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Glass transition temperatures of mixtures of sugars, polymers, and penetrating cryoprotectants
Abstracts / Cryobiology 73 (2016) 399e443
from this study clearly demonstrate the potential of subzero temperature storage to extend kidney storage time and thus the possibility of increased organ availability for transplant purposes. Conflict of interest: O. Kukal and T. Allen are owners of CryoStasis Ltd. Source of funding: This project was funded by CryoStasis Ltd., IRAP, NSERC, and MITACS Accelerate. P27 TOWARD THE CRYOPRESERVATION OF TOLERANCE TO OSMOTIC DEHYDRATION
ZEBRAFISH
EMBRYOS:
M.H. Connolly 1,*, E. Paredes 1, P. Mazur 1, M. Hagedorn 2, 3. 1 University of Tennessee, Biochemistry Cellular and Molecular Biology, Fundamental and Applied Cryobiology Group, Knoxville, Tennessee, United States; 2 Smithsonian Conservation Biology Institute-National Zoological Park, Department of Reproductive Sciences, Washington, District of Columbia, United States; 3 University of Hawaii, Hawaii Institute of Marine Biology, Kaneohe, Hawaii, United States * Corresponding author.
Zebrafish are an important model of vertebrate development, and as such, several hundred genetic lines have been developed. However, the maintenance of an ever-increasing number of unique genetic lines is challenging to major zebrafish resource centers, which would greatly benefit from the cryopreservation of zebrafish embryos. Yet, to date, traditional cryopreservation techniques have not been amenable to zebrafish embryos, due in part to their large yolky eggs which have a low surface to volume ratio and limited permeability to water and cryoprotectants. Therefore, all attempts to cryopreserve zebrafish embryos have consistently resulted in lethal ice crystal formation upon freezing. New techniques such as vitrification and ultra-rapid warming may help overcome these obstacles, but detailed dehydration and rehydration studies are crucial to identify suitable development stages prior to ultra-rapid warming studies. The purpose of this study was to determine the dehydration tolerance of zebrafish (Danio rerio) embryos at various developmental time points. We find that zebrafish embryos undergoing gastrulation (30-e70% epiboly) are particularly sensitive to osmotic dehydration/rehydration. By contrast, a subset of embryos dehydrated during or after segmentation (20-e22 somite, prim 5) survived 3 hours in a 2 M sucrose solution but exhibit developmental delay, edema, and trunk necrosis 2e-4 days post-treatment. Source of funding: NIH grant# R01-OD011201 P28 GLASS TRANSITION TEMPERATURES OF MIXTURES OF SUGARS, POLYMERS, AND PENETRATING CRYOPROTECTANTS E. Burgess 1, A. De La Ree 1, Y. Lee 1, A. Eroglu 2, J.O.M. Karlsson 3, A. Higgins 1, *. 1 Oregon State University, School of Chemical, Biological and Environmental Engineering, Corvallis, Oregon, United States; 2 Augusta University, Medical College of Georgia, Department of Neuroscience and Regenerative Medicine, Augusta, Georgia, United States; 3 Villanova University, Department of Mechanical Engineering, Villanova, Pennsylvania, United States * Corresponding author.
Long-term storage of viable mammalian cells is important for applications ranging from in vitro fertilization to cell therapy. Cryopreservation is currently the most common approach, but storage in liquid nitrogen can be inconvenient in some cases. Desiccation is an alternative strategy with the potential to enable viable cell preservation at more convenient storage temperatures without the need for liquid nitrogen. To achieve stability during storage in the dried state it is necessary to remove enough water that the remaining matrix forms a non-crystalline glassy solid. To facilitate design of cell desiccation procedures, we are investigating the effects of moisture content on the glass transition temperature of aqueous mixtures of sugars, polymers, and penetrating cryoprotectants (CPAs). Aqueous solutions were first prepared by adding different combinations of sugars (trehalose and raffinose), polymers (polyvinylpyrrolidone and ficoll), and
439
CPAs (ethylene glycol, propylene glycol, and dimethyl sulfoxide) to phosphate buffered saline. These solutions were then dried to different moisture contents by equilibrating samples in closed chambers containing saturated salt solutions that produce a range of relative humidity values. The moisture content and glass transition temperature of the resulting dried samples were then measured. Moisture content was determined by completely drying the sample in an oven and measuring the change in sample mass. Glass transition temperatures were determined by differential scanning calorimetry. The resulting data provides insight into the relationship between relative humidity, moisture content, and glass transition temperature for cell desiccation solutions containing sugars, polymers, and CPAs. Source of funding: This work was funded by a grant from the National Institute of Biomedical Imaging and Bioengineering (Grant No. R21EB018538). P29 IDENTIFICATION OF ICE-BINDING ACTIVITY IN THE GALL FLY AND ITS GOLDENROD HOST H. Tomalty 1, *, K. Storey 2, V. Walker 1. 1 Queen's University, Department of Biology, Kingston, Ontario, Canada; 2 Carleton University, Institute of Biochemistry and Department of Biology, Ottawa, Ontario, Canada * Corresponding author.
The overwintering strategy and polyol production in the freeze-tolerant goldenrod gall fly, Eurosta solidaginis, has been well documented. However, it's unknown whether the larvae also produce an ice-binding protein (IBP) as part of their low temperature adaptation. We assessed ice-binding activity in larvae gathered in early autumn and acclimated to three different temperatures (15 C, 4 C, - 15 C), representing the mid-autumn to winter transition period. Ice recrystallization inhibition and ice shaping/ thermal hysteresis activity was only detected in larvae representing a midautumn period (15 C). Hemolymph samples from October field-collected larvae also displayed ice-binding activity. Since it is formally possible that such activity could be “borrowed” from the host plant, the goldenrod hosts collected from late summer to autumn were also assayed for ice-binding activity. October-collected plants were positive. Ice-affinity purification of these ice-binding activities in both the insect larvae and leaves was undertaken, with the purified proteins subjected to liquid chromatographytandem mass spectrometry (LC-MS/MS). Candidate proteins from insect and host were distinct, with pathogenesis-related proteins identified in the leaves, and “unknown” peptide sequences in the fly larvae. Taken together, these results suggest that E. solidaginis expresses an uncharacterized IBP during the autumn months. We speculate that these IBPs could be used as a protective measure against freeze damage when temperatures can sporadically drop below 0 C, and prior to the synthesis of sufficient glycerol and sorbitol for cryoprotection in the insects, and before vegetative senescence in the plants. Source of funding: This work was funded by an NSERC (Canada) grant to V.K. Walker. P30 CRYOPRESERVATION OF ISOLATED PERFORATUM AS A MODEL SYSTEM
PLANT
ROOTS:
HYPERICUM
X. Yang*, E. Popova, M. Shukla, A. Jones, P. Saxena. University of Guelph, Gosling Research Institute for Plant Preservation, Department of Plant Agriculture, Guelph, Ontario, Canada * Corresponding author.
St. John's Wort, Hypericum perforatum L, is a well known medicinal herb widely used to treat depression. Its major active components are hypericin and hyperforin, and it has been reported to contain high amounts of melatonin. It is also one of the best plant models to investigate regenerative processes in isolated root culture systems. This study is focused on developing methods for long-term conservation (biobanking) of isolated roots of wild type and elite lines of St. John's Wort with value for the herbal medicine industry. Root explants (10 mm) were excised from in vitro
from this study clearly demonstrate the potential of subzero temperature storage to extend kidney storage time and thus the possibility of increased organ availability for transplant purposes. Conflict of interest: O. Kukal and T. Allen are owners of CryoStasis Ltd. Source of funding: This project was funded by CryoStasis Ltd., IRAP, NSERC, and MITACS Accelerate. P27 TOWARD THE CRYOPRESERVATION OF TOLERANCE TO OSMOTIC DEHYDRATION
ZEBRAFISH
EMBRYOS:
M.H. Connolly 1,*, E. Paredes 1, P. Mazur 1, M. Hagedorn 2, 3. 1 University of Tennessee, Biochemistry Cellular and Molecular Biology, Fundamental and Applied Cryobiology Group, Knoxville, Tennessee, United States; 2 Smithsonian Conservation Biology Institute-National Zoological Park, Department of Reproductive Sciences, Washington, District of Columbia, United States; 3 University of Hawaii, Hawaii Institute of Marine Biology, Kaneohe, Hawaii, United States * Corresponding author.
Zebrafish are an important model of vertebrate development, and as such, several hundred genetic lines have been developed. However, the maintenance of an ever-increasing number of unique genetic lines is challenging to major zebrafish resource centers, which would greatly benefit from the cryopreservation of zebrafish embryos. Yet, to date, traditional cryopreservation techniques have not been amenable to zebrafish embryos, due in part to their large yolky eggs which have a low surface to volume ratio and limited permeability to water and cryoprotectants. Therefore, all attempts to cryopreserve zebrafish embryos have consistently resulted in lethal ice crystal formation upon freezing. New techniques such as vitrification and ultra-rapid warming may help overcome these obstacles, but detailed dehydration and rehydration studies are crucial to identify suitable development stages prior to ultra-rapid warming studies. The purpose of this study was to determine the dehydration tolerance of zebrafish (Danio rerio) embryos at various developmental time points. We find that zebrafish embryos undergoing gastrulation (30-e70% epiboly) are particularly sensitive to osmotic dehydration/rehydration. By contrast, a subset of embryos dehydrated during or after segmentation (20-e22 somite, prim 5) survived 3 hours in a 2 M sucrose solution but exhibit developmental delay, edema, and trunk necrosis 2e-4 days post-treatment. Source of funding: NIH grant# R01-OD011201 P28 GLASS TRANSITION TEMPERATURES OF MIXTURES OF SUGARS, POLYMERS, AND PENETRATING CRYOPROTECTANTS E. Burgess 1, A. De La Ree 1, Y. Lee 1, A. Eroglu 2, J.O.M. Karlsson 3, A. Higgins 1, *. 1 Oregon State University, School of Chemical, Biological and Environmental Engineering, Corvallis, Oregon, United States; 2 Augusta University, Medical College of Georgia, Department of Neuroscience and Regenerative Medicine, Augusta, Georgia, United States; 3 Villanova University, Department of Mechanical Engineering, Villanova, Pennsylvania, United States * Corresponding author.
Long-term storage of viable mammalian cells is important for applications ranging from in vitro fertilization to cell therapy. Cryopreservation is currently the most common approach, but storage in liquid nitrogen can be inconvenient in some cases. Desiccation is an alternative strategy with the potential to enable viable cell preservation at more convenient storage temperatures without the need for liquid nitrogen. To achieve stability during storage in the dried state it is necessary to remove enough water that the remaining matrix forms a non-crystalline glassy solid. To facilitate design of cell desiccation procedures, we are investigating the effects of moisture content on the glass transition temperature of aqueous mixtures of sugars, polymers, and penetrating cryoprotectants (CPAs). Aqueous solutions were first prepared by adding different combinations of sugars (trehalose and raffinose), polymers (polyvinylpyrrolidone and ficoll), and
439
CPAs (ethylene glycol, propylene glycol, and dimethyl sulfoxide) to phosphate buffered saline. These solutions were then dried to different moisture contents by equilibrating samples in closed chambers containing saturated salt solutions that produce a range of relative humidity values. The moisture content and glass transition temperature of the resulting dried samples were then measured. Moisture content was determined by completely drying the sample in an oven and measuring the change in sample mass. Glass transition temperatures were determined by differential scanning calorimetry. The resulting data provides insight into the relationship between relative humidity, moisture content, and glass transition temperature for cell desiccation solutions containing sugars, polymers, and CPAs. Source of funding: This work was funded by a grant from the National Institute of Biomedical Imaging and Bioengineering (Grant No. R21EB018538). P29 IDENTIFICATION OF ICE-BINDING ACTIVITY IN THE GALL FLY AND ITS GOLDENROD HOST H. Tomalty 1, *, K. Storey 2, V. Walker 1. 1 Queen's University, Department of Biology, Kingston, Ontario, Canada; 2 Carleton University, Institute of Biochemistry and Department of Biology, Ottawa, Ontario, Canada * Corresponding author.
The overwintering strategy and polyol production in the freeze-tolerant goldenrod gall fly, Eurosta solidaginis, has been well documented. However, it's unknown whether the larvae also produce an ice-binding protein (IBP) as part of their low temperature adaptation. We assessed ice-binding activity in larvae gathered in early autumn and acclimated to three different temperatures (15 C, 4 C, - 15 C), representing the mid-autumn to winter transition period. Ice recrystallization inhibition and ice shaping/ thermal hysteresis activity was only detected in larvae representing a midautumn period (15 C). Hemolymph samples from October field-collected larvae also displayed ice-binding activity. Since it is formally possible that such activity could be “borrowed” from the host plant, the goldenrod hosts collected from late summer to autumn were also assayed for ice-binding activity. October-collected plants were positive. Ice-affinity purification of these ice-binding activities in both the insect larvae and leaves was undertaken, with the purified proteins subjected to liquid chromatographytandem mass spectrometry (LC-MS/MS). Candidate proteins from insect and host were distinct, with pathogenesis-related proteins identified in the leaves, and “unknown” peptide sequences in the fly larvae. Taken together, these results suggest that E. solidaginis expresses an uncharacterized IBP during the autumn months. We speculate that these IBPs could be used as a protective measure against freeze damage when temperatures can sporadically drop below 0 C, and prior to the synthesis of sufficient glycerol and sorbitol for cryoprotection in the insects, and before vegetative senescence in the plants. Source of funding: This work was funded by an NSERC (Canada) grant to V.K. Walker. P30 CRYOPRESERVATION OF ISOLATED PERFORATUM AS A MODEL SYSTEM
PLANT
ROOTS:
HYPERICUM
X. Yang*, E. Popova, M. Shukla, A. Jones, P. Saxena. University of Guelph, Gosling Research Institute for Plant Preservation, Department of Plant Agriculture, Guelph, Ontario, Canada * Corresponding author.
St. John's Wort, Hypericum perforatum L, is a well known medicinal herb widely used to treat depression. Its major active components are hypericin and hyperforin, and it has been reported to contain high amounts of melatonin. It is also one of the best plant models to investigate regenerative processes in isolated root culture systems. This study is focused on developing methods for long-term conservation (biobanking) of isolated roots of wild type and elite lines of St. John's Wort with value for the herbal medicine industry. Root explants (10 mm) were excised from in vitro