Equilibrium Vitrification of Mouse Embryos

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Oct 14, 2009 - Laboratory of Animal Science,3 College of Agriculture, Kochi University, Nankoku, Kochi, Japan. Institute of Physical and Chemical Research ...
BIOLOGY OF REPRODUCTION 82, 444–450 (2010) Published online before print 14 October 2009. DOI 10.1095/biolreprod.109.077685

Equilibrium Vitrification of Mouse Embryos1 Bo Jin,3 Keiji Mochida,4 Atsuo Ogura,4 Eri Hotta,3 Yukiko Kobayashi,3 Kaori Ito,3 Go Egawa,3 Shinsuke Seki,3 Hiroshi Honda,3 Keisuke Edashige,3 and Magosaburo Kasai2,3 Laboratory of Animal Science,3 College of Agriculture, Kochi University, Nankoku, Kochi, Japan Institute of Physical and Chemical Research (RIKEN) Bioresource Center,4 Tsukuba, Japan

For the cryopreservation of embryos, vitrification has various advantages, but it also has disadvantages because embryos are vitrified with a considerable supercooling (i.e., in nonequilibrium). Here, we tried to develop a novel method in which embryos are vitrified in near-equilibrium. The extent of equilibrium was assessed by examining whether vitrified embryos survive after being kept at 808C. Two-cell embryos of ICR mice were vitrified with ethylene glycol (EG)-based solutions, either EFSa or EFSc solutions, which were mixtures of EG (30%–40%) and an FSa or FSc solution, respectively. The FSa and FSc solutions were PB1 medium containing 30% Ficoll plus 0.5 or 1.5 M sucrose, respectively. In vitro survival rate was high when embryos vitrified with 30%–40% EG (EFS30a, EFS40a, EFS30c, and EFS40c) were warmed rapidly. When embryos were vitrified and then kept at 808C for 4 days, large proportions survived with EFS30c and EFS40c. When embryos were vitrified with EFS35c or EFS40c, the survival rate was high even for those kept at 808C for 10 days. When embryos of ICR and C57BL/6J mice were vitrified with EFS35c or EFS40c and then kept at 808C for 4 days, the survival rate was high even after recooling in liquid nitrogen; a high proportion (75%) of C57BL/6J embryos vitrified with EFS35c developed to term after transfer. In conclusion, we have developed a novel method by which embryos are vitrified in near-equilibrium. This will be a supreme method for cryopreservation, retaining the advantages of both current vitrification and equilibrium slow freezing. cryobiology, cryopreservation, embryo, vitrification

INTRODUCTION For the cryopreservation of mammalian embryos, various methods have been developed. The method most widely used is interrupted slow freezing, where embryos are cooled slowly to around 308C using a programmable freezer before being cooled rapidly with liquid nitrogen (LN2) [1–3]. The embryos are preserved with considerable supercooling (in nonequilibrium) because slow cooling that promotes dehydration is interrupted, and thus the osmolality of the cytoplasm is not sufficiently high (Fig. 1). Above the glass transition temperature of the cytoplasm (approximately 1308C), the vitrified 1 Supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, and the Ministry of Health, Labor and Welfare of Japan. 2 Correspondence: Magosaburo Kasai, Laboratory of Animal Science, College of Agriculture, Kochi University, Nankoku, Kochi 783-8502, Japan. FAX: 81 88 864 5200; e-mail: [email protected]

Received: 22 March 2009. First decision: 13 April 2009. Accepted: 2 October 2009. Ó 2010 by the Society for the Study of Reproduction, Inc. eISSN: 1529-7268 http://www.biolreprod.org ISSN: 0006-3363

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(solid) cytoplasm would turn to liquid, and the supercooled cytoplasm could devitrify [4]. Devitrification of the cytoplasm is the formation of intracellular ice and is lethal to embryos. To prevent this, the sample needs to be warmed rapidly [1, 2, 5, 6]. With original slow freezing, embryos are cooled slowly to approximately 708C before being cooled in LN2 [7]. Embryos frozen by this method would be dehydrated sufficiently, and thus cryopreserved in LN2 with a minimal extent of supercooling (in near-equilibrium; Fig. 1) [4]. With this equilibrium freezing, embryos survive not only after slow warming [7, 8] but also after preservation at 808C for as long as 6 mo (M. Yokoyama, personal communication). Consequently, the original slow freezing has several advantages; 1) embryos are less likely to be damaged during handling for warming, 2) frozen embryos can be evacuated in a freezer at 808C for a certain period in case of trouble with the LN2 tank or for arrangement of samples, and 3) frozen embryos can be transported short distances using dry ice ( 798C). However, slow freezing has disadvantages in that it requires ice seeding and a programmable freezer for slow cooling, and it takes a long time. Since the pioneering reports on the vitrification of mouse embryos [9, 10], the method has been modified and become an alternative to slow freezing for the cryopreservation of mammalian embryos, because vitrification has many advantages; it requires neither ice seeding nor slow cooling nor a programmable freezer, and thus cooling is instant [11]. In addition, vitrification is expected to achieve a high rate of survival because of the absence of ice [12, 13]. With current vitrification, however, embryos are vitrified with considerable supercooling, and thus cryopreserved in nonequilibrium as in interrupted slow freezing (Fig. 1) [4]. Recently, we have shown that the rate of survival of vitrified mouse morulae decreased when they were kept at 808C for 3 min (25%–75%), 60 min (24%–66%), or 24 h (0–14%) before recovery [14]. This shows that embryos were vitrified with a considerable extent of supercooling (in nonequilibrium), and the osmolality of the vitrification solution is not sufficiently high. Therefore, the cytoplasm of embryos must have devitrified at 808C, because most embryos swelled on recovery in PB1 medium, which is a sign of the formation of intracellular ice [15]. This would also be true for all of the vitrification solutions reported so far for the cryopreservation of embryos and oocytes [4, 16]. To prevent devitrification, embryos need to be warmed rapidly, as in the case of interrupted slow freezing. Essentially, there should be four strategies for cryopreservation: nonequilibrium slow freezing, equilibrium slow freezing, nonequilibrium vitrification, and equilibrium vitrification (Fig. 1). Among them, equilibrium vitrification would be the best strategy, but this strategy is missing. If embryos were vitrified in equilibrium, the method would retain the advantages both of original slow freezing and current vitrification. In the present study, we aimed to develop a novel method in which mouse embryos are vitrified in near-equilibrium (with minimal supercooling) by using a vitrification solution with

ABSTRACT

EQUILIBRIUM VITRIFICATION OF EMBRYOS

445 FIG. 1. Schematic representation of an embryo (circle) during nonequilibrium slow freezing, equilibrium slow freezing, nonequilibrium vitrification, and equilibrium vitrification. Darker representation shows higher osmolality. Small hexagons show ice crystals. Black lines show rapid cooling, and dotted lines show slow cooling. CPA, cryoprotectant.

Collection of Embryos

Vitrification Solutions

ICR mice (8–12 wk old; CLEA Japan Inc., Tokyo, Japan) and C57BL/6J mice (8–12 wk old; Charles River Japan, Yokohama, Japan) were used. Female ICR mice were induced to superovulate with i.p. injections of 5 IU of equine chorionic gonadotropin (Serotropin; Teikokuzoki, Tokyo, Japan) and 5 IU of human chorionic gonadotropin (hCG; Puberogen; Sankyozoki, Tokyo, Japan) given 48 h apart and were housed with ICR male mice. On the other hand, female C57BL/6J mice were housed with male mice of the same strain without superovulation; only three to four embryos (on average) were recovered after superovulation, because the proportion of females having a copulation plug was small, whereas five to six embryos were constantly obtained after natural mating. At 45–48 h after the hCG injection (ICR mice) or after the detection of estrus (C57BL/6J mice), two-cell embryos were flushed from the oviducts of mated animals with PB1 medium [17]. For each experiment, 50–112 and 28–37

We tried to develop new vitrification solutions based on the original EFS solution, PB1 medium containing ethylene glycol, Ficoll, and sucrose [13]. The solution was first developed for the vitrification of mouse morulae and has proven effective for mouse embryos at various developmental stages [18, 19], as well as for embryos of many mammalian species [11, 20]. In the present study, original EFS solutions (renamed EFSa) and modified EFS solutions (named EFSc) were used. EFSa solutions (EFS20a, EFS30a, EFS40a, and EFS50a) were composed by mixing ethylene glycol (20%, 30%, 40%, and 50%, v/v) and an FSa solution (80%, 70%, 60%, and 50%, v/v, respectively) [13, 21]. The FSa solution was PB1 medium containing 30% (w/ v) Ficoll PM-70 (average molecular weight, Mr 70 000; GE Healthcare, BioSciences AB, Uppsala, Sweden) plus 0.5 M sucrose [13]. EFS20a was for the pretreatment of embryos, and the other solutions were for vitrification. For near-equilibrium vitrification, we composed EFSc solutions by increasing the osmolality. EFSc solutions (EFS30c, EFS35c, EFS40c, and EFS50c) were composed by mixing ethylene glycol (30%, 35%, 40%, and 50%, v/v) and an FSc solution (70%, 65%, 60%, and 50%, v/v, respectively). The FSc solution was PB1 medium containing 30% (w/v) Ficoll PM-70 plus a higher concentration (1.5 M) of sucrose to promote dehydration of the cytoplasm before cooling. All of the EFSc solutions were for the vitrification of embryos. The osmolality of EFS solutions is given in Table 1.

TABLE 1. The osmolality (moles/kilogram water) of EFS solutions used for vitrification. Solution

EG*

Sucrose

PB1

Total

EFS30a EFS40a EFS30c EFS35c EFS40c

11.0 17.0 15.8 19.9 24.6

0.7 0.7 3.1 3.1 3.1

0.3 0.3 0.3 0.3 0.3

12.0 18.0 19.2 23.3 28.0

* Ethylene glycol.

Vitrification of Embryos Embryos were vitrified by a two-step method reported previously [11, 19, 22]. Embryos were manipulated in a room at 258C 6 18C. A 0.25-ml plastic

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MATERIALS AND METHODS

embryos were collected from ICR and C57BL/6J mice, respectively. Embryos were washed twice with PB1 medium, and those with a normal morphology were used for experiments. Unless otherwise noted, chemicals were purchased from Wako Pure Chemical Industries (Osaka, Japan). All experiments were approved by the Animal Care and Use Committee of Kochi University.

higher osmolality (Fig. 1). To assess the extent of the supercooling, we examined the rate of survival of vitrified embryos kept at 808C before recovery.

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TABLE 2. Survival of 2-cell embryos of ICR mice vitrified with EFSa and EFSc solutions at 1968C and then kept at 808C for 4 days before being warmed in water at 258C. No. of embryos Treatmenta Fresh control Solution control

Vitrified control

Vitrified and kept at

808C for 4 days

Vitrification solution

Vitrified

Recovered

– EFS30a EFS40a EFS50a EFS30c EFS40c EFS50c EFS30a EFS40a EFS30c EFS40c EFS30a EFS40a EFS30c EFS40c

– – – – – – – 81 81 48 45 50 51 50 51

85 80 80 80 51 50 52 81 81 48 45 50 51 50 51

No. of blastomeres Swelledb (%)c 0 0 0 0 0 0 0 2 2 2 1 100 102 2 2

(0) (0) (0) (0) (0) (0) (0) (1) (1) (2) (1) (100)* (100)* (2) (2)

Morphologically normald (%)c 170 160 160 128 102 100 95 158 160 94 88 0 0 98 100

(100) (100) (100) (80)* (100) (100) (91)* (98) (99) (98) (98) (0)* (0)* (98) (98)

No. of embryos developed into expanded blastocystse (%)c 81 72 70 24 46 44 0 74 73 44 40 0 0 43 45

(95) (90) (88) (30)* (90) (88) (0)* (91) (90) (92) (89) (0)* (0)* (86) (88)

a

Nine to twelve embryos were loaded in each straw or treated for solution control or for fresh control and each datum is the total for 5 to 8 replicates. Embryos were observed just after being transferred into PB1 medium. Percentage of recovered embryos; values with an asterisk are significantly different from the fresh control value (*P , 0.05). d Embryos were observed after 1 h of culture in modified M16 medium. e Embryos were examined after 96 h of culture in modified M16 medium. b c

Warming and Recovery of Vitrified Embryos Ethanol was cooled at 808C by placing a Dewar flask containing ethanol in a freezer at 808C for 24 h or more. The vitrified straw sample was taken out of LN2 ( 1968C) and then immersed in ethanol precooled at 808C. The sample was kept at that temperature for 4, 7, or 10 days and then warmed in water at 258C. As the vitrified control, the vitrified sample was warmed without being kept at 808C. In some experiments, some samples kept at 808C for 4 days were recooled in LN2 directly or after being kept in LN2 gas before recovery. When warmed from 808C, the sample was directly immersed in water at 258C. When warmed from the LN2 temperature, the sample was kept in air at room temperature (258C) for 10 sec to allow it to pass through the glass transition temperature of the cytoplasm (approximately 1308C) slowly, and then it was immersed in water at 258C [23]. When the crystallized S-PB1 medium in the straw began to melt in water (after ;7 sec), the straw was taken out and wiped dry, and both ends were cut off. The contents containing embryos were recovered quickly in an empty watch glass by flushing the straw with 0.8 ml of S-PB1 medium using a 1-ml syringe at 258C and agitated gently to promote dilution of the vitrification solution. Embryos were collected and transferred into fresh S-PB1 medium under paraffin oil. At about 5 min after perfusion, the embryos were transferred into fresh PB1 medium under paraffin oil. Upon transfer to PB1 medium, embryos were observed as to whether they had swelled or not, because swelling is a feature of the formation of intracellular ice [14, 15].

Assessment of In Vitro Survival As the basic medium for culturing embryos, M16 medium [24] supplemented with 10 lM ethylenediaminetetraacetic acid-Na, 1 mM glutamine, and 10 lM bmercaptoethanol [22] was used. Three drops (0.2 ml each) of modified M16 medium were placed in a culture dish under paraffin oil and equilibrated in a humidified CO2 incubator (5% CO2, 95% air) at 378C overnight. Vitrified embryos were recovered and cultured in modified M16 medium. As the solution control, embryos were pretreated with EFS20a for 2 min at 258C, then treated with vitrification solution (EFS30a-EFS50a, EFS30cEFS50c) for 60 sec at 258C as for vitrification, and then recovered without cooling. As the fresh control, fresh embryos were cultured without any treatment. After 1 h of culture, the morphology of each blastomere of the embryos was observed under a dissecting microscope. Morphological survival was expressed as the total number of morphologically normal blastomeres per total number of blastomeres of recovered embryos. The survival of embryos was assessed by their ability to develop into expanded blastocysts during 96 h of culture.

Assessment of In Vivo Survival To examine the developmental potential of vitrified embryos in vivo, a total of 62 two-cell embryos were collected from C57BL/6J mice, vitrified with EFS35c, kept at 808C for 4 days, and recooled in LN2. A total of 8–10 embryos were loaded into each straw, and two to three straw samples were vitrified in each experiment. Embryos were warmed and recovered as described above. The experiment was replicated three times. To prepare recipient mice, 8to 14-wk-old ICR females were housed with a vasectomized male of the same strain to induce pseudopregnancy. A total of 11–13 morphologically normal embryos were transferred into the ampullar portions of each pseudopregnant female in the morning of the day when a copulation plug was found. As the fresh control, 12 intact embryos of C57BL/6J mice were transferred to each recipient mouse. After 20 days, the recipients were euthanized, their uteri were removed, viable offspring were recovered, and the uteri were observed for implantation sites.

Statistical Analysis Statistical difference was analyzed using the chi-square test unless the expected frequency was less than five, in which case Fisher exact probability test was used. Statistical significance was evaluated at the P , 0.05 level.

RESULTS Table 2 shows the survival of embryos vitrified with EFSa solutions (EFS30a, EFS40a) and EFSc solutions (EFS30c,

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straw (IMV, L’Aigle, France) was loaded with ;65 mm of PB1 medium containing 0.5 M sucrose (S-PB1 medium), ;15 mm of air, ;3 mm of EFS solution, ;4 mm of air, and ;13 mm of EFS solution [19]. A total of 5–12 embryos were pretreated with EFS20a at 258C by being suspended and washed in the solution for permeation by ethylene glycol and dehydration by sucrose. After 2 min of pretreatment, the embryos were transferred into the larger (;13mm) column of vitrification solution (EFS30a, EFS40a, EFS30c, EFS35c, or EFS40c) for further dehydration, using a fine pipette with a minimal volume of EFS20a. The straw was sealed with a heat sealer. After the embryos were exposed to the vitrification solution at 258C for 60 sec, the straw was placed horizontally on a Styrofoam boat (thickness, 1.5 cm) floated on LN2 in a Dewar flask (inner diameter, 140 mm; temperature on the boat, approximately 1508C) and was left there for 3 min or more before the straw was immersed in LN2. The average rate of cooling between 258C and 1008C was ;3008C/min, which was determined by recording the change in temperature of the EFS solution in the straw using a thermocouple during cooling in LN2 gas. A total of 5–10 straw samples were vitrified for each experiment, and the experiment was replicated five to eight times except for the experiment for embryo transfer, which was replicated three times with 16–28 embryos for each experiment.

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EQUILIBRIUM VITRIFICATION OF EMBRYOS

TABLE 3. Survival of 2-cell embryos of ICR mice vitrified with EFSc solutions at 1968C and then kept at 808C for various periods before being warmed in water at 258C. No. of embryos Treatmenta Fresh control Solution control Vitrified

No. of blastomeres

Time kept at 808C (day)

Vitrification solution

Vitrified

Recovered

– – – – 0 (control)f

– EFS30c EFS35c EFS40c EFS30c EFS35c EFS40c EFS30c EFS35c EFS40c EFS30c EFS35c EFS40c EFS30c EFS35c EFS40c

– – – – 55 51 53 51 50 50 65 60 64 58 60 60

60 60 58 61 52 51 53 51 50 50 65 60 64 58 60 60

4 7 10

Swelledb (%)c 0 0 0 0 0 0 0 2 0 1 34 5 4 82 8 5

(0) (0) (0) (0) (0) (0) (0) (2) (0) (1) (26)* (4) (3) (71)* (7)* (4)

Morphologically normald (%)c 120 120 116 119 102 101 103 100 100 97 96 112 122 34 110 115

(100) (100) (100) (98) (98) (99) (97) (98) (100) (97) (74)* (93)* (95)* (29)* (92)* (96)

No. of embryos developed into expanded blastocystse (%)c 56 55 56 56 47 49 46 43 46 44 32 51 53 0 50 49

(93) (92) (97) (92) (90) (96) (87) (84) (92) (88) (49)* (85) (83) (0)* (83) (82)

a

Nine to twelve embryos were loaded in each straw or treated for solution control or for fresh control and each datum is the total for 5 to 6 replicates. Embryos were observed just after being transferred into PB1 medium. c Percentage of recovered embryos; values with an asterisk are significantly different from the fresh control value (*P , 0.05). d Embryos were observed after 1 h of culture in modified M16 medium. e Embryos were examined after 96 h of culture in modified M16 medium. f Warmed in water at 258C without being kept at 808C. b

To increase the osmolality of the cytoplasm, embryos were vitrified with EFSc solutions. As shown in Table 2, EFS30c and EFS40c were as effective as EFS30a and EFS40a for the vitrification; 89%–92% of vitrified embryos developed into expanded blastocysts. In addition, the survival rates of embryos vitrified with EFS30c and EFS40c were high (86%–88%), even when the embryos were placed in ethanol at 808C and kept there for 4 days before recovery. The survival rates were not significantly different from the rate for vitrified control embryos (89%–92%) or fresh control embryos (95%). To determine the most effective concentration of ethylene glycol for vitrification with EFSc solutions, embryos were vitrified with EFS30c, EFS35c, and EFS40c in LN2 and then kept at 808C for up to 10 days (Table 3). The swelling rate of

TABLE 4. Survival of 2-cell embryos of ICR mice vitrified with EFSc solutions at 1968C, kept at 808C for 4 days, and then recooled in liquid nitrogen (LN2) before being warmed in water at 258C. No. of embryos Treatmenta Fresh control Vitrified

a

Time kept at 808C (day)

Recooling

– 0 (control)f

– –

4



4

LN2 gasg

4

LN2h

Vitrification solution

Vitrified

Recovered

– EFS35c EFS40c EFS35c EFS40c EFS35c EFS40c EFS35c EFS40c

– 56 54 55 56 80 81 79 81

91 56 54 55 55 80 81 79 81

No. of blastomeres Swelledb (%)c 0 0 0 0 2 3 1 0 4

(0) (0) (0) (0) (2) (2) (1) (0) (2)

Morphologically normald (%)c 180 111 108 110 106 155 161 158 158

(99) (99) (100) (100) (96) (97) (99) (100) (98)

Nine to twelve embryos were loaded in each straw or treated for fresh control and each datum is the total for 5 to 8 replicates. Embryos were observed just after being transferred into PB1 medium. c Percentage of recovered embryos; none of the values are significantly different from the fresh control value (P . 0.05). d Embryos were observed after 1 h of culture in modified M16 medium. e Embryos were examined after 96 h of culture in modified M16 medium. f Warmed in water at 258C without being kept at 808C. g Recooled with LN2 gas and then immersed in LN2. h Recooled in LN2 directly. b

No. of embryos developed into expanded blastocystse (%)c 85 55 48 51 49 72 69 71 70

(93) (98) (89) (93) (89) (90) (85) (90) (86)

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EFS40c) that were kept at 808C for 4 days before recovery. When exposed to EFS30a or EFS40a without cooling (solution control), all the embryos were morphologically normal (100%), and a high proportion (88%–90%) developed to the expanded blastocyst stage in culture. Accordingly, a high proportion of embryos vitrified with EFS30a or EFS40a (vitrified control) were normal (98%–99%) and developed into expanded blastocysts (90%–91%). However, when embryos vitrified with EFS30a or EFS40a were placed in ethanol at 808C and kept there for 4 days, all embryos swelled and were damaged. When embryos were exposed to EFS50a, on the other hand, the proportion that developed to the expanded blastocyst stage decreased markedly (30%) without cooling.

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TABLE 5. Survival of 2-cell embryos of C57BL/6J mice vitrified with EFSc solutions at 1968C, kept at 808C for 4 days, and then recooled in liquid nitrogen (LN2) before being warmed in water at 258C. No. of embryos Treatmenta Fresh control Vitrified

Time kept at 808C (day)

Recooling

– 0 (control)f

– –

4



4

LN2g

Vitrification solution

Vitrified

Recovered

– EFS35c EFS40c EFS35c EFS40c EFS35c EFS40c

– 45 42 47 44 45 42

44 42 42 47 41 45 42

No. of blastomeres Swelledb (%)c 0 0 0 0 1 1 2

(0) (0) (0) (0) (1) (1) (2)

Morphologically normald (%)c 88 84 84 94 78 89 80

(100) (100) (100) (100) (95) (99) (95)

No. of embryos developed into expanded blastocystse (%)c 40 37 37 42 36 39 37

(91) (88) (88) (89) (88) (87) (88)

a

Five to ten embryos were loaded in each straw or treated for fresh control and each datum is the total for 5 to 6 replicates. Embryos were observed just after being transferred into PB1 medium. c Percentage of recovered embryos; none of the values are significantly different from the fresh control value (P . 0.05). d Embryos were observed after 1 h of culture in modified M16 medium. e Embryos were examined after 96 h of culture in modified M16 medium. f Warmed in water at 258C without being kept at 808C. g Recooled directly with LN2. b

DISCUSSION The aim of the present study was to develop a nearequilibrium vitrification method for mouse embryos. The new method has the advantages of vitrification and equilibrium slow freezing. The advantages of vitrification are 1) the cooling process is simple and quick and 2) high survival is expected because extracellular ice is absent. The advantages of equilibrium slow freezing are 1) embryos are less likely to be damaged during handling for warming because they are not susceptible to the warming rate, 2) frozen embryos can be evacuated in a conventional freezer at 808C for a certain period of time in case of trouble with the LN2 tank or for the arrangement of samples, and 3) frozen embryos can be transported short distances using dry ice.

To confirm the vitrification of a solution, a differential scanning calorimetry analysis or an x-ray analysis is necessary [12, 25]. In the present study, however, we observed with the naked eye (to examine) whether the vitrification solution vitrified or not during cooling. In our previous study, we observed that vitrification solutions (EFS30a and EFS40a) in straws were transparent in LN2. When they were placed in ethanol at 808C, they remained transparent after 3 min but became opaque after 24 h [14]. This strongly suggests that small, invisible ice crystals had not formed in the transparent solutions in LN2. If the crystals had formed, the solution would have turned opaque quickly at 808C. Therefore, we consider that visual inspection is a handy, clear, and effective method for defining the vitrification of a solution in LN2. We confirmed that EFSc solutions (EFS30c, EFS35c, and EFS40c) were also transparent in LN2 and after storage at 808C for 10 days. From these observations, we considered that ice crystals did not form in the vitrification solutions used in the present study in LN2 (i.e., the solutions were vitrified). We have confirmed that high proportions (90%–91%) of two-cell embryos (ICR) vitrified with EFS30a and EFS40a developed to the expanded blastocyst stage when warmed rapidly without being kept at 808C, but none did so when kept at 808C for 4 days before recovery (Table 2). This shows that TABLE 6. In vivo development of 2-cell embryos of C57BL/6J mice vitrified with EFS35c at 1968C, kept at 808C for 4 days, and then recooled in liquid nitrogen. No. of embryos Recipient no. Fresh control 1 2 3 4 5 Total Vitrified 1 2 3 4 5 Total

Transferred

Implanted (%)

No. of offspring (%)

13 11 11 12 13 60

13 10 11 10 12 56

(100) (91) (100) (83) (92) (93)

10 7 6 9 9 41

(77) (64) (55) (75) (69) (68)

12 12 12 12 12 60

11 12 12 10 12 57

(92) (100) (100) (83) (100) (95)

10 11 8 6 10 45

(83) (92) (67) (50) (83) (75)

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embryos was significantly higher with EFS30c (26%–71%) than with EFS35c (4%–7%) and EFS40c (3%–4%). Accordingly, survival rates were high with EFS35c and EFS40c (82%–85%) but not with EFS30c (0–49%). In the next experiment, we examined whether embryos vitrified with EFS35c or EFS40c and then kept at 808C for 4 days remain viable after being recooled with LN2 gas or LN2. As shown in Table 4, the swelling rate of the embryos was quite low (1%–2%), and the survival rate of embryos was high (85%–90%) with both LN2 gas and LN2. The survival rates were not significantly different from the rate for vitrified control (89%–98%) or fresh control (93%) embryos. Similar results were obtained with embryos of C57BL/6J mice (Table 5). When the embryos were vitrified with EFS35c and EFS40c, kept at 808C for 4 days, and then recooled in LN2, the swelling rate of the embryos was quite low (1%–2%), and the survival rate was high (87%–88%). The survival rate was not significantly different from the rate for vitrified control embryos (88%) or fresh control embryos (91%). Of 62 embryos vitrified for transfer, 60 embryos (97%) were morphologically normal and were transferred to five recipients (Table 6). All of the recipients became pregnant, and in total, 57 embryos (95%) implanted and 45 embryos (75%) developed to term. In the control, a total of 60 fresh embryos were transferred to five recipient mice. All the recipients became pregnant, and 56 embryos (93%) implanted and 41 embryos (68%) developed to term (Table 6). There were no significant differences in the rates between the two groups.

EQUILIBRIUM VITRIFICATION OF EMBRYOS

4) and then to confirm the efficacy with embryos of C57BL/6J mice (Table 5), because this strain is widely used for genetic engineering. When two-cell embryos of C57BL/6J mice were vitrified with EFS35c or EFS40c and then kept at 808C for 4 days before recovery, the survival rate remained as high (88%–89%; Table 5) as that of fresh control embryos (91%; Table 5). In addition, we have confirmed that a high proportion of two-cell embryos of C57BL/6J mice vitrified and kept at 808C for 4 days survive after being recooled in LN2 (87%–88%; Table 5). When the embryos were transferred to recipient mice, the rates of implantation and offspring (95% and 75%, respectively) were as high as those of fresh control (93% and 68%, respectively). This demonstrates that an equilibrium vitrification method was developed in the present study. In the mouse, a large number of transgenic and knockout variants have been produced, many of which are preserved as embryos in LN2. Consequently, the need to transport cryopreserved embryos is increasing. To maintain their viability during transportation, embryos should be kept at below the glass transition temperature of the cytoplasm (approximately 1308C), preferably in LN2 ( 1968C). In most cases, however, sending specimens in LN2 is not allowed, and thus cryopreserved embryos are transported in a dry shipper, a large Dewar flask designed for the shipment of specimens in LN2 in the gas phase. Unfortunately, the dry shipper is large, heavy, and expensive. Therefore, an advantage of equilibrium vitrification would be that cryopreserved embryos can be transported using dry ice and can be represerved in LN2. In conclusion, we have developed a novel method by which embryos are vitrified with minimal supercooling (i.e., in near equilibrium). This was a missing approach (Fig. 1), and it would become a supreme method because it has clear advantages over existing methods. Practically, the method is as simple and quick as current vitrification. In addition, it has advantages that current vitrification does not have. Embryos preserved by this method are less susceptible to damage during handling. This method would enable the temporary evacuation of vitrified samples into a freezer at 808C in an emergency or for the arrangement of cryopreserved samples, as well as the handy short-range transportation of vitrified embryos using dry ice. ACKNOWLEDGMENT We thank Dr. Minesuke Yokoyama (Niigata University, Niigata, Japan) for valuable information.

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embryos were vitrified with considerable supercooling (in nonequilibrium) and damaged by devitrification of the cytoplasm (i.e., the formation of intracellular ice), because all of the embryos swelled upon recovery in PB1 medium (Table 2). We have already shown in vitrified mouse blastocysts [15] and morulae [14] that embryos injured by intracellular ice swell upon recovery in PB1 medium. The results of the present study show that this characteristic is also true for two-cell embryos, because the swelling rate was inversely related to the survival rate (Tables 2–5). To prevent the formation of intracellular ice during the period at 808C, the embryos need to have been vitrified in near-equilibrium (with minimal supercooling). To vitrify embryos in near-equilibrium, it is necessary to compose a vitrification solution with higher osmolality. One way to increase osmolality in EFS solutions would be to increase the concentration of ethylene glycol. However, the toxicity of the solution would increase as the concentration of permeating cryoprotectant increased [13]. Actually, when twocell embryos were suspended in EFS50a for 1 min at 258C, the survival rate decreased considerably without cooling (30%; Table 2), and none survived after vitrification (data not shown). This is consistent with our recent finding that the survival rate of mouse morulae was quite low after vitrification with EFS50a (0–13%) [14]. Another way to increase osmolality in EFS solutions would be to increase the concentration of sucrose. In the original EFS solution (EFS40a), sucrose is included to promote dehydration before cooling, to reduce the total amount of permeating cryoprotectant (ethylene glycol) in embryos, which will decrease the toxicity, and to prevent excessive swelling of embryos during removal of the cryoprotectant, because sucrose remains outside the cell and increases extracellular osmolality [11, 13]. In EFSa solutions, however, the concentration of sucrose (0.7 mol/kg water) did not appear to be high enough to prevent devitrification of the cytoplasm when kept at 808C (Table 2). Therefore, we composed new vitrification solutions (EFSc solutions) in which the concentration of sucrose was increased to 3.1 mol/kg water. The increase in the sucrose concentration increases not only the molality (moles/kilogram water) of sucrose but also that of ethylene glycol, because the increase in the volume of sucrose decreases the volume of water. Eventually, the osmolality of EFSc solution was increased greatly (Table 1). When two-cell embryos (ICR mice) were vitrified with EFS30c and EFS40c, a high proportion (86%–88%) survived as expected after being kept at 808C for 4 days before recovery (Table 2). In a preliminary experiment, we composed EFSb solutions with the FSb solution, which contained 1.0 M sucrose. However, EFS30b and EFS40b were much less effective than EFS30c and EFS40c (data not shown). With EFS35c and EFS40c, a high proportion (82%–83%) of embryos survived after being kept at 808C even for 10 days. With EFS30c, on the other hand, the swelling rate increased (26%) and the survival rate decreased when embryos were kept at 808C for 7 days (Table 3), probably because the osmolality of the solution was not sufficiently high to prevent the formation of intracellular ice. In EFS30c, EFS35c, and EFS40c, the molality of sucrose is the same (3.1 mol/kg water), but the molality of ethylene glycol is different (15.8, 19.9, and 24.6 mol/kg water, respectively; Table 1). Considering the practical applications of the new method, we used two-cell embryos, because mouse embryos are most commonly cryopreserved at this stage, especially for the preservation of genetically engineered variants [26]. We tried to find suitable conditions for embryos of ICR mice (Tables 2–

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