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Considerations of warming rates in cryopreservation
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
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