J. Mamm. Ova Res. Vol.31 (4), 115 122, 2014 The Safety of Cryopreservation Devices in Protecting Against Liquid Nitrogen Yoko Kumasako, Kyoko Kido, Eiko Otsu, Takafumi Utsunomiya 870-0823 St. Luke Clinic, 1-4-5 Higashi-Omichi, Oita city, Oita 870-0823, Japan 0.25 ml 0.25 ml Rapid-i Rapid-i Abstract: Currently, in Japan, the most commonly used embryo vitrification method is ultra-rapid vitrifi cation which is designed to achieve the maximum cooling rate by exposing embryos directly to LN2 in a tiny droplet of cryoprotectant. Though this method has yielded acceptable embryo viability rates, questions have been raised by various countries regulatory agencies regarding the effects of LN2 exposure. The 0.25 ml plastic straw has long been used as an entirely closed vitrification system. However, the cooling rate is slower using the 0.25 ml straw than when using the ultra-rapid cooling method, resulting in injury to embryos exposed to a high concentration of cryoprotectant. Recently, a new system has been developed which addresses both the issues of rates of cooling and safeguards against LN2. This paper compares the clinical results of blastocyst vitrifi cation and warming using the conventional 0.25 ml straw method with those of the Rapid-i method. The survival rate after warming the group cryopreserved using Rapid-i was significantly higher than that of the group of frozen-thawed cryopreserved using the 0.25 ml straw. Moreover, the on-going pregnancy rate after transfer blastocysts into recipients uteri was higher after cryopreservation with the Rapid-i method. These results suggest that the new closed system is safe to use in cryopreservation. Key words: liquid nitrogen, contamination, vitrifi cation, safety, straw 2014 5 1 2014 6 27 870-0823 1 4-5 e-mail: lukelab@oct-net.ne.jp 2011 1,050,698 32,426 32 1 1.8 595 1 1)
116 J. Mamm. Ova Res. Vol.31 (4), 2014 1 2011 71,422 4,546 106,024 * 95,764 22,465 117,736 102,473 5,415 80,046 269,659 32,426 303,806 24 * 1.37 2) ART 10 24 1) 2011 21.3 34.2 1.5 2.3 3, 4) 1972 Whittingham 5) 6, 7) DMSO 1983 Trounson 8) 1985 Rall 9) 85 Rall 1990 Gordts 10) Yokota 11) 0.25 ml Mukaida 12, 13) 1998 2001 2000 Kuwayama 14) Cryoloop Cryotop 2 1 10
117 2 0.25 ml 2,500 OPS open pulled straw Cryoloop Cryotop 20,000 200,000 3 0.25 ml Rapid-i 0.25 ml DMSO Rapid-i 10 μl 0.05 μl DMSO Yokota 11) 0.25 ml 15) 16) 90 95 17) 50 5 6 0.25 ml Rapid-i Vitrolife Sweden Cryotop SC-N CVM CryoLogic, Austraria Cryopette Origio, Denmark 0.25 ml 0.25 ml Rapid-i 3 0.25 ml 10 μl Rapid-i
118 J. Mamm. Ova Res. Vol.31 (4), 2014 1 0.25 ml 10 ES 5 12.5% 12.5 DMSO 1/2VSED 1 25% 25 DMSO VSED 30 0.5 M 5 0.05 μl Rapid-i DMSO 0.25 ml 3 1-step Rapid-i 2 2012 12013 6 38 0.25 ml Rapid-i 0.25 ml 177219 Rapid-i 165192 32.8 3.6 33.9 3.3P=0.0003 18, 19) 30% ART 4.3 2.4 4.4 3.0 9.7 2.5 mm 9.6 2.2 mm χ 2 1 0.25 ml 1 Ishimori 20) Yokota 11) 20 SSS Serum Substitute Supplement: Irvine Scientific America m-hff 10 ES5 20 SSS m-hff DMSO Sigma America 2:1:1 VSED 5 ES 1/2 VSED 1 VSED 30 20 SSS m-hff 0.5 mol/l Sigma, America load 10 μl load load 30 2 10 27 5 2 24 1 2 Rapid-i TM
119 2 Rapid-i RapidVit Blast Vitri 1 5~20 Vitri 2 2 Vitri 3 45Rapid-i Vitri3 RapidStraw Rapid-i RapidStraw RapidWarm Blast Warm 1 2 Warm 2 3 Warm 3 5~10 1 Vitrolife RapidVit Blast 5 20RapidVit Blast 2 RapidVit Blast 45 Rapid-i Rapid- 2 Rapid-i RapidWarm Blast Rapid-i 2 RapidWarm Blast 3 RapidWarm Blast 5 10 2 24 2 3 0.25 ml Rapid-i 0.25 ml 79.5% 174/219 Rapid-i 90.6% 174/192 0.25 ml 40.7% 66/162 Rapid-i 41.6% 67/161 3 Rapid-i
120 J. Mamm. Ova Res. Vol.31 (4), 2014 4 Rapid-i 5 Rapid-i 4 19.2% 34/177 25.5% 42/165 5 Rapid-i Rapid-i 0.25 ml Rapid-i 0.25 ml Rapid-i 2 12,21-23 RapidVit Blast Rapid-i 0.25ml 4 5 Rapid-i 1654 177 15 24-26) Bielanski
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