Vitrification of one-cell mouse embryos in cryotubes |
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| Institution: | 1. State Key Laboratory of Satellite Ocean Environment Dynamics, Second Institute of Oceanography, State Oceanic Administration, 36 Bochubeilu, Hangzhou 310012, China;2. State Key Laboratory of Information Engineering in Surveying Mapping and Remote Sensing, Wuhan University, Wuhan 430072, China;1. Laboratory Brio Genetics and Biotechnology Ltd., Araguaína, TO, Brazil;2. University of Dschang, Western, Cameroon;3. State University of Ceará, Fortaleza, CE, Brazil;4. Federal University of Tocantins, EMVZ- Campus, Araguaína, TO, Brazil;5. Federal University of Piauí, Teresina, PI, Brazil;6. Federal University of Pará, Castanhal, PA, Brazil;1. Kanagawa Prefectural Livestock Industry Technology Center, Ebina, Kanagawa 243-0417, Japan;2. Faculty of Agriculture, National Livestock Breeding Center, Shirakawa, Fukushima 961-8511, Japan;3. Research Institute for the Functional Peptides, Higashine, Yamagata 999-3766, Japan;4. Misawa Medical Industry, Kasama, Ibaraki 309-1717, Japan;5. National Institute of Animal Health, Tsukuba, Ibaraki 305-0856, Japan;1. National Institute for Basic Biology, Interuniversity Bio-Backup Project, Aichi, 444-8787, Japan;2. Division of Science and Engineering Graduate School of Arts and Science, Iwate University, Iwate, 020-8551, Japan;3. Department of Chemistry and Biological Sciences, Faculty of Science and Engineering, Iwate University, Iwate, 020-8551, Japan;4. Soft-Path Science and Engineering Research Center (SPERC), Iwate University, Iwate, 020-8551, Japan;1. Fundamental and Applied Cryobiology Group, Department of Biochemistry and Cellular and Molecular Biology, The University of Tennessee, Knoxville, TN 37996, USA;2. Present address: Tokyo University of Marine Science and Technology, Tokyo 108-8477, Japan |
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| Abstract: | Preventing intracellular ice formation is essential to cryopreserve cells. Prevention can be achieved by converting cell water into a non-crystalline glass, that is, to vitrify. The prevailing belief is that to achieve vitrification, cells must be suspended in a solution containing a high concentration of glass-inducing solutes and cooled rapidly. In this study, we vitrified 1-cell mouse embryos and examined the effect of the cooling rate, the warming rate, and the concentration of cryoprotectant on cell survival. Embryos were vitrified in cryotubes. The vitrification solutions used were EFS20, EFS30, and EFS40, which contained ethylene glycol (20, 30 and 40% v/v, respectively), Ficoll (24%, 21%, and 18% w/v, respectively) and sucrose (0.4 0.35, and 0.3 M, respectively). A 5-μl EFS solution suspended with 1-cell embryos was placed in a cryotube. After 2 min in an EFS solution at 23 °C, embryos were vitrified by direct immersion into liquid nitrogen. The sample was warmed at 34 °C/min, 4,600 °C/min and 6,600 °C/min. With EFS40, the survival was low regardless of the warming rate. With EFS30 and EFS20, survival was also low when the warming rate was low, but increased with higher warming rates, likely due to prevention of intracellular ice formation. When 1-cell embryos were vitrified with EFS20 and warmed rapidly, almost all of the embryos developed to blastocysts in vitro. Moreover, when vitrified 1-cell embryos were transferred to recipients at the 2-cell stage, 43% of them developed to term. In conclusion, we developed a vitrification method for 1-cell mouse embryos by rapid warming using cryotubes. |
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| Keywords: | Vitrification Mouse 1-cell embryos Cryotubes Rapid warming Intracellular ice formation Recrystallization |
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