Equilibrium Vitrification of Mouse Embryos1

Jin, Bo; Mochida, Keiichi; Ogura, Atsuo; Hotta, Eri; Kobayashi, Yukiko; Ito, Kaori; Egawa, Go; Seki, Shinsuke; Honda, Hiroshi; Edashige, Keisuke; Kasai, Magosaburo

doi:10.1095/biolreprod.109.077685

Cited by 25 publications

(12 citation statements)

References 24 publications

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“…We undertook further fine optimization by extending the storage time to 160 days because the previous method by Jin et al [3] cannot maintain the viability of embryos for more than 30 days at –80°C (Edashige and Kasai, unpublished). The concentration of EG was adjusted to 42.5%, 45%, or 50%.…”

Section: Resultsmentioning

confidence: 99%

“…Currently, vitrification is the technique of first choice for mouse embryo cryopreservation because of its technical simplicity, but it is still under intense research pressure for improvements. In general, vitrification requires neither slow cooling nor a programmable freezer, so the procedure is very rapid [3]. However, to avoid cryodamage to embryos, they should be kept supercooled below –130°C during cryopreservation and need to be warmed rapidly at recovery [3], [4].…”

Section: Introductionmentioning

confidence: 99%

“…In general, vitrification requires neither slow cooling nor a programmable freezer, so the procedure is very rapid [3]. However, to avoid cryodamage to embryos, they should be kept supercooled below –130°C during cryopreservation and need to be warmed rapidly at recovery [3], [4]. If embryos are allowed to stay above the devitrification temperature, intracellular ice crystal formation occurs and this damages the embryos irreversibly.…”

Section: Introductionmentioning

confidence: 99%

“…We have developed a new vitrification solution that has a higher osmolality than the existing vitrification solutions used to prevent devitrification of the cytoplasm at around –80°C [3]. The osmolality of the solution is about 19–28 mol/kg water, provided by high concentrations of ethylene glycol (EG) and sucrose.…”

Section: Introductionmentioning

confidence: 99%

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High Osmolality Vitrification: A New Method for the Simple and Temperature-Permissive Cryopreservation of Mouse Embryos

Mochida

Hasegawa

et al. 2013

PLoS ONE

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Procedures for cryopreserving embryos vary considerably, each having its specific advantages and disadvantages in terms of technical feasibility, embryo survival yield, temperature permissibility and species- or strain-dependent applicability. Here we report a high osmolality vitrification (HOV) method that is advantageous in these respects. Cryopreservation by vitrification is generally very simple, but, unlike slow freezing, embryos should be kept at a supercooling temperature (below –130°C) to avoid cryodamage. We overcame this problem by using an HOV solution containing 42.5% (v/v) ethylene glycol, 17.3% (w/v) Ficoll and 1.0 M sucrose. This solution is more viscous than other cryopreservation solutions, but easy handling of embryos was assured by employing a less viscous equilibration solution before vitrification. Most (>80%) embryos cryopreserved in this solution survived at –80°C for at least 30 days. Normal mice were recovered even after intercontinental transportation in a conventional dry-ice package for 2–3 days, indicating that special containers such as dry shippers with liquid nitrogen vapor are unnecessary. The HOV solution could also be employed for long-term storage in liquid nitrogen, as with other conventional cryoprotectants. Finally, we confirmed that this new vitrification method could be applied successfully to embryos of all six strains of mice we have tested so far. Thus, our HOV method provides an efficient and reliable strategy for the routine cryopreservation of mouse embryos in animal facilities and biomedical laboratories, and for easy and cheap transportation.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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High Osmolality Vitrification: A New Method for the Simple and Temperature-Permissive Cryopreservation of Mouse Embryos

Mochida

Hasegawa

et al. 2013

PLoS ONE

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“…But, if the concentration of sucrose in the vitrification solution was increased three-fold, a high percentage survived being held at −80°C for 4 to 10 days after initial cooling to −196°C. (Jin et al 2010). …”

Section: Discussionmentioning

confidence: 99%

Stability of mouse oocytes at −80 °C: the role of the recrystallization of intracellular ice

Seki¹,

Mazur²

2011

Reproduction

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The germplasm of mutant mice is stored as frozen oocytes/embryos in many facilities world-wide. Their transport to and from such facilities should be easy and inexpensive with Dry Ice at −79 °C. The purpose of our study was to determine the stability of mouse oocytes with time at that temperature. The metaphase II oocytes were cryopreserved with a vitrification solution (EAFS10/10) developed by M. Kasai and colleagues. Two procedures were followed. In one, the samples were cooled at 187 °C/min to −196 °C, warmed to −80 °C, held at −80 °C for 1 hour to three months, and warmed to 25 °C at one of three rates. With the highest warming rate (2,950 °C/min), survival remained at 75% for the first month, but then slowly declined to 40% over the next two months. With the slowest warming (139 °C/min) survival was only ~5% even at 0 time at −80 °C. In the second procedure, the samples were cooled at 294 °C/min to −80 °C (without cooling to −196 °C) and held for up to 3 months before warming at 2,950 °C/min. Survival was ~90% after 7 days and dropped slowly to 35% after 3 months. We believe that small non-lethal quantities of intracellular ice formed during the cooling and that the intracellular crystals increased to a damaging size by recrystallization during the 3-months storage at −80 °C. From the practical point of view, this protocol yields sufficient stability to make it feasible to ship oocytes world-wide in Dry Ice.

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Equilibrium vitrification of mouse embryos at various developmental stages

Jin

Mochida

Ogura

et al. 2012

Molecular Reproduction Devel

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Previously, we developed a new method by which 2-cell mouse embryos can be vitrified in liquid nitrogen in a near-equilibrium state, and then kept at -80°C for several days. In the present study, we examined whether or not the method was effective for mouse embryos at other developmental stages. Eight-cell embryos, morulae, and expanded blastocysts of ICR mice were vitrified with ethylene glycol-based solutions, named EFSc because of their composition of ethylene glycol (30-40%, v/v) and FSc solution. The FSc solution was PB1 medium containing 30% (w/v) Ficoll PM-70 plus 1.5 M sucrose. The extent of equilibrium was assessed by examining how well vitrified embryos survived after being kept at -80°C. When 8-cell embryos and morulae were vitrified with EFS35c or EFS40c and then kept at -80°C, the survival rate was high even after 4 days in storage and remained high after re-cooling in liquid nitrogen. On the other hand, the survival of vitrified-expanded blastocysts kept at -80°C was low. Therefore, 8-cell embryos and morulae can be vitrified in a near-equilibrium state using the same method as for 2-cell embryos. A high proportion of C57BL/6J embryos at the 2-cell, 8-cell, and morula stages vitrified with EFS35c developed to term after transportation on dry ice, re-cooling in liquid nitrogen, and transfer to recipients. In conclusion, the near-equilibrium vitrification method, which is effective for 2-cell mouse embryos, is also effective for embryos at the 8-cell and morula stages. The method would enable handy transportation of vitrified embryos using dry ice.

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Equilibrium Vitrification of Mouse Embryos1

Cited by 25 publications

References 24 publications

High Osmolality Vitrification: A New Method for the Simple and Temperature-Permissive Cryopreservation of Mouse Embryos

High Osmolality Vitrification: A New Method for the Simple and Temperature-Permissive Cryopreservation of Mouse Embryos

Stability of mouse oocytes at −80 °C: the role of the recrystallization of intracellular ice

Equilibrium vitrification of mouse embryos at various developmental stages

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