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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. [ABSTRACT FROM AUTHOR]

To cryopreserve cells, it is essential to avoid intracellular ice formation during cooling and warming. One way to do so is to subject them to procedures that convert cell water into a non-crystalline glass. Current belief is that to achieve this vitrification, cells must be suspended in very high concentrations of glass-inducing solutes (i.e., ⩾6molal) and cooled at very high rates (i.e., ≫1000°C/min). We report here that both these beliefs are incorrect with respect to the vitrification of 8-cell mouse embryos. In this study, precompaction 8-cell embryos were vitrified in several dilutions of EAFS10/10 using various cooling rates and warming rates. Survival was based on morphology, osmotic functionality, and on the ability to develop to expanded blastocysts. With a warming rate of 117,500°C/min, the percentages of embryos vitrified in 1×, 0.75×, and 0.5× EAFS that developed to blastocysts were 93%, 92%, and 83%, respectively. And the percentages of morphological survivors that developed to expanded blastocysts were 100%, 92%, and 97%, respectively. Even when the solute concentration of the EAFS was reduced to 33% of normal, we obtained 40% functional survival of these 8-cell embryos. [ABSTRACT FROM AUTHOR]

Abstract: As a step to develop a cryopreservation method for zebrafish oocytes, we investigated the cryobiological properties of immature oocytes at stage III by examining their ability to mature and to develop into hatching embryos after fertilization. When oocytes were chilled at −5°C for 30min, the maturation rate decreased, but the rates of fertilization and hatching were not significantly different from those of controls. When oocytes were exposed to hypotonic solutions for 60min at 25°C, the rates of maturation, fertilization, and hatching decreased in a solution with 0.16Osm/kg or below. When oocytes were exposed to hypertonic solutions (containing sucrose) at 25°C for 30min, the maturation rate decreased in solution with 0.51Osm/kg, whereas the hatching rate decreased with lower osmolality (0.40Osm/kg). In an experiment on the toxicity of cryoprotectants (∼10%, at 25°C), it was found that glycerol and ethylene glycol were toxic both by the assessment of maturation and hatching. Propylene glycol, DMSO and methanol were less toxic by the assessment of maturation, but were found to be toxic by the assessment of hatching. Methanol was the least toxic, but it was less effective to make a solution vitrify than propylene glycol. Therefore, a portion of methanol was replaced with propylene glycol. The replacement increased the toxicity, but could be effective to reduce chilling injury at −5°C. These results clarified the sensitivity of immature oocytes to various cryobiological properties accurately, which will be useful for realizing cryopreservation of zebrafish oocytes. [Copyright &y& Elsevier]

Abstract: There is great interest in achieving reproducibly high survivals of mammalian oocytes (especially human) after cryopreservation, but the results to date have not matched the interest. A prime cause of cell death is the formation of more than trace amounts of intracellular ice, and one strategy to avoid it is vitrification. In vitrification procedures, cells are loaded with high concentrations of glass-inducing solutes and cooled to −196°C at rates high enough to presumably induce the glassy state. In the last decade, several devices have been developed to achieve very high cooling rates. Nearly all in the field have assumed that the cooling rate is the critical factor. The purpose of our study was to test that assumption by examining the consequences of cooling mouse oocytes in a vitrification solution at four rates ranging from 95 to 69,250°C/min to −196°C and for each cooling rate, subjecting them to five warming rates back above 0°C at rates ranging from 610 to 118,000°C/min. In samples warmed at the highest rate (118,000°C/min), survivals were 70% to 85% regardless of the prior cooling rate. In samples warmed at the lowest rate (610°C/min), survivals were low regardless of the prior cooling rate, but decreased from 25% to 0% as the cooling rate was increased from 95 to 69,000°C/min. Intermediate cooling and warming rates gave intermediate survivals. The especially high sensitivity of survival to warming rate suggests that either the crystallization of intracellular glass during warming or the growth by recrystallization of small intracellular ice crystals formed during cooling are responsible for the lethality of slow warming. [Copyright &y& Elsevier]

Abstract: The formation of more than trace amounts of ice in cells is lethal. The two contrasting routes to avoiding it are slow equilibrium freezing and vitrification. The cryopreservation of mammalian oocytes by either method continues to be difficult, but there seems a slowly emerging consensus that vitrification procedures are somewhat better for mouse and human oocytes. The approach in these latter procedures is to load cells with high concentrations of glass-inducing solutes and cool them at rates high enough to induce the glassy state. Several devices have been developed to achieve very high cooling rates. Our study has been concerned with the relative influences of warming rate and cooling rate on the survival of mouse oocytes subjected to a vitrification procedure. Oocytes suspended in an ethylene glycol–acetamide–Ficoll–sucrose solution were cooled to −196°C at rates ranging from 37 to 1827°C/min between 20 and −120°C, and for each cooling rate, warmed at rates ranging from 139 to 2950°C/min between −70 and −35°C. The results are unambiguous. If the samples were warmed at the highest rate, survivals were >80% over cooling rates of 187–1827°C/min. If the samples were warmed at the lowest rate, survivals were near 0% regardless of the cooling rate. We interpret the lethality of slow warming to be a consequence of it allowing time for the growth of small intracellular ice crystals by recrystallization. [Copyright &y& Elsevier]

Abstract: The occurrence of intracellular ice formation (IIF) during freezing, or the lack there of, is the single most important factor determining whether or not cells survive cryopreservation. One important determinant of IIF is the temperature at which a supercooled cell nucleates. To avoid intracellular ice formation, the cell must be cooled slowly enough so that osmotic dehydration eliminates nearly all cell supercooling before reaching that temperature. This report is concerned with factors that determine the nucleation temperature in mouse oocytes. Chief among these is the concentration of cryoprotective additive (here, glycerol or ethylene glycol). The temperature for IIF decreases from −14°C in buffered isotonic saline (PBS) to −41°C in 1M glycerol/PBS and 1.5M ethylene glycol/PBS. The latter rapidly permeates the oocyte; the former does not. The initial extracellular freezing at −3.9 to −7.8°C, depending on the CPA concentration, deforms the cell. In PBS that deformation often leads to IIF; in CPA it does not. The oocytes are surrounded by a zona pellucida. That structure appears to impede the growth of external ice through it, but not to block it. In most cases, IIF is characterized by an abrupt blackening or flashing during cooling. But in some cases, especially with dezonated oocytes, a pale brown veil abruptly forms during cooling followed by slower blackening during warming. Above −30°C, flashing occurs in a fraction of a second. Below −30°C, it commonly occurs much more slowly. We have observed instances where flashing is accompanied by the abrupt ejection of cytoplasm. During freezing, cells lie in unfrozen channels between the growing external ice. From phase diagram data, we have computed the fraction of water and solution that remains unfrozen at the observed flash temperatures and the concentrations of salt and CPA in those channels. The results are somewhat ambiguous as to which of these characteristics best correlates with IIF. [Copyright &y& Elsevier]

The permeability to water and cryoprotectants of the plasma membrane affects the suitable condition for cryopreservation of the cell. Previously, we showed that water, glycerol, ethylene glycol, Me2SO, and acetamide move through mouse morulae by facilitated diffusion. We also showed that the facilitated diffusion of water, glycerol, and ethylene glycol relies on aquaporin 3, an aquaglyceroporin (Edashige et al., 2007), and suggested that AQP9, another aquaglyceroporin, can transport Me2SO, acetamide, and other cryoprotectants when AQP9 was exogenously expressed in mouse oocytes (Edashige et al., in Cryo 2011). In the present study, we examined the transporting-specificity of AQP9 endogenously expressed in mouse morulae. In addition, we examined whether AQP9 expressed in morulae was involved in the tolerance to cryopreservation. As shown in previous studies, the suppression of the endogenous expression of AQP9 in morulae decreased the permeability to Me2SO and acetamide but not that to other cryoprotectants. And the exogenous expression of AQP9 in mouse oocytes, in which water/cryoprotectant channels are not expressed at the functional level, significantly increased not only the permeability to Me2SO and acetamide but also to glycerol and ethylene glycol. Cystic fibrosis transmembrane conductance regulator (CFTR), which has been reported to modulate the transporting-property of AQP9, was markedly expressed in oocytes but not in morulae. The suppression of the endogenous expression of CFTR in oocytes decreased the permeability to glycerol and ethylene glycol of oocytes enhanced by exogenous expression of AQP9 but not that to Me2SO and acetamide. These results indicate that the transporting-property of AQP9 is modulated by CFTR, and suggest that AQP9 endogenously expressed in morulae transports only Me2SO and acetamide because CFTR is not expressed in morulae. When mouse morulae were vitrified with DFS20, a Me2SO -based solution, by a one-step method, most of intact morulae survived and had the ability to develop into blastocysts after warming, whereas only 40% of AQP9-suppressed morulae survived and no embryos had the ability to develop, suggesting that AQP9 plays an important role in the tolerance to vitrification. These results will provide important information for understanding the movement of cryoprotectants in mouse embryos. Source of funding: The Japan Society for the Promotion of Science. Conflict of interest: None declared. keisuke@cc.kochi-u.ac.jp [Copyright &y& Elsevier]

Although numerous inbred lines and endangered wild populations of medaka exist, the only method currently available for preserving the resources is to rear live individuals, as protocols for cryopreservation of fish eggs or embryos have not yet been successfully established due to their large size. However, rearing live individuals is always associated with the risk of mutation due to mobile elements, and the loss of parent fish through infectious disease or accidents related to rearing conditions and equipment. Our group developed a novel method named surrogate broodstock technology. By transplanting donor germ cells into recipient fish of another species, the donor germ cells mature into sperm or oocytes in recipient gonads (Takeuchi et al., 2004; Okutsu et al., 2006; Yoshizaki et al., 2011). More importantly, the germ cells cryoporeserved can be differentiated into functional oocytes (Okutsu et al., 2007; Lee et al., 2013). In this study, we aimed to cryopreserve spermatogonia that can be differentiated into both sperm and eggs. When medaka spermatogonia were dehydrated with hypertonic solutions at 25°C, the survival was decreased in 0.5 M sucrose solution. However, survival rates were improved, when dehydration was carried out at 0°C. Toxicity assay of cryoprotectant (−10%, 25°C) revealed that propylene glycol (PG) was the least toxic, while ethylene glycol (EG) proved less toxic than glycerol and Me2SO. Based on these results, vitrification solutions containing ficoll and sucrose were based with either PG or EG and the testes were vitrified after exposure to them at 0°C. Although survival of cells treated with PG was low, survival rate of 44.9% was obtained using vitrification solution containing EG. Thus, vitrification method of medaka whole testis includes spermatogonia was established. When vitrified spermatogonia from vasa-GFP transgenic strain (body color: orange) were transplanted into larvae of non-transgenic strain (body color: black), female recipients produced oocytes showing green fluorescence. In their gonads, however, there were recipient-derived oocytes together with donor-derived green fluorescent oocytes. Since recipients producing gametes derived only from transplanted donor germ cells are desirable, infertile triploids were used as the recipients in the next experiment. Triploid medaka was prepared by placing fertilized eggs at 41°C water for 5 min (Naruse et al., 1985). When cryopreserved spermatogonia were transplanted into triploid larvae, the mature recipients produced only sperm and eggs showing green fluorescence, which is the donor-derived characteristic. Moreover, the body colors of the offspring obtained by mating the recipient males and females were orange (100%, 1169/1169), suggesting that all of them were derived from cryopreserved spermatogonia. Thus, we succeeded to produce the donor-derived offspring by allogenic transplantation of cryopreserved spermatogonia in medaka. These techniques will facilitate the establishment of a stable and reliable system for preserving valuable medaka strains semi-permanently. Source of funding: Grant-in-Aid for Research Fellow of the Japan Society for the Promotion of Science. Conflict of interest: None declared. sekishinsuke@hotmail.com [ABSTRACT FROM AUTHOR]

Abstract: In vitrified solutions, ice can form during warming if the concentration of the cryoprotectant is insufficient. For the cryopreservation of cells, ice is innocuous when it remains outside the cell, but intracellular ice (ICI) is lethal. We tried to estimate the conditions in which ICI forms in vitrified mouse morulae during warming. The solutions for the experiments (EFS10–EFS50) contained 10–50% ethylene glycol plus Ficoll plus sucrose. When vitrified EFS20, EFS30, and EFS40 were kept at −80°C, they remained transparent after 3min, but turned opaque after 60min (EFS20, EFS30) or 24h (EFS40). Morulae were vitrified with EFS solutions after exposure for 30–120s at 25°C. They were warmed by various methods and survival was assessed in culture. After rapid warming (control), survival was high with EFS30 (79–93%) and EFS40 (96–99%). After slow warming, survival decreased with both EFS30 (48–62%) and EFS40 (44–64%). This must be from the formation of ICI. To examine the temperature at which ICI formed during slow warming, vitrified embryos were kept at various sub-zero temperatures during warming. Survival with EFS30 and EFS40 decreased on keeping samples for 3min at −80 (25–75%), −60 (7–49%), −40 (0–41%), or −20°C (26–60%). When samples were kept at −80°C for 24h, the survival decreased to 0–14%. These results suggest that ICI forms at a wide range of temperatures including −80 and −20°C, more likely between −60 and −40°C, and the ice forms not only quickly but also slowly. [Copyright &y& Elsevier]

Abstract: The permeability of the plasma membrane plays a crucial role in the successful cryopreservation of oocytes/embryos. To identify a stage feasible for the cryopreservation of teleost oocytes, we investigated the permeability to water and various cryoprotectants of medaka (Oryzias latipes) oocytes at the germinal vesicle (GV) and metaphase II (MII) stages. In sucrose solutions, the volume changes were greater in GV oocytes than MII oocytes. Estimated values for osmotically inactive volume were 0.41 for GV oocytes and 0.74 for MII oocytes. Water-permeability (μm/min/atm) at 25°C was higher in GV oocytes (0.13±0.01) than MII oocytes (0.06±0.01). The permeability of MII oocytes to various cryoprotectants (glycerol, propylene glycol, ethylene glycol, and DMSO) was quite low because the oocytes remained shrunken during 2h of exposure in the cryoprotectant solutions at 25°C. When the chorion of MII oocytes was removed, the volume change was not affected, except in DMSO solution, where dechorionated oocytes shrunk and then regained their volume slowly; the P DMSO value was estimated to be 0.14±0.01×10−3 cm/min. On the other hand, the permeability of GV oocytes to cryoprotectants were markedly high, the P s values (×10−3 cm/min) for propylene glycol, ethylene glycol, and DMSO being 2.21±0.29, 1.36±0.18, and 1.19±0.01, respectively. However, the permeability to glycerol was too low to be estimated, because GV oocytes remained shrunken after 2h of exposure in glycerol solution. These results suggest that, during maturation, medaka oocytes become less permeable to water and to small neutral solutes, probably by acquiring resistance to hypotonic conditions before being spawned in fresh water. Since such changes would make it difficult to cryopreserve mature oocytes, immature oocytes would be more suitable for the cryopreservation of teleosts. [Copyright &y& Elsevier]