to the final 1.5 M PROH solution (in isotonic buffer). This protocol results in less than half the 15 min cryoprotectant exposure time required to load the same amount of PROH using a conventional one-step addition method, while also reducing the magnitude of deleterious volume excursions. A second strategy suggested by computer simulations was to perform cryoprotectant loading at elevated temperatures, to increase the rate of molecular transport. Higher temperatures are typically assumed to result in increased cytotoxicity, to wit, the fertilization rate of mouse oocytes after one-step loading 1.5 M Me2SO at 30 °C (8%) was found to be much lower than the fertilization rate for one-step loading at room temperature (34%). However, this assumption has not previously been tested using experimental protocols based on optimal cryoprotectant exposure times. When using a mathematically optimized two-step loading protocol, fertilizability of mouse oocytes after Me2SO addition at 30 °C was 86%, comparable to that of controls (96%). Combining the temperature elevation technique with the hypotonic diluent strategy, an optimized protocol (2.4 min exposure to 1.4 M Me2SO in 50 mOsmol/L phosphate-buffered saline, followed by an 8-s exposure to isotonic saline with 1.5 M Me2SO) resulted in fertilization and blastocyst formation rates of 92% and 88%, respectively, comparable to those of controls. Applying the same approaches to the development of methods for loading human oocytes with penetrating cryoprotectant, the computer algorithm generated an optimal two-step protocol that is predicted to require only 83 seconds to load 1.5 M PROH. The optimal protocol consists of an initial 79-s exposure at 30 °C to a solution containing 1.4 M PROH and 50 mOsm salt, followed by a 4-s equilibration in the 1.5-M cryoprotectant solution. Source of funding: National Institute of Child Health and Human Development Grant No. R01HD049537, awarded to AE. Conflict of interest: None declared. Email address: [email protected] http://dx.doi.org/10.1016/j.cryobiol.2013.09.041
036 Cryobiology of the Natural World: Linking the laboratory to the Field. Andrew Clarke, British Antarctic Survey, Cambridge, UK Life has been found on Earth wherever water is liquid. Whilst considerable attention has been directed at microbes growing in boiling geysers and hydrothermal vents, much of the natural environment is seasonally or permanently cold. The coldest seawater is 1.96 °C, but highly saline aquatic environments may reach 18 °C. On land the interior of Siberia and Alaska may reach 60 °C, a temperature which poses a significant physiological challenge to plants and animals living there. Laboratory physiology has elucidated how temperature affects physiological performance in all organisms. Of particular importance is the temperature dependence in two important processes: ATP synthesis and contractile protein function. These temperature dependencies underpin much of the thermal physiology of cellular performance. They are not, however generally the cause of chilling injury which can lead to impairment or death of many organisms at temperatures above zero. These appear to be membrane events or neurophysiological functions in more complex organisms. Extra factors come into play at the freezing point, as the phase change of water from liquid to solid presents an extra suite of problems. Key challenges for the cell are the freezeconcentration of extracellular solutes, and the need to avoid ice penetration into the cell. Experimental cryobiology in the laboratory has been important in elucidating how organisms cope with these subzero temperatures. Work with model systems has allowed us to define several fundamental aspects, including the effects of low temperature on the structure and function of cell membranes, the circumstances under which intracellular freezing may occur, and the role of vitrification. Work on organisms in the field has revealed the presence of antifreeze molecules, the importance of small molecular weight cryoprotectants, and the importance of dehydration. Bringing laboratory and field cryobiology together is important to allow us to develop a more complete picture of how organisms survive very low temperatures. Source of funding: British Antarctic Survey. Conflict of interest: None declared. Email address: [email protected] http://dx.doi.org/10.1016/j.cryobiol.2013.09.042