- Email: [email protected]
Novel approaches for cryopreservation: Meeting the needs for cellular therapy, tissue, and organ preservation
418
Abstracts / Cryobiology 73 (2016) 399e443
S071 LESS IS MORE: A NEW CRYOABLATION STRATEGY IN LUNG CANCER MANAGEMENT T. Zhou*, Q. Li, K. Hu. Beijing University of Chinese Medicine, Dongfang Hospital, Beijing, China * Corresponding author.
Lung cancer results in around 2 million deaths worldwide every year. About 70~80% of patients are in advanced-stage at diagnosis. According to NCCN Guidelines, the median overall survivals (MOS) following the standard therapeutic strategy are only 8~10 months. Modern cryoablation has been applied in cancer management for more than two decades. We reviewed 119 lung cancer patients in advanced-stage who received cryoablation with other therapies between 2005 and 2013. It is amazing that combining cryoablation with classic therapies could result in 19 months MOS. Apparently, cryoablation is a promising anticancer therapy. Following the surgical spirits, it seems natural to pursue “radical” in cryosurgery. Thus, most prevalent protocol is 2~3 freezing-thawing cycles with the thawing temperature 20~40 C. Dissimilarly, single cycle with the thawing temperature 0 C was adopted in our protocol, inspired by the concept “less is more” in Chinese Medicine Philosophy. So we conducted this study to determine which was better. In this study, forty-eight C57BL/ 6J mice were grafted GFP-labeled Lewis lung cancer and randomized into four groups. Mice in Group1 were given single freezing-thawing cycle with thawing temperature 0 C; Mice in Group2 and Group 3 were given double or triple cycles, respectively, with thawing temperature 70 C; no interventions for Group4. Fourteen days after the cryoablation, the tumor inhibition rates were 90.43%, 76.96% and 74.13% in the 3 Cryo-groups, respectively. The average tumor weight in Group2 was about twice as it was in Group1, and three times in Group3. The possibility of tumor shrinkage was 67% in Group1, but only 42% in Group2 or Group3. Lung metastases rate was only 17% in Group1 and Group2, contrasted to 25% in Group3 and 20% in the control group. This new strategy is less radical, much simpler, however, it shows better local and remote effects. Source of funding: This study was funded by Beijing University of Chinese Medicine (Grant No. 2013-XRCX-05). S072 MICROFLUIDIC TECHNOLOGIES FOR SELECTION OF CRYOPRESERVED OR UNPROCESSED SPERM U. Demirci*. Stanford University, Stanford, California, United States * Corresponding author.
Micro- and nano-scale technologies can have a significant impact on medicine and biology in the areas of cell manipulation, diagnostics, and monitoring. At the convergence of these new technologies and biology, we search for enabling solutions to the real world problems at the clinic. Emerging nano-scale and microfluidic technologies integrated with biology offer innovative possibilities for creating intelligent, mobile medical lab-chip devices that could transform fertility, diagnostics and monitoring, tissue engineering, and regenerative medicine. Human semen cryopreservation is an indispensable procedure for the management of male infertility. However, current cryopreservation procedures effect sperm in terms of motility, cell morphology, and DNA fragmentation due to the generation of reactive oxygen species. Further, the presence of cryoprotectants lead to alteration of sperm acrosomal content, in-homogeneous nucleus, and disorganized mitochondrial crests. Thus, isolation of healthy sperm from a cryopreserved semen is challenging. While rraditional sperm selection techniques, such as the swim-up and density gradient methods, have been widely used to isolate healthy sperm they are not efficient in isolating sperm with high motility and DNA integrity. Herein, addressing these challenges, we developed an innovative microfluidic-based sperm-sorting device which exploits inherent characteristics of sperm to isolate highly motile, morphologically viable, and sperm with reduced epigenetic abnormalities from the cryopreserved semen samples. We demonstrate that the sorted sperm showed higher motility (98%), trajectory kinetics, high DNA integrity, and reduced epigenetic
abnormalities compared to conventional methods. The microfluidic device has a significant potential to improve routine sperm sorting from both fresh and thawed semen samples. Since ARTs are labor intensive, such an easy-to-use device can assist the sperm selection process, reduce dependence on operator skills, and facilitate repeatable and reliable operational steps. In this talk, we will present an overview of our work in these areas focussed on applications in microfluidics and sperm selection. S073 ALGORITHM OPTIMIZATION OF CRYOPRESERVATION PROTOCOLS TO IMPROVE MESENCHYMAL STEM CELL FUNCTIONALITY K. Pollock*, J. Budenske, D. McKenna, P. Dosa, A. Hubel. University of Minnesota, College of Science and Engineering, Minneapolis, Minnesota, United States * Corresponding author.
Mesenchymal stem cells (MSCs) are used clinically for the treatment of an increasing number of diseases. Cryopreservation of these cells in dimethyl sulfoxide (Me2SO) allows for storage and transport to more places and patients, but can result in poor cell function and adverse reactions upon infusion. There is a need for cryopreservation solutions that maintain post thaw function, but are free of Me2SO. We hypothesize that multiple additives will be needed to replace Me2SO, but optimization of a multicomponent solution can require thousands of experiments. Implementation of a differential evolution (DE) algorithm permits optimization of multicomponent freezing solutions in a rational, accelerated fashion. Mesenchymal stem cells were combined with algorithm dictated solutions of sucrose, glycerol, and creatine or isoleucine (SGC/SGI), incubated for 1 hour, and frozen in a controlled rate freezer at 3 C/min. Samples were thawed and assessed for live cell recovery and attachment, and attachment results were iterated back to the algorithm to dictate the next generation of solutions to test. Cells frozen in SGC/SGI solutions exhibited improved recovery and attachment after multiple iterations of the algorithm, and displayed significantly improved attachment compared to Me2SO (p <0.05). Fresh cells as well as cells frozen in algorithm-optimized solutions of SGC/SGI or Me2SO were assessed quantitatively for actin expression and alignment. Actin alignment was significantly worse in Me2SO frozen cells vs SGC/SGI frozen cells and fresh cells (p<0.05). Proliferation did not vary significantly among the frozen samples tested. This technique can be applied to functionally optimize freezing conditions, which vary by cell type, with significantly fewer experiments than traditional methods. Ultimately, these optimization strategies can improve cellular preservation to advance the quality of patient care. Source of funding: NIH Grant 1 R21 EB016247-01A1, University of Minnesota Doctoral Dissertation Fellowship, 3M Science and Technology Fellowship S074 NOVEL APPROACHES FOR CRYOPRESERVATION: MEETING THE NEEDS FOR CELLULAR THERAPY, TISSUE, AND ORGAN PRESERVATION R. Ben. University of Ottawa, Ottawa, Ontario, Canada Cell-based therapies are rapidly emerging as a critical aspect of modern health care and have shaped new models of care in stem cell therapy, regenerative medicine, and transfusion practices. Novel cell therapies, such as CAR-Ts and mesenchymal adjuvant therapies are nearing full clinical and commercial utility. Cryopreservation is becoming increasing important for these therapies because it will greatly increase the manufacturing, management, and deployment of these products. However, current cryopreservation protocols are suboptimal and improved cryoprotective agents (CPAs) and protocols are urgently required. During the past fifty years, the field of modern cryobiology has seen tremendous advances and much information has been gained with respect to the physical and biochemical effects when freezing cells, tissues, and organs. Despite this understanding, ice formation is the major cause for decreased post-thaw viability and impaired cellular function. While currently utilized CPAs function via a number of different mechanisms, they all fail to control ice growth during cryopreservation and prevent subsequent cellular injury
Abstracts / Cryobiology 73 (2016) 399e443
generating a “bottle neck” for cellular therapies as well as tissue and organ preservation. The Ben laboratory has discovered several classes of ice recrystallization inhibitors that control ice growth during cryopreservation and increase the post-thaw viability and functionality of human red blood cells, hematopoietic stem cells from umbilical cord blood, and hepatocytes. These compounds have also proven effective in primary endothelial cells where they mitigate cellular damage from intracellular ice recrystallization during cryopreservation at high subzero temperatures. This finding is the first critical step towards using these compounds for the successful cryopreservation of complex tissues and perhaps organs. S075 AVOIDING THE ICE CRYOLESION IN TISSUE CRYOPRESERVATION WITH ICE MODULATORS M. Taylor 1,2, 3, *, Z. Chen 1, C. Crossley 1, E. Greene 1, L. Campbell 1, K. Brockbank 1, Y. Rabin 3. 1 Tissue Testing Technologies LLC, United States; 2 Sylvatica Biotech Inc., Charleston, South Carolina, United States; 3 Carnegie Mellon University, Pittsburgh, Pennsylvania, United States * Corresponding author. Tissue Testing Technologies LLC, United States.
Effective methods of cryopreservation that maintain the function and structural integrity of integrated tissues will not only be realized by effectively controlling ice formation, but must also avoid structural damage due to thermo-mechanical stresses. Building upon published prior work this study focused on defining the structural and functional profile of blood vessels vitrified under marginal conditions of cooling and warming as a function of the system volume and cryoprotectant solution design. A cryomacroscope (Type III) was used to vitrify carotid artery segments in DP6 solutions containing a range of synthetic ice modulators (SIMs) using previously determined marginal thermal conditions for vitrification. The application of SIMs enables lowering the CPA concentration, thereby reducing the toxicity potential, while decreasing the critical cooling and rewarming rates, thereby reducing the risk of structural destruction to the tissue as a consequence of thermo-mechanical stress. Using our established baseline for respectable recovery of function of blood vessels after vitrification in small volumes (1 ml), we determine the outcome of scaleup by orders of magnitude as a crucial step towards successful vitreous cryopreservation of bulky tissues and organs. The impact of scaling up by 400 and 1000% was assessed using glass and plastic specimen vials. At 400% scaling in glass vials > 70% metabolic recovery was achieved for carotid artery segments by the alamarBlue assay and >50% by smooth muscle physiology (contractility). At 1000% it was demonstrated that material properties of the glasses formed result in fracturing at this volume that correlates with losses in functional recovery. Fracturing, prevalent when samples were vitrified in glass containers was avoided in plastic containers with concomitant retention of tissue function. This emphasizes that material properties of the packaging as well as the nature of the vitrification solution impacts the risks of thermo-mechanical stresses and functional recovery of the product. Conflict of interest: All authors except Dr. Yoed Rabin are either full or part-time employees of T3-Tissue Testing Technologies, LLC. Source of funding: This work was supported in part by The Department of Defense DHP-H151-013-0162 SBIR Phase I and NIH, NHLBI, R01HL127618. S076 LASER CONTACTLESS CRYOPRESERVATION
ICE
NUCLEATION
FOR
STEM
CELL
L. Lauterboeck 1, *, W. Wieprecht 2, S. Kabelac 1, B. Glasmacher 1. 1 Leibniz University Hannover, Institute for Multiphase Processes, Hannover, Germany; 2 University of Technology Cottbus, Atmospheric Chemistry and Air Quality, Cottbus, Germany * Corresponding author.
Induced nucleation is used in freezing cells in reproductive medicine and transfusion medicine and has been applied to sperm, oocytes, stem cells, and red blood cells. However, all current induction methods need a direct contact with the sample, such as crystalline cholesterol or sample
419
container, such as cryovials. These methods have the potential to compromise the sample through contamination. Thus, contactless methods such as ultrasound or laser induced nucleation offer high potential. A device was developed that uses a highly focused, 532 nm, nanosecond pulsed laser beam that can induce nucleation at the desired temperature in cryovials. The challenge in this project was to induce nucleation inside a standard cryovial without damaging the vial or cells. The device was validated by comparing electro-freezing, seeding, and laser induced nucleation in multipotent stromal cells (MSCs) of the common marmoset monkey from two different origins (amnion and bone marrow) by determining efficiency of re-cultivation and metabolic activity. No negative effect on cell survival was found when cells were exposed to laser light up to 48 hours. At all tested nucleation temperatures between -4 C and -14 C the amount of successfully induced samples was higher for laser induced nucleation as compared to electro-freezing induction. MSCs had the highest outcome after freezing when the samples were nucleated at -10 C using 5% dimethyl sulfoxide and a two-step freezing protocol (7.5 C/min to -30 C followed by 3 C/min to -80 C). This was true for all three applied nucleation methods and for both analysed stem cell types. To sum up, a contactless method for ice nucleation using green laser light does not harm the stem cells and leads to high cryopreservation outcome. S077 ARTIFICIAL HYPOXIA INDUCIBLE FACTOR-1a (HIF-1a) STABILIZATION CAN ENHANCE CELL AND TISSUE CONSTRUCT SURVIVAL W. Ho*, B. Fuller, G. Jell. University College London, London, United Kingdom * Corresponding author.
Stem cell therapy is a promising technology that has the potential to revolutionise modern medicine. There are, however, challenges associated with cell survival and phenotype stability during cryopreservation, transportation and following implantation. Strategies to increase cell survival from resuscitation and after implantation would enhance the translation potential of cell therapy. Current research has demonstrated that placing cells in mild hypoxia insult can enhance cellular adaptation to adverse environments. The success of hypoxia preconditioning is, however, curtailed by the lack of oxygen on cell growth, phenotype stability, and reoxygenation injury. Cell survival may be artificially increased by stabilising HIF-1a through the use of HIF mimetics, in normoxic, prior to implantation or storage. Stabilising HIF-1a in normoxia may activate several pro-survival factors whilst preventing the oxidative stress associated with hypoxia-reoxygenation injury. In a model of adverse conditions (Saltron perfusion fluid at 4 C for 24 hours), we demonstrated that HepG2 (liver cancerous cells) had increased survival and a higher proliferation rate following resuscitation when exposed to HIF mimetics during cold storage (100 mM Co ions and 250 mM DMOG). Following resuscitation, the cells exposed to the HIF mimetics in normoxia had increased survival (P ¼ <0.01 for Co ions and P ¼ <0.01 for DMOG) and increased proliferation (as determined by total DNA) up to 7 days, (P ¼ <0.05). A similar result was observed in ADMSCs (Adipose Mesenchyme Stem Cell) when incubated in the same dosage of hypoxia mimetics. These results demonstrate that hypoxia mimetics increased the cell survival in adverse conditions e.g. cold storage at 4 C. Current studies are underway to understand the mechanism of this increased survival and resuscitation pro-survival factors expressed in HIF stabilised cells, including the role of ROS and Heat Shock Proteins. The incorporation of HIF mimetics into tissue scaffolds may provide a means to increase cell survival following implantation. S078 TRANSCRIPTOMIC PROFILING REVEALED THE REGULATORY MECHANISM OF ARABIDOPSIS RESPONSE TO OXIDATIVE STRESS FROM CRYOPRESERVATION X. Shen. Shanghai Jiao Tong University, School of Agriculture and Biology, Minhang, Shanghai, China The 48-h Arabidopsis seedlings after germination could be restored, whereas 72-h seedlings died after cryopreservation. To understand the mechanism of this correlation between the seedling age and survival,
Abstracts / Cryobiology 73 (2016) 399e443
S071 LESS IS MORE: A NEW CRYOABLATION STRATEGY IN LUNG CANCER MANAGEMENT T. Zhou*, Q. Li, K. Hu. Beijing University of Chinese Medicine, Dongfang Hospital, Beijing, China * Corresponding author.
Lung cancer results in around 2 million deaths worldwide every year. About 70~80% of patients are in advanced-stage at diagnosis. According to NCCN Guidelines, the median overall survivals (MOS) following the standard therapeutic strategy are only 8~10 months. Modern cryoablation has been applied in cancer management for more than two decades. We reviewed 119 lung cancer patients in advanced-stage who received cryoablation with other therapies between 2005 and 2013. It is amazing that combining cryoablation with classic therapies could result in 19 months MOS. Apparently, cryoablation is a promising anticancer therapy. Following the surgical spirits, it seems natural to pursue “radical” in cryosurgery. Thus, most prevalent protocol is 2~3 freezing-thawing cycles with the thawing temperature 20~40 C. Dissimilarly, single cycle with the thawing temperature 0 C was adopted in our protocol, inspired by the concept “less is more” in Chinese Medicine Philosophy. So we conducted this study to determine which was better. In this study, forty-eight C57BL/ 6J mice were grafted GFP-labeled Lewis lung cancer and randomized into four groups. Mice in Group1 were given single freezing-thawing cycle with thawing temperature 0 C; Mice in Group2 and Group 3 were given double or triple cycles, respectively, with thawing temperature 70 C; no interventions for Group4. Fourteen days after the cryoablation, the tumor inhibition rates were 90.43%, 76.96% and 74.13% in the 3 Cryo-groups, respectively. The average tumor weight in Group2 was about twice as it was in Group1, and three times in Group3. The possibility of tumor shrinkage was 67% in Group1, but only 42% in Group2 or Group3. Lung metastases rate was only 17% in Group1 and Group2, contrasted to 25% in Group3 and 20% in the control group. This new strategy is less radical, much simpler, however, it shows better local and remote effects. Source of funding: This study was funded by Beijing University of Chinese Medicine (Grant No. 2013-XRCX-05). S072 MICROFLUIDIC TECHNOLOGIES FOR SELECTION OF CRYOPRESERVED OR UNPROCESSED SPERM U. Demirci*. Stanford University, Stanford, California, United States * Corresponding author.
Micro- and nano-scale technologies can have a significant impact on medicine and biology in the areas of cell manipulation, diagnostics, and monitoring. At the convergence of these new technologies and biology, we search for enabling solutions to the real world problems at the clinic. Emerging nano-scale and microfluidic technologies integrated with biology offer innovative possibilities for creating intelligent, mobile medical lab-chip devices that could transform fertility, diagnostics and monitoring, tissue engineering, and regenerative medicine. Human semen cryopreservation is an indispensable procedure for the management of male infertility. However, current cryopreservation procedures effect sperm in terms of motility, cell morphology, and DNA fragmentation due to the generation of reactive oxygen species. Further, the presence of cryoprotectants lead to alteration of sperm acrosomal content, in-homogeneous nucleus, and disorganized mitochondrial crests. Thus, isolation of healthy sperm from a cryopreserved semen is challenging. While rraditional sperm selection techniques, such as the swim-up and density gradient methods, have been widely used to isolate healthy sperm they are not efficient in isolating sperm with high motility and DNA integrity. Herein, addressing these challenges, we developed an innovative microfluidic-based sperm-sorting device which exploits inherent characteristics of sperm to isolate highly motile, morphologically viable, and sperm with reduced epigenetic abnormalities from the cryopreserved semen samples. We demonstrate that the sorted sperm showed higher motility (98%), trajectory kinetics, high DNA integrity, and reduced epigenetic
abnormalities compared to conventional methods. The microfluidic device has a significant potential to improve routine sperm sorting from both fresh and thawed semen samples. Since ARTs are labor intensive, such an easy-to-use device can assist the sperm selection process, reduce dependence on operator skills, and facilitate repeatable and reliable operational steps. In this talk, we will present an overview of our work in these areas focussed on applications in microfluidics and sperm selection. S073 ALGORITHM OPTIMIZATION OF CRYOPRESERVATION PROTOCOLS TO IMPROVE MESENCHYMAL STEM CELL FUNCTIONALITY K. Pollock*, J. Budenske, D. McKenna, P. Dosa, A. Hubel. University of Minnesota, College of Science and Engineering, Minneapolis, Minnesota, United States * Corresponding author.
Mesenchymal stem cells (MSCs) are used clinically for the treatment of an increasing number of diseases. Cryopreservation of these cells in dimethyl sulfoxide (Me2SO) allows for storage and transport to more places and patients, but can result in poor cell function and adverse reactions upon infusion. There is a need for cryopreservation solutions that maintain post thaw function, but are free of Me2SO. We hypothesize that multiple additives will be needed to replace Me2SO, but optimization of a multicomponent solution can require thousands of experiments. Implementation of a differential evolution (DE) algorithm permits optimization of multicomponent freezing solutions in a rational, accelerated fashion. Mesenchymal stem cells were combined with algorithm dictated solutions of sucrose, glycerol, and creatine or isoleucine (SGC/SGI), incubated for 1 hour, and frozen in a controlled rate freezer at 3 C/min. Samples were thawed and assessed for live cell recovery and attachment, and attachment results were iterated back to the algorithm to dictate the next generation of solutions to test. Cells frozen in SGC/SGI solutions exhibited improved recovery and attachment after multiple iterations of the algorithm, and displayed significantly improved attachment compared to Me2SO (p <0.05). Fresh cells as well as cells frozen in algorithm-optimized solutions of SGC/SGI or Me2SO were assessed quantitatively for actin expression and alignment. Actin alignment was significantly worse in Me2SO frozen cells vs SGC/SGI frozen cells and fresh cells (p<0.05). Proliferation did not vary significantly among the frozen samples tested. This technique can be applied to functionally optimize freezing conditions, which vary by cell type, with significantly fewer experiments than traditional methods. Ultimately, these optimization strategies can improve cellular preservation to advance the quality of patient care. Source of funding: NIH Grant 1 R21 EB016247-01A1, University of Minnesota Doctoral Dissertation Fellowship, 3M Science and Technology Fellowship S074 NOVEL APPROACHES FOR CRYOPRESERVATION: MEETING THE NEEDS FOR CELLULAR THERAPY, TISSUE, AND ORGAN PRESERVATION R. Ben. University of Ottawa, Ottawa, Ontario, Canada Cell-based therapies are rapidly emerging as a critical aspect of modern health care and have shaped new models of care in stem cell therapy, regenerative medicine, and transfusion practices. Novel cell therapies, such as CAR-Ts and mesenchymal adjuvant therapies are nearing full clinical and commercial utility. Cryopreservation is becoming increasing important for these therapies because it will greatly increase the manufacturing, management, and deployment of these products. However, current cryopreservation protocols are suboptimal and improved cryoprotective agents (CPAs) and protocols are urgently required. During the past fifty years, the field of modern cryobiology has seen tremendous advances and much information has been gained with respect to the physical and biochemical effects when freezing cells, tissues, and organs. Despite this understanding, ice formation is the major cause for decreased post-thaw viability and impaired cellular function. While currently utilized CPAs function via a number of different mechanisms, they all fail to control ice growth during cryopreservation and prevent subsequent cellular injury
Abstracts / Cryobiology 73 (2016) 399e443
generating a “bottle neck” for cellular therapies as well as tissue and organ preservation. The Ben laboratory has discovered several classes of ice recrystallization inhibitors that control ice growth during cryopreservation and increase the post-thaw viability and functionality of human red blood cells, hematopoietic stem cells from umbilical cord blood, and hepatocytes. These compounds have also proven effective in primary endothelial cells where they mitigate cellular damage from intracellular ice recrystallization during cryopreservation at high subzero temperatures. This finding is the first critical step towards using these compounds for the successful cryopreservation of complex tissues and perhaps organs. S075 AVOIDING THE ICE CRYOLESION IN TISSUE CRYOPRESERVATION WITH ICE MODULATORS M. Taylor 1,2, 3, *, Z. Chen 1, C. Crossley 1, E. Greene 1, L. Campbell 1, K. Brockbank 1, Y. Rabin 3. 1 Tissue Testing Technologies LLC, United States; 2 Sylvatica Biotech Inc., Charleston, South Carolina, United States; 3 Carnegie Mellon University, Pittsburgh, Pennsylvania, United States * Corresponding author. Tissue Testing Technologies LLC, United States.
Effective methods of cryopreservation that maintain the function and structural integrity of integrated tissues will not only be realized by effectively controlling ice formation, but must also avoid structural damage due to thermo-mechanical stresses. Building upon published prior work this study focused on defining the structural and functional profile of blood vessels vitrified under marginal conditions of cooling and warming as a function of the system volume and cryoprotectant solution design. A cryomacroscope (Type III) was used to vitrify carotid artery segments in DP6 solutions containing a range of synthetic ice modulators (SIMs) using previously determined marginal thermal conditions for vitrification. The application of SIMs enables lowering the CPA concentration, thereby reducing the toxicity potential, while decreasing the critical cooling and rewarming rates, thereby reducing the risk of structural destruction to the tissue as a consequence of thermo-mechanical stress. Using our established baseline for respectable recovery of function of blood vessels after vitrification in small volumes (1 ml), we determine the outcome of scaleup by orders of magnitude as a crucial step towards successful vitreous cryopreservation of bulky tissues and organs. The impact of scaling up by 400 and 1000% was assessed using glass and plastic specimen vials. At 400% scaling in glass vials > 70% metabolic recovery was achieved for carotid artery segments by the alamarBlue assay and >50% by smooth muscle physiology (contractility). At 1000% it was demonstrated that material properties of the glasses formed result in fracturing at this volume that correlates with losses in functional recovery. Fracturing, prevalent when samples were vitrified in glass containers was avoided in plastic containers with concomitant retention of tissue function. This emphasizes that material properties of the packaging as well as the nature of the vitrification solution impacts the risks of thermo-mechanical stresses and functional recovery of the product. Conflict of interest: All authors except Dr. Yoed Rabin are either full or part-time employees of T3-Tissue Testing Technologies, LLC. Source of funding: This work was supported in part by The Department of Defense DHP-H151-013-0162 SBIR Phase I and NIH, NHLBI, R01HL127618. S076 LASER CONTACTLESS CRYOPRESERVATION
ICE
NUCLEATION
FOR
STEM
CELL
L. Lauterboeck 1, *, W. Wieprecht 2, S. Kabelac 1, B. Glasmacher 1. 1 Leibniz University Hannover, Institute for Multiphase Processes, Hannover, Germany; 2 University of Technology Cottbus, Atmospheric Chemistry and Air Quality, Cottbus, Germany * Corresponding author.
Induced nucleation is used in freezing cells in reproductive medicine and transfusion medicine and has been applied to sperm, oocytes, stem cells, and red blood cells. However, all current induction methods need a direct contact with the sample, such as crystalline cholesterol or sample
419
container, such as cryovials. These methods have the potential to compromise the sample through contamination. Thus, contactless methods such as ultrasound or laser induced nucleation offer high potential. A device was developed that uses a highly focused, 532 nm, nanosecond pulsed laser beam that can induce nucleation at the desired temperature in cryovials. The challenge in this project was to induce nucleation inside a standard cryovial without damaging the vial or cells. The device was validated by comparing electro-freezing, seeding, and laser induced nucleation in multipotent stromal cells (MSCs) of the common marmoset monkey from two different origins (amnion and bone marrow) by determining efficiency of re-cultivation and metabolic activity. No negative effect on cell survival was found when cells were exposed to laser light up to 48 hours. At all tested nucleation temperatures between -4 C and -14 C the amount of successfully induced samples was higher for laser induced nucleation as compared to electro-freezing induction. MSCs had the highest outcome after freezing when the samples were nucleated at -10 C using 5% dimethyl sulfoxide and a two-step freezing protocol (7.5 C/min to -30 C followed by 3 C/min to -80 C). This was true for all three applied nucleation methods and for both analysed stem cell types. To sum up, a contactless method for ice nucleation using green laser light does not harm the stem cells and leads to high cryopreservation outcome. S077 ARTIFICIAL HYPOXIA INDUCIBLE FACTOR-1a (HIF-1a) STABILIZATION CAN ENHANCE CELL AND TISSUE CONSTRUCT SURVIVAL W. Ho*, B. Fuller, G. Jell. University College London, London, United Kingdom * Corresponding author.
Stem cell therapy is a promising technology that has the potential to revolutionise modern medicine. There are, however, challenges associated with cell survival and phenotype stability during cryopreservation, transportation and following implantation. Strategies to increase cell survival from resuscitation and after implantation would enhance the translation potential of cell therapy. Current research has demonstrated that placing cells in mild hypoxia insult can enhance cellular adaptation to adverse environments. The success of hypoxia preconditioning is, however, curtailed by the lack of oxygen on cell growth, phenotype stability, and reoxygenation injury. Cell survival may be artificially increased by stabilising HIF-1a through the use of HIF mimetics, in normoxic, prior to implantation or storage. Stabilising HIF-1a in normoxia may activate several pro-survival factors whilst preventing the oxidative stress associated with hypoxia-reoxygenation injury. In a model of adverse conditions (Saltron perfusion fluid at 4 C for 24 hours), we demonstrated that HepG2 (liver cancerous cells) had increased survival and a higher proliferation rate following resuscitation when exposed to HIF mimetics during cold storage (100 mM Co ions and 250 mM DMOG). Following resuscitation, the cells exposed to the HIF mimetics in normoxia had increased survival (P ¼ <0.01 for Co ions and P ¼ <0.01 for DMOG) and increased proliferation (as determined by total DNA) up to 7 days, (P ¼ <0.05). A similar result was observed in ADMSCs (Adipose Mesenchyme Stem Cell) when incubated in the same dosage of hypoxia mimetics. These results demonstrate that hypoxia mimetics increased the cell survival in adverse conditions e.g. cold storage at 4 C. Current studies are underway to understand the mechanism of this increased survival and resuscitation pro-survival factors expressed in HIF stabilised cells, including the role of ROS and Heat Shock Proteins. The incorporation of HIF mimetics into tissue scaffolds may provide a means to increase cell survival following implantation. S078 TRANSCRIPTOMIC PROFILING REVEALED THE REGULATORY MECHANISM OF ARABIDOPSIS RESPONSE TO OXIDATIVE STRESS FROM CRYOPRESERVATION X. Shen. Shanghai Jiao Tong University, School of Agriculture and Biology, Minhang, Shanghai, China The 48-h Arabidopsis seedlings after germination could be restored, whereas 72-h seedlings died after cryopreservation. To understand the mechanism of this correlation between the seedling age and survival,