- Email: [email protected]
Vitrification and ultra-rapid laser warming of yeast Saccharomyces cerevisiae
Abstracts / Cryobiology 73 (2016) 399e443
type. IBP-knockdown lines also demonstrated a significant decrease in viability following freezing to 8 C, and depending on the particular line, showed only two-thirds the survival seen in control lines. These results underscore the vital role IBPs play in the development of a freeze-tolerant phenotype and that expression of these proteins in frost-susceptible plants could be valuable for the production of more hardy crops. S082 REGULATION OF OXIDATIVE STRESS AND PROGRAMMED CELL DEATH IN AGAPANTHUS PRAECOX CRYOPRESERVATION G. Chen*, D. Zhang, L. Ren, X. Shen. Jiao Tong University, Minhang, Shanghai, China * Corresponding author.
Our research work suggested that oxidative stress and programmed cell death (PCD) induced by ROS seriously impact cell viability during Agapanthus praecox embryogenic callus cryopreservation. Application of GSH to cryoprotectant could improve cell survival rate from 49.14% to 86.85%. GSH, acted as H2O2 decomposer, enhanced antioxidative activity, and suppressed cell death through AsA-GSH and GPX cycles. PCD events including autophagy, apoptosis-like, and necrosis occurred at later stages of cryopreservation. Cryopreservation-induced delayed onset cell death (CIDOCD) was also detected within 48 h-recovery. Further, apoptosis-like and necrosis were burst in recovery for 9~18 h. Transcriptomic and proteomic analysis showed that the PCD-related proteases, e.g. cathepsin B, subtilisin-like protease, were up-regulated at dehydration and recovery stage. They were down-regulated at pre-culture and dilution stages. Interestingly, protease inhibitor gene expressed negatively. This phenomenon suggested that the interaction between protease and protease inhibitor might control the cell fate under conditions of stress. Therefore, we applied PCD inhibitors, e.g. Ac-DEVD-CHO (Caspase inhibitor), Antimycin A3 (Bcl-2 inhibitor), and 3-Amin (PARP inhibitor) to cryoprotectant solution. Ac-DEVD-CHO improved cell viability from 49.14% to 89.91%. The addition of recombinant protease inhibitor of A. praecox was also beneficial to cell viability. Our research provided a theoretical basis and new viewpoint to application of new exogenous substances (botanical protease inhibitor) in cryoprotectant solution and recovery medium for higher survival. Source of funding: This work was supported by the National Natural Science Funding of China (No. 31170655, 31300580).
421
S084 CONSIDERATIONS OF WARMING RATES IN CRYOPRESERVATION P. Kilbride 1, *, K. Mahbubani 2, 3, J. Baboo 4, M. Delahaye 4, N. Gaddum 4, J. Morris 1. 1 Asymptote Ltd., Cambridge, United Kingdom; 2 University of Cambridge, Department of Chemical Engineering and Biotechnology; 3 Department of Surgery, Cambridge, United Kingdom; 4 Cell and Gene Therapy Catapult, London, United Kingdom * Corresponding author.
The significance of cooling rates in traditional cryopreservation has been well documented. Thawing rates on the other hand have received considerably less attention. It is generally assumed that rapid thawing is necessary to achieve high viability on thawing. However, with increasing interest in the cryopreservation of larger tissue volumes such as is required for T-cell therapy and devices such as bioartificial livers, rapid thawing (>10 C/min) are difficult to achieve practically. A common concern with thawing profiles is recrystallization, a phenomenon where smaller ice crystals develop or merge into larger crystals. This study examined the impact of slow (1 C/min) and faster (10 and 100 C/min) warming rates on biologics and ice structure after either slow cooling at 1 C/min, where the water-solute phase diagram was followed, and faster cooling rates (10 C/ min), where the water-solute phase diagram was not followed. We found that after slow cooling, the post-thaw outcome of biologics (T- cells) did not depend on the thawing rate. The same was true of ice structure, if the phase diagram was followed on cooling no re-crystallization was observed on warming as the system was already in an equilibrium state. During slow cooling ice crystals were observed to increase in size during cooling in the high sub-zero zone (above - 25 C). After fast cooling, post-thaw viability was found to be influenced by thawing rate, with fast warming giving optimised results. Ice-structure was observed to change on slow warming after fast cooling, in contrast to slowly cooled samples. In conclusion, slow warming is acceptable in biological systems after slow cooling as no change in ice structure occurs. This is significant for cryopreservation of large volume samples where ice forms in an equilibrium manner and rapid rates of cooling cannot be achieved practically. S085 VITRIFICATION AND ULTRA-RAPID SACCHAROMYCES CEREVISIAE
LASER WARMING OF
YEAST
E. Paredes. University of Tennessee, Knoxville, Tennessee, United States S083 ULTRA-RAPID WARMING TECHNOLOGY WITH A LASER PULSE FOR VITRIFIED MOUSE/HUMAN OOCYTES AND EMBRYOS B. Jin*, J. Qiu, C. Ma, X. Shao. Dalian Municipal Women’s and Children Medical Center, Reproductive and Genetic Center, Fundamental and Applied Cryobiology Group, Dalian, China * Corresponding author.
Vitrification is now the main route to the cryopreservation of human and animal oocytes and embryos. A central belief is that for success, the cells must be placed in very high concentrations of cryoprotective solutes and must be cooled extremely rapidly. We have shown recently that these beliefs are incorrect. Over 90% of mouse oocytes and embryos survive being cooled relatively slowly even in solutions containing only one-third the normal solute concentrations, provided that they are warmed ultrarapidly by a laser pulse. Nearly all vitrification solutions contain both permeating and non-permeating solutes, and an important question is whether the former protect because they permeate the cells and promote intracellular vitrification (as is almost universally believed), or because they osmotically withdraw a large fraction of intracellular water prior to cooling. The answer for the mouse system is clearly the latter. We got similar results for human oocytes/embryos.
Mazur and collaborators' work on vitrification of mouse oocytes and embryos produced a series of findings with implications in the understanding and foundation of vitrification. Like the high sensitivity to warming rate that strongly suggests that the lethality of slow warming is a consequence of crystallisation of intracellular glassy water during warming. A second important finding was that the survival of oocytes seemed to be more dependent on the osmotic withdrawal of much of the intracellular water before vitrification than it is on the penetration of cryoprotective solutes into the cells. The development and application of vitrification plus ultrarapid laser warming to mice oocytes and embryos produced survivals ranging from 80 to 100%. However, it remains to be seen how widely these findings will be applicable to other types of cells and tissues from other species. Yeast are fundamental models of study due to their ease of culture, manipulation, and the well-studied genome. The aim of this work was to explore the possibility of cryopreserving yeast by vitrification and ultrarapid laser warming. Saccharomyces cerevisiae cells were exposed to several permeating and non-permeating cryoprotectants in low concentration prior to vitrification (cooling rate 69,000 C/min) by immersion of a 0.1 mL drop of cells culture in liquid nitrogen, cells were subsequently warmed ultra-rapidly with a laser (warming rate 107 C/min). When using 0.33x EAFS as cryoprotecting solution, survival was 79.93% ± 16.15; using only non permeating solutes (1 M sucrose) as cryoprotecting solution results are slightly lower 60.75% ± 26.39. When the cells are suspended only
422
Abstracts / Cryobiology 73 (2016) 399e443
in distilled water prior to vitrification, survivals are 12.68% ± 4.70. This high survival, post vitrification adds information to the study of the effect of numerous cooling and warming rates of the baker's yeast and provides further examples of the application of vitrification and ultra-fast laser warming. Source of funding: The research reported here was supported by NIH grant 5R01OD011201. S086 NANOWARMING OF TISSUES N. Manuchehrabadi 1, *, Z. Gao 2, J. Zhang 3, H. Ring 2, Q. Shao 1, F. Liu 1, Y. Chen 4, M. Mcdermott 2, A. Fok 4, K. Brockbank 5, M. Garwood 6, C. Haynes 2, J. Bischof 1. 1 University of Minnesota, Department of Mechanical Engineering, Minneapolis, Minnesota, United States; 2 University of Minnesota, Department of Chemistry, Minneapolis, Minnesota, United States; 3 University of Minnesota, Center of Magnetic Resonance Research, Minneapolis, Minnesota, United States; 4 Minnesota Dental Research Center for Biomaterials and Biomechanics, Minneapolis, Minnesota, United States; 5 Clemson University, Department of Bioengineering, Clemson, South Carolina, United States; 6 University of Minnesota, Department of Radiology, Minneapolis, Minnesota, United States * Corresponding author.
Ice-free vitrification as a means of preservation for tissue has many potential benefits including indefinite storage and banking. Unfortunately, current convective warming methods fail in larger tissue systems due to speed (i.e., too slow in center) or non-uniformity (edge to center temperature gradient). Thus both fast and uniform warming rates are necessary to avoid devitrification and cracking. Here we present a scalable technology for rewarming bulk vitrified tissues called: “nanowarming”, which can both rapidly and uniformly rewarm vitrified tissues regardless of size. Nanowarming uses radiofrequency (RF) excited mesoporous silicacoated iron oxide nanoparticles deployed within vitrification solutions to create uniform heat generation. The current study shows successful biological and functional outcomes when nanowarming VS55 loaded vitrified cell systems (human dermal fibroblast) and porcine cardiovascular tissues (carotid arteries and heart valve leaflets). These systems have the benefits of small scale and a high degree of technical reproducibility while avoiding the requirements for complex vascular reconstruction. Using a 1 mL vial system we imposed cooling rates (10 e 15 C/min) with annealing at - 115 C and maintained vitrified tissue at liquid nitrogen temperature. Advanced imaging techniques (mCT and SWIFT MRI) verified the loading and unloading of cryoprotectant and nanoparticle in addition to homogeneous vitrification. The tissues in 1 ml vials were then heated rapidly and uniformly in a 1 kW RF system (>200 C/min) by nanowarming. The viability results post-rewarming match gold standard fast convective warming, and demonstrate significant improvements compared to slow warmed (i.e. devitrified) samples representing warming failure at the center of a larger convectively warmed system (critical warming rate <1.5 C/min). Importantly, experimental and modeling results from a larger 15 kW system demonstrate our ability to scale nanowarming up to 80 ml volumes and beyond (>Di:5 cm). Together these results demonstrate that nanowarming is a scalable technology for bulk vitrified tissue rewarming. Source of funding: Acknowledgments: We thank Paul Iaizzo and the Visible Heart Lab for access to porcine carotid arteries and Connie Chung for help with initial cell culture. Funding: NSF/CBET (1066343,1133285,1336659), NIH (R43HL123317, P41EB015894), ARMY W81XWH-15-C-0173, UMN (MN Futures grant and IEM Seed Grant), the Kuhrmeyer Chair to JCB and the US Army Medical Research and Materiel Command under Contract W81XWH-15-C-0173 to KGMB are gratefully acknowledged. S087 ULTRARAPID INDUCTIVE REWARMING BIOMATERIALS WITH METAL FOAMS
OF
VITRIFIED
BULK
M. Shi 1,*, N. Manuchehrabadi 2, A. Clopton 2, Q. Jinbin 3, F. Xu 3, T. Lu 3, J. Bischof 2. 1 Xi’an Jiaotong University, School of Energy and Power
Engineering, Xi'an, Shaanxi, China; 2 University of Minnesota, Department of Mechanical Engineering, Minneapolis, Minnesota, United States; 3 Xi’an Jiaotong University, Bioinspired Engineering and Biomechanics Center (BEBC), Xi'an, Shaanxi, China * Corresponding author.
Tissues have been successfully vitrified by loading high molarity (6e8.4 M) cryoprotective agents (CPA) such as VS55, DP6, and even glycerol at critical cooling rates of 2.5, 40 and 85 C/min respectively. However, successful rewarming from the vitrified state remains challenging with standard convective methods as it requires critical warming rates (CWR) of 55, 185 and 3.2x104 C/min respectively to avoid devitrification. Furthermore, these rates must be sufficiently uniform to avoid thermal stresses yield stress (2.5 MPa), which can crack the tissue. By achieving faster yet sufficiently uniform rates, the CPA concentration can be reduced (lower chemical toxicity). Here we present a new method that may achieve this goal by inductively warming metal foams embedded in vitrified samples utilizing radiofrequency (RF) fields. We initially tested copper foam due to its high electrical and thermal conductivity. This foam (porosity:94%, density:0.3 g/cm3) was deployed directly into a cryovial filled with VS55 (8.4M). Controlled cooling to the vitrified state was achieved at rates between 10 - 150 C/min by exposure to either gaseous or liquid nitrogen environments. Once vitrified, standard convective warming was compared directly to RF rewarming. RF warming rates up to 1000 C/min and specific absorption rates (SAR) 40 W/cm3 were experimentally measured (RF settings: 20 kA/m and 360 kHz). This compares to only 100 C/min, < CWR of DP6 and glycerol, by convective thawing in 37 C water bath. Computational modeling suggests that tissues such as arteries, heart valves or skin tissues up to 4 mm thick can be warmed by this approach without cracking. In the future we will study the physical and biological (i.e. viability) limits of this technology, which may well be on the cooling side, by finding the lowest possible concentrations of CPA that still allow vitrification prior to deploying this ultra-rapid warming technology. Source of funding: JCB gratefully acknowledges the Kuhrmeyer Chair in Mechanical Engineering for support and NSF CBET 1336659. S088 ORIGIN OF THE CONVERGENTLY EVOLVED FLOUNDER ANTIFREEZE PROTEIN NEOGENE L.A. Graham*, P.L. Davies. Queen’s University, Department of Biomedical and Molecular Sciences, Kingston, Ontario, Canada * Corresponding author.
Some ocean fish produce antifreeze proteins (AFPs), which inhibit the growth of ice, thereby allowing them to inhabit icy niches. The four types of AFPs made by fish demonstrate all major evolutionary mechanisms. Type III AFP shows the standard pattern whereby a gene arose and remained within a single lineage. The phylogenetic distribution of the other three types is scattered and for type II AFP, lateral gene transfer occurred between three very distantly-related fish groups. Convergent evolution explains the presence of AFGPs in two lineages, but this has been taken to extremes with the type I AFP, as this alanine-rich, alpha-helical AFP has independently evolved in four separate lineages. We have traced the origin of one of these, the type I AFP neogene of flatfish, by sequencing loci from the starry flounder, Platichthys stellatus. The AFP gene arose from the functionally and structurally unrelated gig2 locus following gene duplication. The first to arise was the 3-kDa skin isoform and following tandem duplications to increase gene number, internal duplications of the 11-a.a. repeat and the gain of a signal sequence gave rise to a 32-kDa circulating isoform, Maxi. Later, a 3.3-kDa portion of Maxi became the dominant internally secreted isoform as a result of massive gene amplification. Present day starry flounder are found along the Pacific Rim from California to Japan. There is a strong correlation between latitude and AFP gene dosage as Alaskan fish have many AFP gene copies, Californian fish have only a few and fish from British Columbia have an intermediate number. Taken together, these results suggest that the ocean cooling that took place during the Cenozoic era led to AFP acquisition in numerous naïve fish lineages and that a decrease in gene copy number will be the
type. IBP-knockdown lines also demonstrated a significant decrease in viability following freezing to 8 C, and depending on the particular line, showed only two-thirds the survival seen in control lines. These results underscore the vital role IBPs play in the development of a freeze-tolerant phenotype and that expression of these proteins in frost-susceptible plants could be valuable for the production of more hardy crops. S082 REGULATION OF OXIDATIVE STRESS AND PROGRAMMED CELL DEATH IN AGAPANTHUS PRAECOX CRYOPRESERVATION G. Chen*, D. Zhang, L. Ren, X. Shen. Jiao Tong University, Minhang, Shanghai, China * Corresponding author.
Our research work suggested that oxidative stress and programmed cell death (PCD) induced by ROS seriously impact cell viability during Agapanthus praecox embryogenic callus cryopreservation. Application of GSH to cryoprotectant could improve cell survival rate from 49.14% to 86.85%. GSH, acted as H2O2 decomposer, enhanced antioxidative activity, and suppressed cell death through AsA-GSH and GPX cycles. PCD events including autophagy, apoptosis-like, and necrosis occurred at later stages of cryopreservation. Cryopreservation-induced delayed onset cell death (CIDOCD) was also detected within 48 h-recovery. Further, apoptosis-like and necrosis were burst in recovery for 9~18 h. Transcriptomic and proteomic analysis showed that the PCD-related proteases, e.g. cathepsin B, subtilisin-like protease, were up-regulated at dehydration and recovery stage. They were down-regulated at pre-culture and dilution stages. Interestingly, protease inhibitor gene expressed negatively. This phenomenon suggested that the interaction between protease and protease inhibitor might control the cell fate under conditions of stress. Therefore, we applied PCD inhibitors, e.g. Ac-DEVD-CHO (Caspase inhibitor), Antimycin A3 (Bcl-2 inhibitor), and 3-Amin (PARP inhibitor) to cryoprotectant solution. Ac-DEVD-CHO improved cell viability from 49.14% to 89.91%. The addition of recombinant protease inhibitor of A. praecox was also beneficial to cell viability. Our research provided a theoretical basis and new viewpoint to application of new exogenous substances (botanical protease inhibitor) in cryoprotectant solution and recovery medium for higher survival. Source of funding: This work was supported by the National Natural Science Funding of China (No. 31170655, 31300580).
421
S084 CONSIDERATIONS OF WARMING RATES IN CRYOPRESERVATION P. Kilbride 1, *, K. Mahbubani 2, 3, J. Baboo 4, M. Delahaye 4, N. Gaddum 4, J. Morris 1. 1 Asymptote Ltd., Cambridge, United Kingdom; 2 University of Cambridge, Department of Chemical Engineering and Biotechnology; 3 Department of Surgery, Cambridge, United Kingdom; 4 Cell and Gene Therapy Catapult, London, United Kingdom * Corresponding author.
The significance of cooling rates in traditional cryopreservation has been well documented. Thawing rates on the other hand have received considerably less attention. It is generally assumed that rapid thawing is necessary to achieve high viability on thawing. However, with increasing interest in the cryopreservation of larger tissue volumes such as is required for T-cell therapy and devices such as bioartificial livers, rapid thawing (>10 C/min) are difficult to achieve practically. A common concern with thawing profiles is recrystallization, a phenomenon where smaller ice crystals develop or merge into larger crystals. This study examined the impact of slow (1 C/min) and faster (10 and 100 C/min) warming rates on biologics and ice structure after either slow cooling at 1 C/min, where the water-solute phase diagram was followed, and faster cooling rates (10 C/ min), where the water-solute phase diagram was not followed. We found that after slow cooling, the post-thaw outcome of biologics (T- cells) did not depend on the thawing rate. The same was true of ice structure, if the phase diagram was followed on cooling no re-crystallization was observed on warming as the system was already in an equilibrium state. During slow cooling ice crystals were observed to increase in size during cooling in the high sub-zero zone (above - 25 C). After fast cooling, post-thaw viability was found to be influenced by thawing rate, with fast warming giving optimised results. Ice-structure was observed to change on slow warming after fast cooling, in contrast to slowly cooled samples. In conclusion, slow warming is acceptable in biological systems after slow cooling as no change in ice structure occurs. This is significant for cryopreservation of large volume samples where ice forms in an equilibrium manner and rapid rates of cooling cannot be achieved practically. S085 VITRIFICATION AND ULTRA-RAPID SACCHAROMYCES CEREVISIAE
LASER WARMING OF
YEAST
E. Paredes. University of Tennessee, Knoxville, Tennessee, United States S083 ULTRA-RAPID WARMING TECHNOLOGY WITH A LASER PULSE FOR VITRIFIED MOUSE/HUMAN OOCYTES AND EMBRYOS B. Jin*, J. Qiu, C. Ma, X. Shao. Dalian Municipal Women’s and Children Medical Center, Reproductive and Genetic Center, Fundamental and Applied Cryobiology Group, Dalian, China * Corresponding author.
Vitrification is now the main route to the cryopreservation of human and animal oocytes and embryos. A central belief is that for success, the cells must be placed in very high concentrations of cryoprotective solutes and must be cooled extremely rapidly. We have shown recently that these beliefs are incorrect. Over 90% of mouse oocytes and embryos survive being cooled relatively slowly even in solutions containing only one-third the normal solute concentrations, provided that they are warmed ultrarapidly by a laser pulse. Nearly all vitrification solutions contain both permeating and non-permeating solutes, and an important question is whether the former protect because they permeate the cells and promote intracellular vitrification (as is almost universally believed), or because they osmotically withdraw a large fraction of intracellular water prior to cooling. The answer for the mouse system is clearly the latter. We got similar results for human oocytes/embryos.
Mazur and collaborators' work on vitrification of mouse oocytes and embryos produced a series of findings with implications in the understanding and foundation of vitrification. Like the high sensitivity to warming rate that strongly suggests that the lethality of slow warming is a consequence of crystallisation of intracellular glassy water during warming. A second important finding was that the survival of oocytes seemed to be more dependent on the osmotic withdrawal of much of the intracellular water before vitrification than it is on the penetration of cryoprotective solutes into the cells. The development and application of vitrification plus ultrarapid laser warming to mice oocytes and embryos produced survivals ranging from 80 to 100%. However, it remains to be seen how widely these findings will be applicable to other types of cells and tissues from other species. Yeast are fundamental models of study due to their ease of culture, manipulation, and the well-studied genome. The aim of this work was to explore the possibility of cryopreserving yeast by vitrification and ultrarapid laser warming. Saccharomyces cerevisiae cells were exposed to several permeating and non-permeating cryoprotectants in low concentration prior to vitrification (cooling rate 69,000 C/min) by immersion of a 0.1 mL drop of cells culture in liquid nitrogen, cells were subsequently warmed ultra-rapidly with a laser (warming rate 107 C/min). When using 0.33x EAFS as cryoprotecting solution, survival was 79.93% ± 16.15; using only non permeating solutes (1 M sucrose) as cryoprotecting solution results are slightly lower 60.75% ± 26.39. When the cells are suspended only
422
Abstracts / Cryobiology 73 (2016) 399e443
in distilled water prior to vitrification, survivals are 12.68% ± 4.70. This high survival, post vitrification adds information to the study of the effect of numerous cooling and warming rates of the baker's yeast and provides further examples of the application of vitrification and ultra-fast laser warming. Source of funding: The research reported here was supported by NIH grant 5R01OD011201. S086 NANOWARMING OF TISSUES N. Manuchehrabadi 1, *, Z. Gao 2, J. Zhang 3, H. Ring 2, Q. Shao 1, F. Liu 1, Y. Chen 4, M. Mcdermott 2, A. Fok 4, K. Brockbank 5, M. Garwood 6, C. Haynes 2, J. Bischof 1. 1 University of Minnesota, Department of Mechanical Engineering, Minneapolis, Minnesota, United States; 2 University of Minnesota, Department of Chemistry, Minneapolis, Minnesota, United States; 3 University of Minnesota, Center of Magnetic Resonance Research, Minneapolis, Minnesota, United States; 4 Minnesota Dental Research Center for Biomaterials and Biomechanics, Minneapolis, Minnesota, United States; 5 Clemson University, Department of Bioengineering, Clemson, South Carolina, United States; 6 University of Minnesota, Department of Radiology, Minneapolis, Minnesota, United States * Corresponding author.
Ice-free vitrification as a means of preservation for tissue has many potential benefits including indefinite storage and banking. Unfortunately, current convective warming methods fail in larger tissue systems due to speed (i.e., too slow in center) or non-uniformity (edge to center temperature gradient). Thus both fast and uniform warming rates are necessary to avoid devitrification and cracking. Here we present a scalable technology for rewarming bulk vitrified tissues called: “nanowarming”, which can both rapidly and uniformly rewarm vitrified tissues regardless of size. Nanowarming uses radiofrequency (RF) excited mesoporous silicacoated iron oxide nanoparticles deployed within vitrification solutions to create uniform heat generation. The current study shows successful biological and functional outcomes when nanowarming VS55 loaded vitrified cell systems (human dermal fibroblast) and porcine cardiovascular tissues (carotid arteries and heart valve leaflets). These systems have the benefits of small scale and a high degree of technical reproducibility while avoiding the requirements for complex vascular reconstruction. Using a 1 mL vial system we imposed cooling rates (10 e 15 C/min) with annealing at - 115 C and maintained vitrified tissue at liquid nitrogen temperature. Advanced imaging techniques (mCT and SWIFT MRI) verified the loading and unloading of cryoprotectant and nanoparticle in addition to homogeneous vitrification. The tissues in 1 ml vials were then heated rapidly and uniformly in a 1 kW RF system (>200 C/min) by nanowarming. The viability results post-rewarming match gold standard fast convective warming, and demonstrate significant improvements compared to slow warmed (i.e. devitrified) samples representing warming failure at the center of a larger convectively warmed system (critical warming rate <1.5 C/min). Importantly, experimental and modeling results from a larger 15 kW system demonstrate our ability to scale nanowarming up to 80 ml volumes and beyond (>Di:5 cm). Together these results demonstrate that nanowarming is a scalable technology for bulk vitrified tissue rewarming. Source of funding: Acknowledgments: We thank Paul Iaizzo and the Visible Heart Lab for access to porcine carotid arteries and Connie Chung for help with initial cell culture. Funding: NSF/CBET (1066343,1133285,1336659), NIH (R43HL123317, P41EB015894), ARMY W81XWH-15-C-0173, UMN (MN Futures grant and IEM Seed Grant), the Kuhrmeyer Chair to JCB and the US Army Medical Research and Materiel Command under Contract W81XWH-15-C-0173 to KGMB are gratefully acknowledged. S087 ULTRARAPID INDUCTIVE REWARMING BIOMATERIALS WITH METAL FOAMS
OF
VITRIFIED
BULK
M. Shi 1,*, N. Manuchehrabadi 2, A. Clopton 2, Q. Jinbin 3, F. Xu 3, T. Lu 3, J. Bischof 2. 1 Xi’an Jiaotong University, School of Energy and Power
Engineering, Xi'an, Shaanxi, China; 2 University of Minnesota, Department of Mechanical Engineering, Minneapolis, Minnesota, United States; 3 Xi’an Jiaotong University, Bioinspired Engineering and Biomechanics Center (BEBC), Xi'an, Shaanxi, China * Corresponding author.
Tissues have been successfully vitrified by loading high molarity (6e8.4 M) cryoprotective agents (CPA) such as VS55, DP6, and even glycerol at critical cooling rates of 2.5, 40 and 85 C/min respectively. However, successful rewarming from the vitrified state remains challenging with standard convective methods as it requires critical warming rates (CWR) of 55, 185 and 3.2x104 C/min respectively to avoid devitrification. Furthermore, these rates must be sufficiently uniform to avoid thermal stresses yield stress (2.5 MPa), which can crack the tissue. By achieving faster yet sufficiently uniform rates, the CPA concentration can be reduced (lower chemical toxicity). Here we present a new method that may achieve this goal by inductively warming metal foams embedded in vitrified samples utilizing radiofrequency (RF) fields. We initially tested copper foam due to its high electrical and thermal conductivity. This foam (porosity:94%, density:0.3 g/cm3) was deployed directly into a cryovial filled with VS55 (8.4M). Controlled cooling to the vitrified state was achieved at rates between 10 - 150 C/min by exposure to either gaseous or liquid nitrogen environments. Once vitrified, standard convective warming was compared directly to RF rewarming. RF warming rates up to 1000 C/min and specific absorption rates (SAR) 40 W/cm3 were experimentally measured (RF settings: 20 kA/m and 360 kHz). This compares to only 100 C/min, < CWR of DP6 and glycerol, by convective thawing in 37 C water bath. Computational modeling suggests that tissues such as arteries, heart valves or skin tissues up to 4 mm thick can be warmed by this approach without cracking. In the future we will study the physical and biological (i.e. viability) limits of this technology, which may well be on the cooling side, by finding the lowest possible concentrations of CPA that still allow vitrification prior to deploying this ultra-rapid warming technology. Source of funding: JCB gratefully acknowledges the Kuhrmeyer Chair in Mechanical Engineering for support and NSF CBET 1336659. S088 ORIGIN OF THE CONVERGENTLY EVOLVED FLOUNDER ANTIFREEZE PROTEIN NEOGENE L.A. Graham*, P.L. Davies. Queen’s University, Department of Biomedical and Molecular Sciences, Kingston, Ontario, Canada * Corresponding author.
Some ocean fish produce antifreeze proteins (AFPs), which inhibit the growth of ice, thereby allowing them to inhabit icy niches. The four types of AFPs made by fish demonstrate all major evolutionary mechanisms. Type III AFP shows the standard pattern whereby a gene arose and remained within a single lineage. The phylogenetic distribution of the other three types is scattered and for type II AFP, lateral gene transfer occurred between three very distantly-related fish groups. Convergent evolution explains the presence of AFGPs in two lineages, but this has been taken to extremes with the type I AFP, as this alanine-rich, alpha-helical AFP has independently evolved in four separate lineages. We have traced the origin of one of these, the type I AFP neogene of flatfish, by sequencing loci from the starry flounder, Platichthys stellatus. The AFP gene arose from the functionally and structurally unrelated gig2 locus following gene duplication. The first to arise was the 3-kDa skin isoform and following tandem duplications to increase gene number, internal duplications of the 11-a.a. repeat and the gain of a signal sequence gave rise to a 32-kDa circulating isoform, Maxi. Later, a 3.3-kDa portion of Maxi became the dominant internally secreted isoform as a result of massive gene amplification. Present day starry flounder are found along the Pacific Rim from California to Japan. There is a strong correlation between latitude and AFP gene dosage as Alaskan fish have many AFP gene copies, Californian fish have only a few and fish from British Columbia have an intermediate number. Taken together, these results suggest that the ocean cooling that took place during the Cenozoic era led to AFP acquisition in numerous naïve fish lineages and that a decrease in gene copy number will be the