The Experts below are selected from a list of 255 Experts worldwide ranked by ideXlab platform
Manfred S. Weiss - One of the best experts on this subject based on the ideXlab platform.
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trimethylamine n oxide as a versatile Cryoprotective Agent in macromolecular crystallography
Journal of Applied Crystallography, 2011Co-Authors: Christoph Muellerdieckmann, Brice Kauffmann, Manfred S. WeissAbstract:The surge of macromolecular crystallography is intimately linked to the advent of methods for cryoprotecting macromolecular crystals. Only if crystals are kept cold during data collection can they withstand the effects of radiation damage during a diffraction experiment, especially at third-generation synchrotron sources. While a number of different Cryoprotective Agents and procedures have been described in the literature over the past three decades, it is still a time- and crystal-consuming process to establish and optimize a good cryo-condition for a specific crystal. In this study, trimethylamine N-oxide (TMAO) has been identified as a very versatile cryoprotectant for macromolecular crystals. In a few test cases it was shown that diffraction data collected from crystals treated with TMAO are of very good quality.
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Trimethylamine N‐oxide as a versatile Cryoprotective Agent in macromolecular crystallography
Journal of Applied Crystallography, 2011Co-Authors: Christoph Mueller-dieckmann, Brice Kauffmann, Manfred S. WeissAbstract:The surge of macromolecular crystallography is intimately linked to the advent of methods for cryoprotecting macromolecular crystals. Only if crystals are kept cold during data collection can they withstand the effects of radiation damage during a diffraction experiment, especially at third-generation synchrotron sources. While a number of different Cryoprotective Agents and procedures have been described in the literature over the past three decades, it is still a time- and crystal-consuming process to establish and optimize a good cryo-condition for a specific crystal. In this study, trimethylamine N-oxide (TMAO) has been identified as a very versatile cryoprotectant for macromolecular crystals. In a few test cases it was shown that diffraction data collected from crystals treated with TMAO are of very good quality.
Suonghyu Hyon - One of the best experts on this subject based on the ideXlab platform.
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carboxylated e poly l lysine a Cryoprotective Agent is an effective partner of ethylene glycol for the vitrification of embryos at various preimplantation stages
Cryobiology, 2020Co-Authors: Yuki Kawasaki, Kazuaki Matsumura, Suonghyu Hyon, Natsuki Kohaya, Maki Kamoshita, Katsuyoshi Fujiwara, Yurie Shibao, Ayumi Suyama, Atsuko Kageyama, Junya ItoAbstract:Abstract It has been known that different protocols are used for embryo preservation at different stages due to different sensitivity to the physical and physiological stress caused by vitrification. In this study, we developed a common vitrification protocol using carboxlated e-poly- l -lysine (COOH-PLL), a new Cryoprotective Agent for the vitrification of mouse embryos at different stages. The IVF-derived Crl:CD1(ICR) x B6D2F1/Crl pronuclear, 2-cell, 4-cell, and 8-cell, morula and blastocyst stage embryos were vitrified with 15% (v/v) ethylene glycol (EG) and 10% (w/v) COOH-PLL (E15P15) or 15% (v/v) EG and 15% (v/v) dimethyl sulfoxide (E15D15) using the minimal volume cooling method. The survival of vitrified embryos from pronuclear to blastocyst stages was equivalent between E15P15 and E15D15 groups. However, the rate of development to blastocysts was significantly lower in E15P15 than E15D15. The rates of survival and development to blastocysts were dramatically improved by a slight modification of EG and COOH-PLL concentrations (E20P10). After transferring 17 (E20P10) and 15 (E15D15) vitrified/warmed blastocysts, 8 and 7 pups were obtained (47.1% and 46.7%, respectively). Taken together, these results indicate that our vitrification protocol is appropriate for the vitrification of mouse embryos at different stages.
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successful vitrification of pronuclear stage pig embryos with a novel Cryoprotective Agent carboxylated e poly l lysine
PLOS ONE, 2017Co-Authors: Maki Kamoshita, Kazuaki Matsumura, Suonghyu Hyon, Katsuyoshi Fujiwara, Junya Ito, Tsubasa Kato, Takafumi Namiki, Naomi KashiwazakiAbstract:Vitrification is a powerful tool for the efficient production of offspring derived from cryopreserved oocytes or embryos in mammalian species including domestic animals. Genome editing technologies such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated (Cas)9 are now available even for domestic species, suggesting that the vitrification of embryos at the pronuclear stage (PN) will be more important because they could provide genomic host cells to be targeted by TALENs or CRISPR/Cas9. Although we reported the successful production of piglets derived from vitrified PN embryos by a solid-surface vitrification method with glutathione supplementation, further improvements are required. The Cryoprotective Agent (CPA) carboxylated e-poly-L-lysine (COOH-PLL) was introduced in 2009. COOH-PLL reduces the physical and physiological damage caused by cryopreservation in mammalian stem cells and the vitrification of mouse oocytes and embryos. Those results suggested that vitrification of COOH-PLL may help improve the developmental ability of pig embryos vitrified at the PN stage. However, it remains unclear whether COOH-PLL is available as a CPA for the vitrification of embryos in domestic species. In this study, we evaluated COOH-PLL as a CPA with ethylene glycol (EG) and Cryotop as a device for the vitrification of PN pig embryos. Exposure to vitrification solution supplemented with COOH-PLL up to 30% did not decrease developmental ability to the 2-cell stage and the blastocyst stage. After warming, most of the vitrified embryos survived regardless of the concentration of COOH-PLL (76.0 ± 11.8% to 91.8 ± 4.6%). However, the vitrified embryos without COOH-PLL showed a lower development rate up to the blastocyst stage (1.3 ± 1.0%) compared to the fresh embryos (28.4 ± 5.0%) (p<0.05). In contrast, supplementation of 20% (w/v) COOH-PLL in the vitrification solution dramatically improved the developmental ability to blastocysts of the vitrified embryos (19.4 ± 4.6%) compared to those without COOH-PLL (p<0.05). After the transfer of embryos vitrified with 30% (v/v) EG and 20% (w/v) COOH-PLL, we successfully obtained 15 piglets from 8 recipients. Taken together, our present findings demonstrate for the first time that COOH-PLL is an effective CPA for embryo vitrification in the pig. COOH-PLL is a promising CPA for further improvements in the vitrification of oocytes and embryos in mammalian species.
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Efficient Production of Live Offspring from Mouse Oocytes Vitrified with a Novel Cryoprotective Agent,
2016Co-Authors: Carboxylated E-poly-l-lysine, Kazuaki Matsumura, Suonghyu Hyon, Hitomi Watanabe, Natsuki Kohaya, Maki Kamoshita, Katsuyoshi Fujiwara, Junya Ito, Naomi KashiwazakiAbstract:In cryopreservation of mammalian germ cells, unfertilized oocytes are one of the most available stages because these cryopreserved oocytes can be used for assisted reproductive technologies, including in vitro fertilization (IVF) and intracytoplasmic sperm injection. However, it has been generally reported that the fertility and developmental ability of the oocytes are reduced by cryopreservation. Therefore further improvement will be required. Very recently, a new Cryoprotective Agent (CPA), called as carboxylated e-poly-L-lysine (COOH-PLL), has been developed to reduce physical and physiological damage by cryopreservation in mammalian stem cells. However, it is unclear the effect of COOH-PLL on fertility and developmental ability of vitrified oocytes. In this study, we used COOH-PLL as a CPA with ethylene glycol (EG) for vitrification of mouse oocytes. Cumulus-oocyte complexes (COCs) were collected from ICR mice and then vitrified with Cryotop using different concentration of COOH-PLL and EG. A combined treatment with COOH-PLL and EG showed high survival rate (more than 90%) of vitrified-warmed COCs after in vitro fertilization. In addition, the fertility and developmental ability of COCs vitrified with E20P10 [EG 20 % (v/v) and COOH-PLL 10 % (w/v)] or E15P15 group (EG 15 % and COOH-PLL 15%) were significantly higher than those with E10P20 (EG10 % and COOH-PLL 20%) or P30 group (PLL30%). The vitrified COCs in E20P10 group developed to term at a high success rate (46.2%) and it was significantly higher than that in control (E30) group (34.8%). Our present study demonstrated for the first time that COOH-PLL is effective for vitrification of mous
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the effect of a novel Cryoprotective Agent carboxylated e poly l lysine on the developmental ability of re vitrified mouse embryos at the pronuclear stage
Cryobiology, 2014Co-Authors: Yurie Shibao, Kazuaki Matsumura, Suonghyu Hyon, Katsuyoshi Fujiwara, Junya Ito, Yuki Kawasaki, Naomi KashiwazakiAbstract:Transgenic animals are generally produced by microinjection of exogeneous DNA into embryos at the pronuclear (PN) stage. PN embryos also can be used for knockout animals because artificial nucleases such as zinc-finger nuclease or transcription activator-like effector nuclease are now available for modification of the targeted gene. If the embryos can be vitrified with multiple rounds, the remaining embryos without microinjection can be reused. In this study, we examined the developmental competence of repetitively vitrified mouse embryos at the PN stage using Cryotop. It was also examined whether a new Cryoprotective Agent (CPA), carboxylated e-poly-l-lysine (COOH-PLL), is available for vitrification of mouse embryos. PN embryos were vitrified with dimethyl sulfoxide (DMSO) and ethylene glycol (EG) as CPAs. After warming, some embryos were re-vitrified up to three times. The re-vitrification did not affect survival and in vitro developmental ability. PN embryos were also vitrified with COOH-PLL instead of DMSO up to three times. The embryos re-vitrified with COOH-PLL and EG also maintained high survival and developmental ability. However embryos vitrified with COOH-PLL and EG at three times significantly showed higher developmental ability (61.2±3.1%) than those vitrified with DMSO and EG at three times (44.2±2.7%) which was equivalent to that of fresh embryos (70.0±3.6%). Taken together, our results show that re-vitrification of mouse PN embryos did not have a detrimental effect on the in vitro and in vivo development of the embryos. In addition, COOH-PLL is available as a CPA for vitrification of mouse PN embryos.
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efficient production of live offspring from mouse oocytes vitrified with a novel Cryoprotective Agent carboxylated e poly l lysine
PLOS ONE, 2013Co-Authors: Hitomi Watanabe, Kazuaki Matsumura, Suonghyu Hyon, Natsuki Kohaya, Maki Kamoshita, Katsuyoshi Fujiwara, Junya Ito, Naomi KashiwazakiAbstract:In cryopreservation of mammalian germ cells, unfertilized oocytes are one of the most available stages because these cryopreserved oocytes can be used for assisted reproductive technologies, including in vitro fertilization (IVF) and intracytoplasmic sperm injection. However, it has been generally reported that the fertility and developmental ability of the oocytes are reduced by cryopreservation. Therefore further improvement will be required. Very recently, a new Cryoprotective Agent (CPA), called as carboxylated e-poly-L-lysine (COOH-PLL), has been developed to reduce physical and physiological damage by cryopreservation in mammalian stem cells. However, it is unclear the effect of COOH-PLL on fertility and developmental ability of vitrified oocytes. In this study, we used COOH-PLL as a CPA with ethylene glycol (EG) for vitrification of mouse oocytes. Cumulus-oocyte complexes (COCs) were collected from ICR mice and then vitrified with Cryotop using different concentration of COOH-PLL and EG. A combined treatment with COOH-PLL and EG showed high survival rate (more than 90%) of vitrified-warmed COCs after in vitro fertilization. In addition, the fertility and developmental ability of COCs vitrified with E20P10 [EG 20% (v/v) and COOH-PLL 10% (w/v)] or E15P15 group (EG 15% and COOH-PLL 15%) were significantly higher than those with E10P20 (EG10% and COOH-PLL 20%) or P30 group (PLL30%). The vitrified COCs in E20P10 group developed to term at a high success rate (46.2%) and it was significantly higher than that in control (E30) group (34.8%). Our present study demonstrated for the first time that COOH-PLL is effective for vitrification of mouse oocytes.
Naomi Kashiwazaki - One of the best experts on this subject based on the ideXlab platform.
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successful vitrification of pronuclear stage pig embryos with a novel Cryoprotective Agent carboxylated e poly l lysine
PLOS ONE, 2017Co-Authors: Maki Kamoshita, Kazuaki Matsumura, Suonghyu Hyon, Katsuyoshi Fujiwara, Junya Ito, Tsubasa Kato, Takafumi Namiki, Naomi KashiwazakiAbstract:Vitrification is a powerful tool for the efficient production of offspring derived from cryopreserved oocytes or embryos in mammalian species including domestic animals. Genome editing technologies such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated (Cas)9 are now available even for domestic species, suggesting that the vitrification of embryos at the pronuclear stage (PN) will be more important because they could provide genomic host cells to be targeted by TALENs or CRISPR/Cas9. Although we reported the successful production of piglets derived from vitrified PN embryos by a solid-surface vitrification method with glutathione supplementation, further improvements are required. The Cryoprotective Agent (CPA) carboxylated e-poly-L-lysine (COOH-PLL) was introduced in 2009. COOH-PLL reduces the physical and physiological damage caused by cryopreservation in mammalian stem cells and the vitrification of mouse oocytes and embryos. Those results suggested that vitrification of COOH-PLL may help improve the developmental ability of pig embryos vitrified at the PN stage. However, it remains unclear whether COOH-PLL is available as a CPA for the vitrification of embryos in domestic species. In this study, we evaluated COOH-PLL as a CPA with ethylene glycol (EG) and Cryotop as a device for the vitrification of PN pig embryos. Exposure to vitrification solution supplemented with COOH-PLL up to 30% did not decrease developmental ability to the 2-cell stage and the blastocyst stage. After warming, most of the vitrified embryos survived regardless of the concentration of COOH-PLL (76.0 ± 11.8% to 91.8 ± 4.6%). However, the vitrified embryos without COOH-PLL showed a lower development rate up to the blastocyst stage (1.3 ± 1.0%) compared to the fresh embryos (28.4 ± 5.0%) (p<0.05). In contrast, supplementation of 20% (w/v) COOH-PLL in the vitrification solution dramatically improved the developmental ability to blastocysts of the vitrified embryos (19.4 ± 4.6%) compared to those without COOH-PLL (p<0.05). After the transfer of embryos vitrified with 30% (v/v) EG and 20% (w/v) COOH-PLL, we successfully obtained 15 piglets from 8 recipients. Taken together, our present findings demonstrate for the first time that COOH-PLL is an effective CPA for embryo vitrification in the pig. COOH-PLL is a promising CPA for further improvements in the vitrification of oocytes and embryos in mammalian species.
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Efficient Production of Live Offspring from Mouse Oocytes Vitrified with a Novel Cryoprotective Agent,
2016Co-Authors: Carboxylated E-poly-l-lysine, Kazuaki Matsumura, Suonghyu Hyon, Hitomi Watanabe, Natsuki Kohaya, Maki Kamoshita, Katsuyoshi Fujiwara, Junya Ito, Naomi KashiwazakiAbstract:In cryopreservation of mammalian germ cells, unfertilized oocytes are one of the most available stages because these cryopreserved oocytes can be used for assisted reproductive technologies, including in vitro fertilization (IVF) and intracytoplasmic sperm injection. However, it has been generally reported that the fertility and developmental ability of the oocytes are reduced by cryopreservation. Therefore further improvement will be required. Very recently, a new Cryoprotective Agent (CPA), called as carboxylated e-poly-L-lysine (COOH-PLL), has been developed to reduce physical and physiological damage by cryopreservation in mammalian stem cells. However, it is unclear the effect of COOH-PLL on fertility and developmental ability of vitrified oocytes. In this study, we used COOH-PLL as a CPA with ethylene glycol (EG) for vitrification of mouse oocytes. Cumulus-oocyte complexes (COCs) were collected from ICR mice and then vitrified with Cryotop using different concentration of COOH-PLL and EG. A combined treatment with COOH-PLL and EG showed high survival rate (more than 90%) of vitrified-warmed COCs after in vitro fertilization. In addition, the fertility and developmental ability of COCs vitrified with E20P10 [EG 20 % (v/v) and COOH-PLL 10 % (w/v)] or E15P15 group (EG 15 % and COOH-PLL 15%) were significantly higher than those with E10P20 (EG10 % and COOH-PLL 20%) or P30 group (PLL30%). The vitrified COCs in E20P10 group developed to term at a high success rate (46.2%) and it was significantly higher than that in control (E30) group (34.8%). Our present study demonstrated for the first time that COOH-PLL is effective for vitrification of mous
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the effect of a novel Cryoprotective Agent carboxylated e poly l lysine on the developmental ability of re vitrified mouse embryos at the pronuclear stage
Cryobiology, 2014Co-Authors: Yurie Shibao, Kazuaki Matsumura, Suonghyu Hyon, Katsuyoshi Fujiwara, Junya Ito, Yuki Kawasaki, Naomi KashiwazakiAbstract:Transgenic animals are generally produced by microinjection of exogeneous DNA into embryos at the pronuclear (PN) stage. PN embryos also can be used for knockout animals because artificial nucleases such as zinc-finger nuclease or transcription activator-like effector nuclease are now available for modification of the targeted gene. If the embryos can be vitrified with multiple rounds, the remaining embryos without microinjection can be reused. In this study, we examined the developmental competence of repetitively vitrified mouse embryos at the PN stage using Cryotop. It was also examined whether a new Cryoprotective Agent (CPA), carboxylated e-poly-l-lysine (COOH-PLL), is available for vitrification of mouse embryos. PN embryos were vitrified with dimethyl sulfoxide (DMSO) and ethylene glycol (EG) as CPAs. After warming, some embryos were re-vitrified up to three times. The re-vitrification did not affect survival and in vitro developmental ability. PN embryos were also vitrified with COOH-PLL instead of DMSO up to three times. The embryos re-vitrified with COOH-PLL and EG also maintained high survival and developmental ability. However embryos vitrified with COOH-PLL and EG at three times significantly showed higher developmental ability (61.2±3.1%) than those vitrified with DMSO and EG at three times (44.2±2.7%) which was equivalent to that of fresh embryos (70.0±3.6%). Taken together, our results show that re-vitrification of mouse PN embryos did not have a detrimental effect on the in vitro and in vivo development of the embryos. In addition, COOH-PLL is available as a CPA for vitrification of mouse PN embryos.
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efficient production of live offspring from mouse oocytes vitrified with a novel Cryoprotective Agent carboxylated e poly l lysine
PLOS ONE, 2013Co-Authors: Hitomi Watanabe, Kazuaki Matsumura, Suonghyu Hyon, Natsuki Kohaya, Maki Kamoshita, Katsuyoshi Fujiwara, Junya Ito, Naomi KashiwazakiAbstract:In cryopreservation of mammalian germ cells, unfertilized oocytes are one of the most available stages because these cryopreserved oocytes can be used for assisted reproductive technologies, including in vitro fertilization (IVF) and intracytoplasmic sperm injection. However, it has been generally reported that the fertility and developmental ability of the oocytes are reduced by cryopreservation. Therefore further improvement will be required. Very recently, a new Cryoprotective Agent (CPA), called as carboxylated e-poly-L-lysine (COOH-PLL), has been developed to reduce physical and physiological damage by cryopreservation in mammalian stem cells. However, it is unclear the effect of COOH-PLL on fertility and developmental ability of vitrified oocytes. In this study, we used COOH-PLL as a CPA with ethylene glycol (EG) for vitrification of mouse oocytes. Cumulus-oocyte complexes (COCs) were collected from ICR mice and then vitrified with Cryotop using different concentration of COOH-PLL and EG. A combined treatment with COOH-PLL and EG showed high survival rate (more than 90%) of vitrified-warmed COCs after in vitro fertilization. In addition, the fertility and developmental ability of COCs vitrified with E20P10 [EG 20% (v/v) and COOH-PLL 10% (w/v)] or E15P15 group (EG 15% and COOH-PLL 15%) were significantly higher than those with E10P20 (EG10% and COOH-PLL 20%) or P30 group (PLL30%). The vitrified COCs in E20P10 group developed to term at a high success rate (46.2%) and it was significantly higher than that in control (E30) group (34.8%). Our present study demonstrated for the first time that COOH-PLL is effective for vitrification of mouse oocytes.
Kazuaki Matsumura - One of the best experts on this subject based on the ideXlab platform.
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carboxylated e poly l lysine a Cryoprotective Agent is an effective partner of ethylene glycol for the vitrification of embryos at various preimplantation stages
Cryobiology, 2020Co-Authors: Yuki Kawasaki, Kazuaki Matsumura, Suonghyu Hyon, Natsuki Kohaya, Maki Kamoshita, Katsuyoshi Fujiwara, Yurie Shibao, Ayumi Suyama, Atsuko Kageyama, Junya ItoAbstract:Abstract It has been known that different protocols are used for embryo preservation at different stages due to different sensitivity to the physical and physiological stress caused by vitrification. In this study, we developed a common vitrification protocol using carboxlated e-poly- l -lysine (COOH-PLL), a new Cryoprotective Agent for the vitrification of mouse embryos at different stages. The IVF-derived Crl:CD1(ICR) x B6D2F1/Crl pronuclear, 2-cell, 4-cell, and 8-cell, morula and blastocyst stage embryos were vitrified with 15% (v/v) ethylene glycol (EG) and 10% (w/v) COOH-PLL (E15P15) or 15% (v/v) EG and 15% (v/v) dimethyl sulfoxide (E15D15) using the minimal volume cooling method. The survival of vitrified embryos from pronuclear to blastocyst stages was equivalent between E15P15 and E15D15 groups. However, the rate of development to blastocysts was significantly lower in E15P15 than E15D15. The rates of survival and development to blastocysts were dramatically improved by a slight modification of EG and COOH-PLL concentrations (E20P10). After transferring 17 (E20P10) and 15 (E15D15) vitrified/warmed blastocysts, 8 and 7 pups were obtained (47.1% and 46.7%, respectively). Taken together, these results indicate that our vitrification protocol is appropriate for the vitrification of mouse embryos at different stages.
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successful vitrification of pronuclear stage pig embryos with a novel Cryoprotective Agent carboxylated e poly l lysine
PLOS ONE, 2017Co-Authors: Maki Kamoshita, Kazuaki Matsumura, Suonghyu Hyon, Katsuyoshi Fujiwara, Junya Ito, Tsubasa Kato, Takafumi Namiki, Naomi KashiwazakiAbstract:Vitrification is a powerful tool for the efficient production of offspring derived from cryopreserved oocytes or embryos in mammalian species including domestic animals. Genome editing technologies such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated (Cas)9 are now available even for domestic species, suggesting that the vitrification of embryos at the pronuclear stage (PN) will be more important because they could provide genomic host cells to be targeted by TALENs or CRISPR/Cas9. Although we reported the successful production of piglets derived from vitrified PN embryos by a solid-surface vitrification method with glutathione supplementation, further improvements are required. The Cryoprotective Agent (CPA) carboxylated e-poly-L-lysine (COOH-PLL) was introduced in 2009. COOH-PLL reduces the physical and physiological damage caused by cryopreservation in mammalian stem cells and the vitrification of mouse oocytes and embryos. Those results suggested that vitrification of COOH-PLL may help improve the developmental ability of pig embryos vitrified at the PN stage. However, it remains unclear whether COOH-PLL is available as a CPA for the vitrification of embryos in domestic species. In this study, we evaluated COOH-PLL as a CPA with ethylene glycol (EG) and Cryotop as a device for the vitrification of PN pig embryos. Exposure to vitrification solution supplemented with COOH-PLL up to 30% did not decrease developmental ability to the 2-cell stage and the blastocyst stage. After warming, most of the vitrified embryos survived regardless of the concentration of COOH-PLL (76.0 ± 11.8% to 91.8 ± 4.6%). However, the vitrified embryos without COOH-PLL showed a lower development rate up to the blastocyst stage (1.3 ± 1.0%) compared to the fresh embryos (28.4 ± 5.0%) (p<0.05). In contrast, supplementation of 20% (w/v) COOH-PLL in the vitrification solution dramatically improved the developmental ability to blastocysts of the vitrified embryos (19.4 ± 4.6%) compared to those without COOH-PLL (p<0.05). After the transfer of embryos vitrified with 30% (v/v) EG and 20% (w/v) COOH-PLL, we successfully obtained 15 piglets from 8 recipients. Taken together, our present findings demonstrate for the first time that COOH-PLL is an effective CPA for embryo vitrification in the pig. COOH-PLL is a promising CPA for further improvements in the vitrification of oocytes and embryos in mammalian species.
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Efficient Production of Live Offspring from Mouse Oocytes Vitrified with a Novel Cryoprotective Agent,
2016Co-Authors: Carboxylated E-poly-l-lysine, Kazuaki Matsumura, Suonghyu Hyon, Hitomi Watanabe, Natsuki Kohaya, Maki Kamoshita, Katsuyoshi Fujiwara, Junya Ito, Naomi KashiwazakiAbstract:In cryopreservation of mammalian germ cells, unfertilized oocytes are one of the most available stages because these cryopreserved oocytes can be used for assisted reproductive technologies, including in vitro fertilization (IVF) and intracytoplasmic sperm injection. However, it has been generally reported that the fertility and developmental ability of the oocytes are reduced by cryopreservation. Therefore further improvement will be required. Very recently, a new Cryoprotective Agent (CPA), called as carboxylated e-poly-L-lysine (COOH-PLL), has been developed to reduce physical and physiological damage by cryopreservation in mammalian stem cells. However, it is unclear the effect of COOH-PLL on fertility and developmental ability of vitrified oocytes. In this study, we used COOH-PLL as a CPA with ethylene glycol (EG) for vitrification of mouse oocytes. Cumulus-oocyte complexes (COCs) were collected from ICR mice and then vitrified with Cryotop using different concentration of COOH-PLL and EG. A combined treatment with COOH-PLL and EG showed high survival rate (more than 90%) of vitrified-warmed COCs after in vitro fertilization. In addition, the fertility and developmental ability of COCs vitrified with E20P10 [EG 20 % (v/v) and COOH-PLL 10 % (w/v)] or E15P15 group (EG 15 % and COOH-PLL 15%) were significantly higher than those with E10P20 (EG10 % and COOH-PLL 20%) or P30 group (PLL30%). The vitrified COCs in E20P10 group developed to term at a high success rate (46.2%) and it was significantly higher than that in control (E30) group (34.8%). Our present study demonstrated for the first time that COOH-PLL is effective for vitrification of mous
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the effect of a novel Cryoprotective Agent carboxylated e poly l lysine on the developmental ability of re vitrified mouse embryos at the pronuclear stage
Cryobiology, 2014Co-Authors: Yurie Shibao, Kazuaki Matsumura, Suonghyu Hyon, Katsuyoshi Fujiwara, Junya Ito, Yuki Kawasaki, Naomi KashiwazakiAbstract:Transgenic animals are generally produced by microinjection of exogeneous DNA into embryos at the pronuclear (PN) stage. PN embryos also can be used for knockout animals because artificial nucleases such as zinc-finger nuclease or transcription activator-like effector nuclease are now available for modification of the targeted gene. If the embryos can be vitrified with multiple rounds, the remaining embryos without microinjection can be reused. In this study, we examined the developmental competence of repetitively vitrified mouse embryos at the PN stage using Cryotop. It was also examined whether a new Cryoprotective Agent (CPA), carboxylated e-poly-l-lysine (COOH-PLL), is available for vitrification of mouse embryos. PN embryos were vitrified with dimethyl sulfoxide (DMSO) and ethylene glycol (EG) as CPAs. After warming, some embryos were re-vitrified up to three times. The re-vitrification did not affect survival and in vitro developmental ability. PN embryos were also vitrified with COOH-PLL instead of DMSO up to three times. The embryos re-vitrified with COOH-PLL and EG also maintained high survival and developmental ability. However embryos vitrified with COOH-PLL and EG at three times significantly showed higher developmental ability (61.2±3.1%) than those vitrified with DMSO and EG at three times (44.2±2.7%) which was equivalent to that of fresh embryos (70.0±3.6%). Taken together, our results show that re-vitrification of mouse PN embryos did not have a detrimental effect on the in vitro and in vivo development of the embryos. In addition, COOH-PLL is available as a CPA for vitrification of mouse PN embryos.
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efficient production of live offspring from mouse oocytes vitrified with a novel Cryoprotective Agent carboxylated e poly l lysine
PLOS ONE, 2013Co-Authors: Hitomi Watanabe, Kazuaki Matsumura, Suonghyu Hyon, Natsuki Kohaya, Maki Kamoshita, Katsuyoshi Fujiwara, Junya Ito, Naomi KashiwazakiAbstract:In cryopreservation of mammalian germ cells, unfertilized oocytes are one of the most available stages because these cryopreserved oocytes can be used for assisted reproductive technologies, including in vitro fertilization (IVF) and intracytoplasmic sperm injection. However, it has been generally reported that the fertility and developmental ability of the oocytes are reduced by cryopreservation. Therefore further improvement will be required. Very recently, a new Cryoprotective Agent (CPA), called as carboxylated e-poly-L-lysine (COOH-PLL), has been developed to reduce physical and physiological damage by cryopreservation in mammalian stem cells. However, it is unclear the effect of COOH-PLL on fertility and developmental ability of vitrified oocytes. In this study, we used COOH-PLL as a CPA with ethylene glycol (EG) for vitrification of mouse oocytes. Cumulus-oocyte complexes (COCs) were collected from ICR mice and then vitrified with Cryotop using different concentration of COOH-PLL and EG. A combined treatment with COOH-PLL and EG showed high survival rate (more than 90%) of vitrified-warmed COCs after in vitro fertilization. In addition, the fertility and developmental ability of COCs vitrified with E20P10 [EG 20% (v/v) and COOH-PLL 10% (w/v)] or E15P15 group (EG 15% and COOH-PLL 15%) were significantly higher than those with E10P20 (EG10% and COOH-PLL 20%) or P30 group (PLL30%). The vitrified COCs in E20P10 group developed to term at a high success rate (46.2%) and it was significantly higher than that in control (E30) group (34.8%). Our present study demonstrated for the first time that COOH-PLL is effective for vitrification of mouse oocytes.
Dayong Gao - One of the best experts on this subject based on the ideXlab platform.
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theoretical investigation of a novel microwave antenna aided cryovial for rapid and uniform rewarming of frozen Cryoprotective Agent solutions
Applied Thermal Engineering, 2015Co-Authors: Tao Wang, Zhongshan Deng, Dayong Gao, Gang Zhao, Cai Gao, Yunxia CaoAbstract:Abstract The most challenging issue in cryopreservation of mass biomaterials is to rewarm the frozen sample in a fast and uniform manner, so that the dangerous devitrification and recrystallization may be avoided. In this study, a conceptual innovation is the design of a novel cryovial, and we investigate the effects of the microwave heating after embedding superparamagnetic nanoparticles on the rewarming processes of the cell suspensions encapsulated in the cryovial. The electromagnetic field and the heat transfer during the hybrid rewarming processes of frozen EC2 solution with temperature-dependent properties were calculated. During the rewarming process of the sample in the cryovial in a traditional 37 °C water bath, the rewarming rate was 72.15 °C/min and the maximum temperature gradient in the sample was 20.5 °C/mm. After a slot antenna was included in the cryovial, the rewarming rate was 83.71 °C/min without nanoparticles and 106.41 °C/min after nanoparticles are embedded, the maximum temperature gradient in the sample was 40.2 °C/mm without nanoparticles and 28.7 °C/mm after nanoparticles embedded, respectively. This indicates that the rewarming rate and the uniformity of the temperature distribution increased after embedding nanoparticles. This could be because nanoparticles homogeneously generate heat in the sample and improve the time-dependent parameters of the sample.
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Cryoprotective Agent cpa removal with dilution filtration method and cpa concentration monitoring with electrical conductivity measurements
ASME 2014 International Mechanical Engineering Congress and Exposition IMECE 2014, 2014Co-Authors: Zhiquan Shu, Cifeng Fang, Xiaoming Zhou, Dayong GaoAbstract:In cell/tissue cryopreservation, Cryoprotective Agents (CPAs) should be added before freezing and removed after thawing. Nowadays people mainly apply centrifugation for CPA removal, which may cause many problems. Meanwhile, a simple and cheap method for real-time monitoring of the residual CPA concentration during processing remains an unfilled need. In this work, a “dilution-filtration” system with hollow fiber dialyzer was implemented and approved to remove dimethyl sulfoxide (Me2SO) effectively. Compared to other methods (centrifugation and microfluidic approach), this one holds advantages of low time and labor consumption, low osmotic injury to the cells, high effectiveness, ease to control the final suspension volume and low risk of contamination. The “dilution-filtration” system can also be easily modified for CPA addition and cell suspension volume control (concentration or dilution of cell suspension). Meanwhile, a method of electrical conductivity (EC) measurement was applied to monitor the residual CPA concentration. The results showed EC measurements of waste solution can convey the CPA concentrations in cell suspension. This validates the feasibility of a safer and easier way to on-line and real-time monitoring of CPA concentration in cell suspension by measuring the EC of waste solution.Copyright © 2014 by ASME
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statistical estimation of red blood cell osmotic damage during Cryoprotective Agent removal from cryopreserved blood
Biopreservation and Biobanking, 2013Co-Authors: Liangquan Gong, Weiping Ding, Sijie Sun, Gang Zhao, Dayong GaoAbstract:Statistical estimation of the osmotic damage of red blood cells (RBCs) during the removal of Cryoprotective Agents (CPAs) from cryopreserved blood has been a very difficult issue. In this paper, the discrete mass transfer model developed in our previous work is modified to study the volume variation of individual RBCs and thereby to estimate the osmotic damage of all RBCs statistically during CPA removal by the dilution-concentration method we proposed recently. The model is validated with respect to the experimental results either with or without RBCs. Then, it is used to investigate the effects of blood volume, hematocrit, blood and diluent flow rates on the osmotic damage of RBCs, as well as the washing time of CPAs. Our results show that both the increase of blood flow rates and the decrease of diluent flow rates can bring about a reduction in osmotic damage of RBCs; however, only the former can cause a decrease in the washing time of CPAs. The blood volume could also affect the osmotic damage of RBCs...
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andrology prevention of osmotic injury to human spermatozoa during addition and removal of glycerol
Human Reproduction, 1995Co-Authors: Dayong Gao, Peter Mazur, J Liu, Chi Liu, L E Mcgann, P F Watson, F W KleinhansAbstract:Use of a Cryoprotective Agent is indispensable to prevent injury to human spermatozoa during the cryopreservation process. However, addition of Cryoprotective Agents to spermatozoa before cooling and their removal after warming may create severe osmotic stress for the cells, resulting in injury. The objective of this study was to test the hypothesis that the degree (or magnitude) of human sperm volume excursion can be used as an independent indicator to evaluate and predict possible osmotic injury to spermatozoa during the addition and removal of Cryoprotective Agents. Glycerol was used as a model Cryoprotective Agent in the present study. To test this hypothesis, first the tolerance limits of spermatozoa to swelling in hypo-osmotic solutions (iso-osmotic medium diluted with water) and to shrinkage in hyperosmotic solutions (iso-osmotic medium with sucrose) were determined. Sperm plasma membrane integrity was measured by fluorescent staining, and sperm motility was assessed by computer-assisted semen analysis before, during and after the anisosmotic exposure. The results indicate firstly that motility was much more sensitive to anisosmotic conditions than membrane integrity, and secondly that motility was substantially more sensitive to hypotonic than to hypertonic conditions. Based on the experimental data, osmotic injury as a function of sperm volume excursion (swelling or shrinking) was determined. The second step, using these sperm volume excursion limits and previously measured glycerol and water nermeability coefficients of human spermatozoa. was to predict, by computer simulation, the cell osmotic injury caused by different procedures for the addition and removal of glycerol. The predicted sperm injury was confirmed by experiment. Based on this study, an analytical methodology has been developed for predicting optimal protocols to reduce osmotic injury associated with the addition and removal of hypertonic concentrations of glycerol in human spermatozoa.