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Initiation of intracellular ice formation during freezing of micropatterned endothelial tissue
Abstracts / Cryobiology 73 (2016) 399e443
S015 FREEZE-DRIED DECELLULARIZED HEART VALVES FOR HEART VALVE REPLACEMENT W. Wolkers. Leibniz University Hannover, Hannover, Germany Decellularized heart valves can be used for heart valve replacement therapies. If heart valves could be freeze-dried without damaging the tissue structure, this would allow long-term storage at room temperature. We have used sucrose as lyoprotectant for freeze-drying of decellularized heart valves. Diffusion studies were done using Fourier transform infrared spectroscopy (FTIR) in order to estimate incubation times needed to infiltrate heart valves with sucrose. It was estimated that 4-hour incubation at 37 C is sufficient to infiltrate heart valves with sucrose. Sucrose protects decellularized heart valves against damage caused by freezedrying. Ice crystal damage was found to be one of the main damaging events during freeze-drying resulting in pores in the tissue after rehydration. The extent of pore formation in rehydrated tissue decreased with increasing sucrose concentration in the freeze-drying formulation. High sucrose concentrations were needed to minimize ice crystal damage. Exposure to an 80% sucrose solution prior to freeze-drying almost completely diminished pore formation and the matrix histoarchitecture of rehydrated tissue closely resembled that of control tissue not subjected to freeze-drying. The protein denaturation profile of rehydrated tissue was found to be nearly identical compared to that of control tissue. The overall protein secondary structure, studied by FTIR, also showed that tissue proteins were little affected by the freeze-drying process. In vivo tests with freeze-dried pulmonary heart valves (ovine as well as porcine origin) in sheep demonstrated the efficacy of freeze-dried valves. Freeze-dried valves showed similar durability and repopulation with cells compared to control valves not exposed to freeze-drying. Source of funding: DFG: Cluster of Excellence REBIRTH “From regenerative biology to reconstructive therapy” S016 ICE-FREE CRYOPRESERVATION OF NATURAL AND ENGINEERED TISSUES K.G.M. Brockbank. Tissue Testing Technologies LLC, Charleston, South Carolina, United States Natural and tissue engineered allogeneic tissues potentially have a huge impact on orthopedic, wound care, urinary, cardiac, and vascular surgery applications. Preserved tissues using traditional freezing methods of cryopreservation are subject to ice damage. We have developed methods for preservation using ice-free vitrification. The original vitrification protocol has evolved over time. Ice-free cryopreservation using VS55 with multistep addition/removal steps and storage below -135 C is limited to small samples where relatively rapid cooling and warming rates are possible. This method typically provides extracellular matrix preservation with at least 80% cell viability and tissue function compared with fresh untreated tissues. It has proven effective on natural cardiovascular tissue samples and cartilage, and a variety of engineered tissues including blood vessel and epithelial tissue constructs. In contrast, preservation of larger samples with retention of cell viability has been limited due to cryoprotectant cytotoxicity, inability to rewarm fast enough to avoid recrystallization, and stresses that occur during vitrification. Initially, for large tissues where the focus is preservation of extracellular matrix without cell viability, we employed high concentration, ±83%, cryoprotectant formulations. Since cell viability is not retained these methods employ simpler addition and removal protocols with long term storage at -80 C. In vitro experiments indicate that ice-free cryopreservation impacts the innate immune response reducing T-cell proliferation and release of cytokines from peripheral blood mononuclear cells. In vivo functions are also improved. The field is returning to development of methods for viable large tissues. This is due to new technologies, including analytical methods such as cryomacroscopy, micro-computed tomography imaging and micro-magnetic resonance imaging, as well as innovative rewarming methods such as “nanowarming” where low radiofrequency alternating magnetic fields and Fe nanoparticles are used to warm rapidly. These technologies promise to facilitate the development of methods for retention of function in large tissues and organs.
403
Conflict of interest: Kelvin Brockbank is an employee and owner of Tissue Testing Technologies LLC. Source of funding: The National Institutes of Health grants 5R44GM106732, 1R43HL120404, and 1R43AI114486; and the US Army Medical Research and Materiel Command under Contract W81XWH-15-C0173. The views, opinions, and/or findings contained in this report are those of the author and should not be construed as an official National Institutes of Health or Department of the Army position, policy, or decision unless so designated by other documentation. S017 QUALITY AND IMMUNOGENICITY OF SKIN TISSUE ALLOGRAFTS FOR TRANSPLANT H. Manhani 1, A. Halpin 1, L. Hidalgo 1, B. Motyka 1, J. Pearcey 1, L. West 1, T. K. Worton 2, M. Bentley 2, G. Dowling 2, J. Mokoena 2, Holovati 1,2, *. 1 University of Alberta, Edmonton, Alberta, Canada; 2 Alberta Health Services, Edmonton, Alberta, Canada * Corresponding author. University of Alberta, Edmonton, Alberta, Canada.
Skin allografts are currently recovered, processed, and distributed by the Comprehensive Tissue Centre (CTC) for transplant applications. The aim of this study was to evaluate the effects of refrigeration, cryopreservation, long-term storage, and glutaraldehyde treatment on structural integrity, cellular viability and alloimmunogenic antigen expression of skin allografts distributed by CTC for transplant applications. Cadaveric skin tissue was recovered according to CTC standard operating procedures. After 14 days of refrigerated storage, skin allografts were cryopreserved in 10% dimethyl sulfoxide and stored in liquid nitrogen for up to 7 years. Glutaraldehyde (0.1%) treatment was either applied pre-freeze or postthaw. Allograft structural integrity was assessed by H&E staining and brightfield microscopy. The viability of allografts was assessed by mitochondrial cell viability assay (MTT). The immunogenicity potential of skin allografts was assessed by immunohistochemistry (IHC) staining for ABH and HLA Class I antigenic expression. Histological assessment of skin allografts revealed no statistically significant difference in microanatomy quality scores of refrigerated, cryopreserved, and glutaraldehyde treated skin allografts. Cellular viability of skin allografts significantly decreases during refrigerated storage (p<0.001), while the cryopreservation process maintains the pre-freeze cell viability levels. The length of liquid nitrogen storage was not a statistically significant predictor of post-thaw skin viability. IHC revealed that ABOeA or B, and HLA class I antigens are expressed in all experimental groups of skin allografts. There was no statistically significant effect of glutaraldehyde treatment on skin structural quality, viability or immunogenicity. CTC skin processing procedures maintain allograft structural integrity, for both refrigerated and cryopreserved tissue allografts. The length of hypothermic storage prior to freezing is an important parameter to consider for maintaining tissue viability properties. The length of cryopreservation storage for up to 7 years in liquid nitrogen temperatures does not negatively influence skin structural integrity or viability. Source of funding: Alberta Health Services S018 INITIATION OF INTRACELLULAR ICE FORMATION DURING FREEZING OF MICROPATTERNED ENDOTHELIAL TISSUE S.E. Harhen, J.O.M. Karlsson*. Department of Mechanical Engineering, Villanova University, Villanova, Pennsylvania, United States * Corresponding author.
Previous models of intracellular ice formation (IIF) in monoculture tissue have assumed that all cells within an unfrozen tissue construct are subject to the same mechanisms of IIF initiation (such as the recently discovered IIF initiation mechanism associated with the paracellular ice penetration phenomenon), implying that the probability of IIF is the same in every cell within an unfrozen construct, and independent of tissue scale. If this assumption is valid, then it is possible to predict IIF kinetics in macroscale tissue using scaling laws to extrapolate measurements from microscale
404
Abstracts / Cryobiology 73 (2016) 399e443
constructs (e.g., micropatterned cells and cell pairs). The hypothesis has previously been validated in paucicellular 1D arrangements of up to four cells, but has not been extensively tested in 2D constructs. We now report observations of IIF initiation in 2D tissue consisting of bovine pulmonary artery endothelial cells adherent to micropatterned circular fibronectin islands of diameter 80, 140, and 225 mm; the largest constructs contained on the order of 50 cells each. The cells were cultured for 24 h after attachment to the substrate, before use in high-speed video cryomicroscopy experiments. Video was acquired at an image acquisition rate of 2000 frames/s during freezing at a constant cooling rate of 150 C/min, in the presence of extracellular ice. The cell-specific hazard rate for the initiating IIF event in 80-, 140-, and 225-mm islands 0.059 ± 0.003, 0.021 ± 0.001, and 0.011 ± 0.002 events/s/cell, respectively. The results indicate that IIF initiation kinetics are not independent of construct size, and that the spatial distribution of IIF initiation sites is not uniform within a construct of a given size. Source of funding: This work was funded in part by the Villanova University College of Engineering and Department of Mechanical Engineering, as well as National Science Foundation Award No. CBET-0954587. S019 RFTCIVIVSCM FOR CRYOPRESERVATION OF HUMAN ADIPOSE TISSUE AND MESENCHYMAL STROMAL CELLS S. Sumida 1, *, A. Rowe 2. 1 Sajio Sumida Clinic, Ginza, Tokyo, Japan; 2 New York University, School of Medicine, New York, New York, United States * Corresponding author.
Human adipose tissue will be a precious source of MSCs for regenerative therapy, however problems in tissue culture remain as for foreign proteins, recombinant stimulants, and the resources for MSCs. Venous wall and the perivascular “adipose” explants (0.1~0.2 gr) obtained by surgery (93 cases) were cultured in a special media including human sera, human platelet lysate, and limulus (bradytelic species) lysate (for limited cases) in a 5% CO2, -37 C, and formed confluent colonies of MSCs in 12.4 ± 3.8 (n ¼ 29) for venous wall, and in 7.1 ± 3 days (n ¼ 19) for adipose tissue. On the other hand, frozen adipose explants in 10% Me2SO in IMDM formed the MSCs colonies overall in 14.5 ± 6.5 days (n ¼ 16). Interestingly, a human skin with adipose tissues (1015around 5 mm thickness) which had been frozen in a 15% glycerol in TC199 at -196 C for 33 years gave buds of fibroblast-mesenchymal cell in 4 days and formed confluent in 11 days. Thus, thawed adipose tissue explants, either non-frozen or frozen, mostly formed MSCs confluent colonies in two weeks. The confluent MSCs were detached by 0.1% trypsin-IMDM or PBS-D solution, and cryopreserved again by RFTIVIVSCM (repetitive freeze-thaw-culture in vivo and in vitro subculture method) for days or months. The thawed MSCs were possible to form confluent colonies of MSCs as well as for the thawed bone marrow stromal cells reported previously. Limulus lysate suppressed the appearance of MSCs either of non-frozen or frozen adipose tissues in culture as well as in the result of bone marrow stem cells. The thawed adipose tissue and vein explants formed MSCs-confluent by culture in 14~40 days. The adipose tissues cryopreserved at -196 C for 33 years also budded fibroblast-like MSCs and formed confluent by culture after thawing. These adipocyte derived MSCs could be preserved by RFTCIVIVSCM and formed passage-2 confluent colonies. S020 CRYOPRESERVATION OF SHOOT TIPS OF FRAGARIA SPP. AND VIRUS ELIMINATION THROUGH CRYOTHERAPY S. Gupta. Tissue Culture and Cryopreservation Unit (TCCU), Indian Council of Agricultural Research-National Bureau of Plant Genetic Resources (ICARNBPGR), New Delhi, India Genus Fragaria L. of family Rosaceae is a native to temperate zones of northern hemisphere and South America. It can occur up to 4000 m altitude. Cultivars of Fragaria x ananassa Duch., the most common cultivated strawberry, is grown around the world in suitable temperate climate. The crop is economically important with the world production 4,594,539 MT in
2011. USA is the top producer of strawberries followed by European countries while many other countries are trying to increase production. Strawberries are produced in 73 countries worldwide. Strawberry is rich in vitamin C and a good source of manganese, and vitamin B, E, K and dietary minerals. It is consumed fresh as well as processed. Fragaria spp. is found growing wild in the Indian Himalayas and Nilgiri hills. Mostly, exotic cultivars are in cultivation. Conservation of cultivated and wild species is important for future strawberry improvement. Apart from ex situ conservation of Fragaria germplasm in the field, under screen, in green house, in vitro cultures, the cryopreservation of plant propagules is the only available method for long-term conservation. Virus-free germplasm should be conserved in the in vitro genebank and cryobank. In the literature, more than 30 viruses and virus-like disease have been reported in the strawberry. Cryotherapy is one of the best known methods used for virus elimination in vitro. In vitro genebank in NBPGR, India is conserving more than 80 accessions of Fragaria including exotic accessions procured from USDA-ARS-NCGR, Corvallis, Oregon, USA along with the several accessions collected from field genbanks in India. In vitro-grown shoot tips are used for cryopreservation as long term conservation strategy. The meristem, the ideal explant for virus elimination, is being used for cryotherapy. Research has been undergoing in our lab on cryopreservation. The outcome of the research work will be discussed in this paper. S021 OXIDATIVE STRESS IS CLOSELY RELATED TO THE POLLEN VIABILITY REDUCTION AFTER CRYOPRESERVATION M. Jia*, Y. Shi, Y. Liu, X. Jiang, J. Peng, W. Di. Beijing Forestry University, Beijing, China * Corresponding author.
Oxidative stress induced by excessive accumulation of reactive oxidative species (ROS) during cryopreservation may be another major contributing factor for cryoinjury of biological materials. In order to explore the role of oxidative stress in the cryopreservation of plant pollen, the germination capacity, ROS level, and malondialdehyde (MDA) content of pollens before and after cryopreservation from 20 common ornamental plants were comparatively studied. The results showed that germinability of cryopreserved pollen from 12 out of 20 species showed no significant difference with fresh pollen (group 1), only 1 increased, while the other 7 declined significantly (group 2). ROS in cryopreserved pollens from 75% species in group 1 remained unchanged compared to fresh pollen, 85.7% species in group 2 increased significantly, suggesting that pollen viability was negatively correlated to ROS. MDA content in pollens after cryopreservation from 66.7% species in group 1 showed no significant difference from fresh pollen, 85.7% species in group 2 rose significantly, which suggested that pollen viability and MDA level were also negatively correlated. Additionally, both ROS and MDA levels in pollens from 71.4% of species in group 2 increased significantly. In conclusion, plant pollens from most species possess a certain cryostorage tolerance, but pollens from some species are damaged severely. Pollen viability is closely related to the level of MDA and ROS. And, it shows a high correlation between ROS and MDA level as well. In addition, oxidative stress in pollens can be induced by cryostorage in liquid nitrogen, and is the main cause for the viability loses following cryopreservation. Source of funding: This work was supported by the National Natural Science Foundation of China (No. 31370693). S022 CELLULAR STRESS RESPONSES DURING CRYO-INDUCED STRESS IN ARABIDOPSIS SHOOT TIPS J. Stock*, M. Nagel, E.R.J. Keller, H.P. Mock. Leibniz Institute of Plant Genetics and Crop Plant Research, Seeland, Germany * Corresponding author.
During cryopreservation, meristematic tissues have to cope with different stresses ranging from desiccation to cold stress and rehydration. As current
S015 FREEZE-DRIED DECELLULARIZED HEART VALVES FOR HEART VALVE REPLACEMENT W. Wolkers. Leibniz University Hannover, Hannover, Germany Decellularized heart valves can be used for heart valve replacement therapies. If heart valves could be freeze-dried without damaging the tissue structure, this would allow long-term storage at room temperature. We have used sucrose as lyoprotectant for freeze-drying of decellularized heart valves. Diffusion studies were done using Fourier transform infrared spectroscopy (FTIR) in order to estimate incubation times needed to infiltrate heart valves with sucrose. It was estimated that 4-hour incubation at 37 C is sufficient to infiltrate heart valves with sucrose. Sucrose protects decellularized heart valves against damage caused by freezedrying. Ice crystal damage was found to be one of the main damaging events during freeze-drying resulting in pores in the tissue after rehydration. The extent of pore formation in rehydrated tissue decreased with increasing sucrose concentration in the freeze-drying formulation. High sucrose concentrations were needed to minimize ice crystal damage. Exposure to an 80% sucrose solution prior to freeze-drying almost completely diminished pore formation and the matrix histoarchitecture of rehydrated tissue closely resembled that of control tissue not subjected to freeze-drying. The protein denaturation profile of rehydrated tissue was found to be nearly identical compared to that of control tissue. The overall protein secondary structure, studied by FTIR, also showed that tissue proteins were little affected by the freeze-drying process. In vivo tests with freeze-dried pulmonary heart valves (ovine as well as porcine origin) in sheep demonstrated the efficacy of freeze-dried valves. Freeze-dried valves showed similar durability and repopulation with cells compared to control valves not exposed to freeze-drying. Source of funding: DFG: Cluster of Excellence REBIRTH “From regenerative biology to reconstructive therapy” S016 ICE-FREE CRYOPRESERVATION OF NATURAL AND ENGINEERED TISSUES K.G.M. Brockbank. Tissue Testing Technologies LLC, Charleston, South Carolina, United States Natural and tissue engineered allogeneic tissues potentially have a huge impact on orthopedic, wound care, urinary, cardiac, and vascular surgery applications. Preserved tissues using traditional freezing methods of cryopreservation are subject to ice damage. We have developed methods for preservation using ice-free vitrification. The original vitrification protocol has evolved over time. Ice-free cryopreservation using VS55 with multistep addition/removal steps and storage below -135 C is limited to small samples where relatively rapid cooling and warming rates are possible. This method typically provides extracellular matrix preservation with at least 80% cell viability and tissue function compared with fresh untreated tissues. It has proven effective on natural cardiovascular tissue samples and cartilage, and a variety of engineered tissues including blood vessel and epithelial tissue constructs. In contrast, preservation of larger samples with retention of cell viability has been limited due to cryoprotectant cytotoxicity, inability to rewarm fast enough to avoid recrystallization, and stresses that occur during vitrification. Initially, for large tissues where the focus is preservation of extracellular matrix without cell viability, we employed high concentration, ±83%, cryoprotectant formulations. Since cell viability is not retained these methods employ simpler addition and removal protocols with long term storage at -80 C. In vitro experiments indicate that ice-free cryopreservation impacts the innate immune response reducing T-cell proliferation and release of cytokines from peripheral blood mononuclear cells. In vivo functions are also improved. The field is returning to development of methods for viable large tissues. This is due to new technologies, including analytical methods such as cryomacroscopy, micro-computed tomography imaging and micro-magnetic resonance imaging, as well as innovative rewarming methods such as “nanowarming” where low radiofrequency alternating magnetic fields and Fe nanoparticles are used to warm rapidly. These technologies promise to facilitate the development of methods for retention of function in large tissues and organs.
403
Conflict of interest: Kelvin Brockbank is an employee and owner of Tissue Testing Technologies LLC. Source of funding: The National Institutes of Health grants 5R44GM106732, 1R43HL120404, and 1R43AI114486; and the US Army Medical Research and Materiel Command under Contract W81XWH-15-C0173. The views, opinions, and/or findings contained in this report are those of the author and should not be construed as an official National Institutes of Health or Department of the Army position, policy, or decision unless so designated by other documentation. S017 QUALITY AND IMMUNOGENICITY OF SKIN TISSUE ALLOGRAFTS FOR TRANSPLANT H. Manhani 1, A. Halpin 1, L. Hidalgo 1, B. Motyka 1, J. Pearcey 1, L. West 1, T. K. Worton 2, M. Bentley 2, G. Dowling 2, J. Mokoena 2, Holovati 1,2, *. 1 University of Alberta, Edmonton, Alberta, Canada; 2 Alberta Health Services, Edmonton, Alberta, Canada * Corresponding author. University of Alberta, Edmonton, Alberta, Canada.
Skin allografts are currently recovered, processed, and distributed by the Comprehensive Tissue Centre (CTC) for transplant applications. The aim of this study was to evaluate the effects of refrigeration, cryopreservation, long-term storage, and glutaraldehyde treatment on structural integrity, cellular viability and alloimmunogenic antigen expression of skin allografts distributed by CTC for transplant applications. Cadaveric skin tissue was recovered according to CTC standard operating procedures. After 14 days of refrigerated storage, skin allografts were cryopreserved in 10% dimethyl sulfoxide and stored in liquid nitrogen for up to 7 years. Glutaraldehyde (0.1%) treatment was either applied pre-freeze or postthaw. Allograft structural integrity was assessed by H&E staining and brightfield microscopy. The viability of allografts was assessed by mitochondrial cell viability assay (MTT). The immunogenicity potential of skin allografts was assessed by immunohistochemistry (IHC) staining for ABH and HLA Class I antigenic expression. Histological assessment of skin allografts revealed no statistically significant difference in microanatomy quality scores of refrigerated, cryopreserved, and glutaraldehyde treated skin allografts. Cellular viability of skin allografts significantly decreases during refrigerated storage (p<0.001), while the cryopreservation process maintains the pre-freeze cell viability levels. The length of liquid nitrogen storage was not a statistically significant predictor of post-thaw skin viability. IHC revealed that ABOeA or B, and HLA class I antigens are expressed in all experimental groups of skin allografts. There was no statistically significant effect of glutaraldehyde treatment on skin structural quality, viability or immunogenicity. CTC skin processing procedures maintain allograft structural integrity, for both refrigerated and cryopreserved tissue allografts. The length of hypothermic storage prior to freezing is an important parameter to consider for maintaining tissue viability properties. The length of cryopreservation storage for up to 7 years in liquid nitrogen temperatures does not negatively influence skin structural integrity or viability. Source of funding: Alberta Health Services S018 INITIATION OF INTRACELLULAR ICE FORMATION DURING FREEZING OF MICROPATTERNED ENDOTHELIAL TISSUE S.E. Harhen, J.O.M. Karlsson*. Department of Mechanical Engineering, Villanova University, Villanova, Pennsylvania, United States * Corresponding author.
Previous models of intracellular ice formation (IIF) in monoculture tissue have assumed that all cells within an unfrozen tissue construct are subject to the same mechanisms of IIF initiation (such as the recently discovered IIF initiation mechanism associated with the paracellular ice penetration phenomenon), implying that the probability of IIF is the same in every cell within an unfrozen construct, and independent of tissue scale. If this assumption is valid, then it is possible to predict IIF kinetics in macroscale tissue using scaling laws to extrapolate measurements from microscale
404
Abstracts / Cryobiology 73 (2016) 399e443
constructs (e.g., micropatterned cells and cell pairs). The hypothesis has previously been validated in paucicellular 1D arrangements of up to four cells, but has not been extensively tested in 2D constructs. We now report observations of IIF initiation in 2D tissue consisting of bovine pulmonary artery endothelial cells adherent to micropatterned circular fibronectin islands of diameter 80, 140, and 225 mm; the largest constructs contained on the order of 50 cells each. The cells were cultured for 24 h after attachment to the substrate, before use in high-speed video cryomicroscopy experiments. Video was acquired at an image acquisition rate of 2000 frames/s during freezing at a constant cooling rate of 150 C/min, in the presence of extracellular ice. The cell-specific hazard rate for the initiating IIF event in 80-, 140-, and 225-mm islands 0.059 ± 0.003, 0.021 ± 0.001, and 0.011 ± 0.002 events/s/cell, respectively. The results indicate that IIF initiation kinetics are not independent of construct size, and that the spatial distribution of IIF initiation sites is not uniform within a construct of a given size. Source of funding: This work was funded in part by the Villanova University College of Engineering and Department of Mechanical Engineering, as well as National Science Foundation Award No. CBET-0954587. S019 RFTCIVIVSCM FOR CRYOPRESERVATION OF HUMAN ADIPOSE TISSUE AND MESENCHYMAL STROMAL CELLS S. Sumida 1, *, A. Rowe 2. 1 Sajio Sumida Clinic, Ginza, Tokyo, Japan; 2 New York University, School of Medicine, New York, New York, United States * Corresponding author.
Human adipose tissue will be a precious source of MSCs for regenerative therapy, however problems in tissue culture remain as for foreign proteins, recombinant stimulants, and the resources for MSCs. Venous wall and the perivascular “adipose” explants (0.1~0.2 gr) obtained by surgery (93 cases) were cultured in a special media including human sera, human platelet lysate, and limulus (bradytelic species) lysate (for limited cases) in a 5% CO2, -37 C, and formed confluent colonies of MSCs in 12.4 ± 3.8 (n ¼ 29) for venous wall, and in 7.1 ± 3 days (n ¼ 19) for adipose tissue. On the other hand, frozen adipose explants in 10% Me2SO in IMDM formed the MSCs colonies overall in 14.5 ± 6.5 days (n ¼ 16). Interestingly, a human skin with adipose tissues (1015around 5 mm thickness) which had been frozen in a 15% glycerol in TC199 at -196 C for 33 years gave buds of fibroblast-mesenchymal cell in 4 days and formed confluent in 11 days. Thus, thawed adipose tissue explants, either non-frozen or frozen, mostly formed MSCs confluent colonies in two weeks. The confluent MSCs were detached by 0.1% trypsin-IMDM or PBS-D solution, and cryopreserved again by RFTIVIVSCM (repetitive freeze-thaw-culture in vivo and in vitro subculture method) for days or months. The thawed MSCs were possible to form confluent colonies of MSCs as well as for the thawed bone marrow stromal cells reported previously. Limulus lysate suppressed the appearance of MSCs either of non-frozen or frozen adipose tissues in culture as well as in the result of bone marrow stem cells. The thawed adipose tissue and vein explants formed MSCs-confluent by culture in 14~40 days. The adipose tissues cryopreserved at -196 C for 33 years also budded fibroblast-like MSCs and formed confluent by culture after thawing. These adipocyte derived MSCs could be preserved by RFTCIVIVSCM and formed passage-2 confluent colonies. S020 CRYOPRESERVATION OF SHOOT TIPS OF FRAGARIA SPP. AND VIRUS ELIMINATION THROUGH CRYOTHERAPY S. Gupta. Tissue Culture and Cryopreservation Unit (TCCU), Indian Council of Agricultural Research-National Bureau of Plant Genetic Resources (ICARNBPGR), New Delhi, India Genus Fragaria L. of family Rosaceae is a native to temperate zones of northern hemisphere and South America. It can occur up to 4000 m altitude. Cultivars of Fragaria x ananassa Duch., the most common cultivated strawberry, is grown around the world in suitable temperate climate. The crop is economically important with the world production 4,594,539 MT in
2011. USA is the top producer of strawberries followed by European countries while many other countries are trying to increase production. Strawberries are produced in 73 countries worldwide. Strawberry is rich in vitamin C and a good source of manganese, and vitamin B, E, K and dietary minerals. It is consumed fresh as well as processed. Fragaria spp. is found growing wild in the Indian Himalayas and Nilgiri hills. Mostly, exotic cultivars are in cultivation. Conservation of cultivated and wild species is important for future strawberry improvement. Apart from ex situ conservation of Fragaria germplasm in the field, under screen, in green house, in vitro cultures, the cryopreservation of plant propagules is the only available method for long-term conservation. Virus-free germplasm should be conserved in the in vitro genebank and cryobank. In the literature, more than 30 viruses and virus-like disease have been reported in the strawberry. Cryotherapy is one of the best known methods used for virus elimination in vitro. In vitro genebank in NBPGR, India is conserving more than 80 accessions of Fragaria including exotic accessions procured from USDA-ARS-NCGR, Corvallis, Oregon, USA along with the several accessions collected from field genbanks in India. In vitro-grown shoot tips are used for cryopreservation as long term conservation strategy. The meristem, the ideal explant for virus elimination, is being used for cryotherapy. Research has been undergoing in our lab on cryopreservation. The outcome of the research work will be discussed in this paper. S021 OXIDATIVE STRESS IS CLOSELY RELATED TO THE POLLEN VIABILITY REDUCTION AFTER CRYOPRESERVATION M. Jia*, Y. Shi, Y. Liu, X. Jiang, J. Peng, W. Di. Beijing Forestry University, Beijing, China * Corresponding author.
Oxidative stress induced by excessive accumulation of reactive oxidative species (ROS) during cryopreservation may be another major contributing factor for cryoinjury of biological materials. In order to explore the role of oxidative stress in the cryopreservation of plant pollen, the germination capacity, ROS level, and malondialdehyde (MDA) content of pollens before and after cryopreservation from 20 common ornamental plants were comparatively studied. The results showed that germinability of cryopreserved pollen from 12 out of 20 species showed no significant difference with fresh pollen (group 1), only 1 increased, while the other 7 declined significantly (group 2). ROS in cryopreserved pollens from 75% species in group 1 remained unchanged compared to fresh pollen, 85.7% species in group 2 increased significantly, suggesting that pollen viability was negatively correlated to ROS. MDA content in pollens after cryopreservation from 66.7% species in group 1 showed no significant difference from fresh pollen, 85.7% species in group 2 rose significantly, which suggested that pollen viability and MDA level were also negatively correlated. Additionally, both ROS and MDA levels in pollens from 71.4% of species in group 2 increased significantly. In conclusion, plant pollens from most species possess a certain cryostorage tolerance, but pollens from some species are damaged severely. Pollen viability is closely related to the level of MDA and ROS. And, it shows a high correlation between ROS and MDA level as well. In addition, oxidative stress in pollens can be induced by cryostorage in liquid nitrogen, and is the main cause for the viability loses following cryopreservation. Source of funding: This work was supported by the National Natural Science Foundation of China (No. 31370693). S022 CELLULAR STRESS RESPONSES DURING CRYO-INDUCED STRESS IN ARABIDOPSIS SHOOT TIPS J. Stock*, M. Nagel, E.R.J. Keller, H.P. Mock. Leibniz Institute of Plant Genetics and Crop Plant Research, Seeland, Germany * Corresponding author.
During cryopreservation, meristematic tissues have to cope with different stresses ranging from desiccation to cold stress and rehydration. As current