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Simple and efficient methods for vitrification of mammalian embryos
SCIENCE Animal Reproduction Science 42 (1996) 67-75
Simple and efficient methods for vitrification of mammalian embryos Magosaburo Kasai
I
Laboratory of Animal Science, College of Agriculture, Kochi University, Nankoku, Kochi 783, Japan
Abstract greatly simplifies the cooling process for embryo cryopreservation, and eliminates caused by extracellular ice. However, embryos may be injured by various factors, toxicity of the solution, intracellular ice, fracture damage and osmotic swelling. By the effect of each of these factors, refined procedures for simple and efficient have been developed for mouse embryos.
Vitrification
any injuries such as the minimising vitrification
Keywords: Vitrification; Cryopreservation; Embryo; Mouse; Ethylene glycol
1. Introduction
If embryos can be cryopreserved efficiently, the technique can be applied to a wide range of mammalian species, i.e. farm animals, laboratory animals, wild animals and human. However, during the processing and cooling of embryos, or during recovery of stored embryos into a physiological solution, they are at risk of injury by various factors, including: (1) toxicity of cryoprotectants; (2) chilling injury; (3) physical injury by extracellular ice; (4) toxicity of concentrated electrolytes; (5) formation and growth of intracellular ice; (6) fracture damage; (7) osmotic swelling. By circumventing these obstacles, various mammalian embryos at various stages of preimplantation have been successfully cryopreserved using several methods. However, there is a need for improvement in the method because post-thawing survival is variable depending on the species, the strain, the developmental stage and the quality of the embryo. In addition, a simpler method would be preferable. Using the controlled slow freezing method established by Whittingham et al. (19721, embryos equilibrated in l-l.5 M cryoprotectant must be cooled slowly so that cellular ’Tel.: 8 I-888-5 194; fax: 8 I-888-64-5200. 0378-4320/%/$15.00
6 1996 Elsevier Science B.V. All rights reserved.
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M. Kasai/Animal Reproduction Science 42 (1996) 67-75
contents are concentrated by gradual dehydration caused by the growth of extracellular ice. When the sample is cooled in liquid nitrogen, the cytoplasm of the embryos, together with the extracellular concentrated fraction, will turn to glassy solids without ice formation by an extreme elevation in viscosity, i.e. they will vitrify. The disadvantage of this slow freezing method is that controlled cooling requires time and elaborate equipment. If embryos are suspended, at 0°C or above, in a concentrated solution which will vitrify in liquid nitrogen, they are expected to survive direct cooling in liquid nitrogen, with both extracellular solution and cytoplasm being vitrified. Based on this idea, Rall and Fahy (1985) devised a vitrification method for mouse embryos. This method not only greatly simplifies the cooling process but also eliminates all the physical and chemical injuries caused by extracellular ice. In addition, it may even lessen the chilling injury, which is observed in embryos of some species, by passing through critical temperatures very rapidly. However, embryos can still be injured by toxicity, intracellular ice formation, fracture and osmosis. By minimising the chances of damage to the embryos by each of these mechanisms, simple and efficient vitrification methods can be developed. The primary obstacle in vitrification is toxicity of the cryoprotectant.
2. Low toxicity vitrification solutions consisting of three categories of agents 2.1. Permeating cryoprotectants The essential component of a vitrification solution is the permeating agent. Rall and Fahy (1985) used a solution containing 6.5 M (in total) of dimethyl sulphoxide (DMSO), acetamide and propylene glycol as permeating agents. Since their pioneering report, however, numerous vitrification solutions have been devised using various cryoprotectants in combination or as a single permeating component (Table 1). Because the permeating agent is responsible for the toxicity (Kasai et al., 1992a), we have tested the relative toxicity of five such agents on mouse morulae by suspending them in phosphate-buffered saline (PBS) containing 30% (v/v> cryoprotectant for 20 min at 20°C. Survival rates assessed by the developmental potential in vitro showed that ethylene glycol is the least toxic (98%), followed by glycerol (88%) and then DMSO (68%). Propylene glycol (16%) and acetamide (0%) are very toxic (Kasai, 1994). Another important characteristic for evaluating the cryoprotectant is its permeating property. In general, rapidly permeating agents are favoured, because the exposure time before rapid cooling can be shortened, and because they are more likely to diffuse out of the cell rapidly, thus preventing osmotic injury. To compare the permeating speed of cryoprotectants, we observed the volume change of mouse morulae during 5 min of suspension in five cryoprotectant solutions. As shown in Fig. l(a), embryos in ethylene glycol shrank the least and regained their original volume most quickly, indicating that of the five agents, ethylene glycol permeates the most rapidly. When toxicity and permeating property of the cryoprotectants are considered, ethylene glycol was the best candidate tested for the permeating component of a vitrification solution and glycerol may be the second choice, at least for mouse morulae.
M. Kasai/Animal Reproduction Science 42 (1996) 67-75
69
Table I Vitrification solutions used for the cryopmservation of mammalian embryos Reference
Cryoprotective additives
Rall and Fahy, 1985 Rail and Fahy, 1985 Scheffen et al., 1986 Rall, 1987 Rail, 1987 Nakagata, 1989 Smorag et al., 1989 Liehman et al., 1990 Landa and Tepla, 1990 Kasai et al., 1990 Schiewe et al., 1991 Ishimori et al., 1992 Leibo and &la, 1993 Tachikawa et al., 1993 Yoshino et al., 1993 Zhu et al., 1993 Tada et al., 1993 Ah and Shelton, 1993 Saito et al., 1994
Permeating agents
Non-permeating agents
(%, v/v)
Macromolecules a (%, w/v)
18.6D+13.4A+9.6P 16.7 D+ 12 A+8.7 P 25G+25P 40.3 P 47.4 G 14.2D+5.1 A+22P 35G+35P 50 G 30G 4OE 47.4 G 25E+25D 44E 4QG 40E 30E 19.5 D+ 20.2 P 31 E 20E+20G
6 PEG 5.4 PEG
Saccharides (MI
6 PEG 6 PEG -
18 Ficoll 6 BSA 7.5 PVP 18 Ficoll 18 Ficoll 21 Ficoll
1.o sue 0.3 sue
0.3 sue 0.3 Treha 0.35 sue 1.o sue 1.o sue 0.75 (Sue + Glc)
a Serum and O.l-0.3% BSA am not shown. D, dimethyl sulphoxide; A, acetamide; P, propylene glycol; G. glycerol; E, ethylene glycol; PEG, polyethylene glycol; BSA, bovine serum albumin; Sue, sucrose; Treha, trehalose; Glc, glucose.
a) YORUlA
“V
I--
b) ZYGOTE
EG
-AA -
DMSO
-
GL
iu _I
or2345012345 Exposure time (min)
Fig. 1. The volume change of mouse (a) morulae and (b) one-cell zygotes in various cryoprotectant solutions at 20°C. Bach embryo was held by a holding pipette connected to a micromanipulator and intrcduced into a cryoprotectant solution with the aid of a covering pipette. The volume change was calculated from the images recorded by a time-lapse video tape recorder. EG, 10% (v/v) ethylene glycol; AA, 1.5 M acetamide; DMSO, 10% (v/v) dimethyl sulphoxide; PG, 10% (v/v) propylene glycol; GL, 10% (v/v) glycerol (Pedro et al., unpublished data).
70
M. Kasai/Animal Reproduction Science 42 (1996) 67-75
2.2. Macromolecules
It is known that incorporation of a macromolecule promotes vitrification of a solution (Fahy et al., 1984). Although macromolecules are generally less toxic, we have found in a toxicity test that Ficoll 70 was less toxic than polyethylene glycol when it was mixed with 40% (v/v) ethylene glycol (Kasai, 1994). Of. other macromolecules, polyvinyl pyrrolidone (Fahy et al., 1984; Leibo and Oda, 1993) and bovine serum albumin (BSA) (Schiewe et al., 1991) have been used. 2.3. Small saccharides As a non-permeating agent, sucrose exerts a considerable osmotic effect. In a vitrification solution containing 40% (v/v> ethylene glycol and 18% (w/v) Ficoll, it was found that incorporation of sucrose reduces the toxicity, probably because the amount of intracellular cryoprotectant is reduced (Kasai et al., 1990). The beneficial effect is not specific to sucrose, with other saccharides, such as trehalose (Yoshino et al., 1993) also being effective. It was shown that sucrose and other saccharides such as glucose, fructose and galactose are virtually non-toxic (Kasai, 1986). 2.4. Composition of vitrijkation solutions After considering the different properties of agents in each category, we developed a vitrification solution, designated EFS40, containing 40% (v/v> ethylene glycol, 18% (w/v) Ficoll and 0.3 M sucrose (Kasai et al., 1990). A precise protocol for preparing this solution has been described elsewhere (Kasai, 1995). A similar solution, CFS40, in which 40% ethylene glycol was replaced by 40% glycerol, has also been developed (Tachikawa et al., 1993; Zhu et al., 1994). A lower toxicity solution, EFS30, with a reduced proportion (30%) of ethylene glycol and with constant molalities of Ficoll and sucrose has also been described (Zhu et al., 1993). However, this solution may not be called a true vitrification solution because it devitrifies during warming.
3. Toxicity of the solution %mdintracellular ice formation 3.1. Time of exposure A strategy to avoid toxicity of a vitrification solution will be to shorten the exposure time of embryos to the solution. If the exposure is too short, however, permeation of the cryoprotectant will not be sufficient, and intracellular ice can be formed even if extracellular ice is absent. Therefore, optimal exposure time for successful vitrification must be a compromise between preventing toxic injury and preventing intracellular ice formation: We vitrified mouse morulae after exposure to EFS40 at 2O”C, and quite high survival was obtained with a wide range of exposure periods (30 s-5 min) (Fig. 2(a); Kasai, 1994). This contrasts with the results obtained with a high toxicity solution containing
M. Kasai/Animal Reproduction Science 42 (1996) 67-75
71
ii~lyq~~i 0 0
30
60
90
120
0
30
60
90
120
0
30
60
90
120
Exposure time (set)
Fig. 2. Percentage survival of mouse morulae vitrified in (a) EFS40, (b) EFS30 and (c) GFS40 after exposure for various times at 20°C (closed symbols) and 25°C (opensymbols)(Pedro et al., unpublished data).
DMSO, acetamide and propylene glycol, in which embryos can survive only about 10 s of exposure (Nakagata, 1989). Our results show that the cytoplasm of mouse morulae can be concentrated rapidly and that these cells are less sensitive to the toxicity of EFS4O. On the other hand, when expanded mouse blastocysts were vitrified using the same procedure, the maximal survival rate was still 57% (Miyake et al., 19931, suggesting that they are injured by the toxicity before enough ethylene glycol permeates, possibly into the blastocoel. In this case, a two step procedure, in which embryos were first equilibrated in a dilute ethylene glycol solution before a brief exposure to EFS40, improved survival (Zhu et al., 1993). Thus, the optimal time and procedure of exposure differ depending on the stage of development at which vitrification is carried out. Further evidence for the importance of the developmental stage was obtained from experiments on the permeating property of various cryoprotectants used on mouse one-cell zygotes. As shown in Fig. I(b), the permeating speed of most agents was much slower than when used on morulae; furthermore, the permeating pattern was quite different from that observed in morulae, suggesting that even adequate cryoprotectants can behave differently depending on the stage of development. The optimal conditions for vitrifying embryos will also differ between species, because embryo characteristics, such as size, shape, membrane properties and sensitivity to cryoprotectant toxicity, will differ. The efficacy of EFS40 has essentially been confirmed for mouse embryos at the one-cell stage to expanded blastocysts (Miyake et al., 1993; Zhu et al., 1993), rabbit morulae (Kasai et al., 1992b), in vitro derived bovine blastocysts (Tachikawa et al., 1993; Mahmoudzadeh et al., 1993) and horse blastocysts (Hochi et al., 1994). 3.2. Temperature of exposure Permeation and toxicity of a cryoprotectant is largely influenced by temperature. One strategy to reduce toxicity is to lower the temperature (Rall and Fahy, 1985). Optimal exposure time in EFS40 for vitrifying mouse morulae was extended at 5°C (Kasai et al., 1992a); however, in expanded mouse blastocysts survival was not improved by lowering the temperature of exposure, probably because sufficient permeation of ethylene glycol was not attained (Zhu et al., 1993). In this case, a rather brief exposure at an elevated
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M. Kasai/Animal
Reproduction Science 42 (1996) 67-75
temperature seems preferable, because 93% (108/116) of blastocysts survived after vitrification with 30 s of exposure at 3O”C, although survival decreased sharply with the extension of exposure time (Zhu, 1995). 3.3. Vitri~cation solutions other than EFS40 When mouse morulae were vitrified in EFS30 after exposure for 2 min at 2O“C,they survived as well as in EFS40. As expected, EFS30 has lower toxicity than EFS4O but longer exposure times or higher temperatures were needed to attain high survival (Fig. 2(b)). In GFS40, which was based on a less permeating cryoprotectant than EFS40, results similar to those using EFS30 were observed (Fig. 2(c)). 4. Fracture damage
After cryopreservation, cracked embryos are occasionally found. This physical injury is thought to be caused by non-uniform volume change of the solution during phase change, and is called fracture damage (Rail and Meyer, 1989). In conventional freezing, more than 50% of embryos can be damaged physically, and efforts have been made to reduce this (Rail and Meyer, 1989; Landa, 1982). In vitrification, the incidence of physical damage is considerably lower than with conventional freezing despite extremely rapid cooling and warming, probably because of the absence of ice. In our vitrification experiments using EFS40, incidences of zona damage were 1.6% in mouse morulae and 3.6% in rabbit morulae (Kasai et al., 1992b). In order to study fracture damage during vitrification, mouse blastocysts were subjected to 10 cycles of rapid vitrification and warming. As a result, 75% of the embryos were found to have an injured zona pellucida. Because rapid phase change is caused by rapid cooling and warming, samples in the next experiment were passed somewhat more slowly through the temperature zone at which the phase change occurs (- 110 to - 130°C) (Rall et al., 1984; Rall, 1987) by suspending samples in liquid nitrogen gas (3 min) or room temperature air (15 s). With slow cooling followed by rapid warming or with rapid cooling followed by slow warming, the respective percentages of embryos with an injured zona after ten vitrification-warming cycles were 41% and 16%, suggesting that the fracture damage occurs during both processes but more damage occurs during warming. Finally, when both cooling and warming were performed slowly, 100% of the embryos had intact zona even after ten cycles of vitrification and warming. Therefore, the fracture damage can be completely prevented by buffering the cooling and warming velocities during passage through the critical temperature zone. This method will be especially effective for rabbit embryos, where an intact zona is essential for in vivo development. 5. Osmotic injury Because of the toxicity of the solution, quick dilution of the vitrification solution after warming is necessary. When embryos permeated by a cryoprotectant are directly recovered into an isotonic solution, they are threatened by injury from osmotic swelling,
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Reproduction Science 42 (1996) 67-75
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because water permeates far more rapidly than the cryoprotectant diffuses out (Jackowski et al., 1980). To counteract excess water inflow, embryos are often suspended in a sucrose solution (Kasai et al., 1980; Leibo, 1983). However, the molality of sucrose has to be high enough to prevent any swelling upon suspension. An effective method to prevent this is to make embryos shrink before they are transferred to a sucrose solution by suspending them in a solution containing the cryoprotectant plus sucrose (Kasai et al., 1980). In the vitrification using EFS solutions, sucrose contained in the solution promoted shrinkage and reduced the intracellular amount of ethylene glycol before dilution. The rapid permeating property of ethylene glycol allows its rapid diffusion out of the cell. Therefore, embryos would be effectively protected from swelling upon direct dilution in 0.5 M sucrose solution. Mazur and Schneider (1986) showed that fresh mouse and bovine embryos can survive swelling to 200% of their volume in isotonic solution. We have also observed that more than 80% of fresh mouse blastocysts can survive 30 min of exposure in a 0.3 X isotonic solution at 25°C. When vitrified mouse blastocysts after successful recovery in an isotonic solution were suspended in hypotonic solution, only 12% of them survived. The results show that cryopreserved embryos are more sensitive to osmotic swelling than fresh embryos. The embryos of some species have not been cryopreserved efficiently at some stages of development, and oocytes are more sensitive to cryopreservation than embryos (Parks and Ruffing, 1992). Osmotic injury would presumably contribute to the difficulties in successful vitrification of these embryos and oocytes, because apparently normal cells, just after recovery in isotonic solution, are sometimes observed to lose their clear outline and degenerate. 6. Conclusions Low toxicity vitrification solutions, designated EFS, comprise representatives of three different categories of agents, that is, ethylene glycol as a rapidly permeating low toxic agent, Ficoll as a macromolecule, and sucrose as a disaccharide. The solutions are efficient for vitrifying various mammalian embryos after brief exposure at room temperature. However, the optimal time of exposure depends on (1) the concentration of ethylene glycol, (2) the temperature, and (3) the developmental stage and species of embryo. If embryos are likely to be injured before sufficient permeation of the cryoprotectant, a two-step method will improve the survival. Fracture damage can be prevented completely by slow cooling and warming. In many embryos, permeated ethylene glycol can be removed efficiently in a sucrose solution, because sucrose contained in the EFS solution promotes shrinkage. In some embryos or oocytes, however, osmotic damage may still be an obstacle, because cryopreserved cells are more sensitive to osmotic swelling than fresh cells. Acknowledgements
I thank Dr. A. Bartke, Southern Illinois University, USA, for critical reading of the manuscript.
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References Ah, J. and Shelton, J.N., 1993. Vitrification of preimplantation stages of mouse embryos. J. Reprod. Fertil., 98: 459-465. Fahy. GM., MacFarlane, D.R., Angell. C.A. and Meryman, H.T., 1984. Vitrification as an approach to cryopreservation. Cryobiology, 21: 407-426. Hochi, S., Fujimoto, T., Braun, J. and Oguri, N., 1994. Pregnancies following transfer of equine embryos cryopteserved by vitritication. Theriogenology, 42: 483-488. Ishimori, H., Takahashi, Y. and Kanagawa, H., 1992. Viability of vitrified mouse embryos using various cryoprotectant mixtures. Theriogenology, 37: 481-487. Jackowski, S., Leibo, S.P. and Mazur, P., 1980. Glycerol permeabilities of fertilized and unfertilized mouse ova. J. Exp. Zool., 212: 329-341. Kasai, M., 1986. Nonfreezing technique for short-term storage of mouse embryos. J. In Vitro Fertil. Embryo Transfer, 3: 10-14. Kasai, M., 1994. Cryopreservation of mammalian embryos by vitrification. In: T. Mori, T. Aono, T. Tominaga and M. Hiroi (Editors), Perspectives on Assisted Reproduction. Frontiers in Endocrinology, Vol. 4. Ares-Serono Symposia Publications, Rome, pp. 481-487. Kasai, M., 1995. Cryopreservation of mammalian embryos-vitrification. In: J.G. Day and M.R. McLellan (Editors), Methods in Molecular Biology. Cryopreservation and Freeze-Drying Protocols, Vol. 38. Humana Press Inc., Totowa, NJ, pp. 211-219. Kasai, M., Niwa, K. and Iritani, A., 1980. Survival of mouse embryos frozen and thawed rapidly. J. Reprod. Fertil., 59: 51-56. Kasai, M., Komi, J.H., Takakamo, A., Tsudera, H., Sakurai, T. and Machida, T., 1990. A simple method for mouse embryo cryopreservation in a low toxicity vitrification solution, without appreciable loss of viability. J. Reprod. Fertil., 89: 91-97. Kasai, M., Nishimori, M., Zhu, S.E., Sakurai, T. and Machida, T., 1992a. Survival of mouse morulae vitrified in an ethylene glycol-based solution after exposure to the solution at various temperatures. Biol. Reprod., 47: 1134-1139. Kasai, M., Hamaguchi, Y., Zhu, SE., Miyake, T., Sakurai, T. and Machida, T., 1992b. High survival of rabbit morulae after vitrification in an ethylene glycol-based solution by a simple method. Biol. Reprod., 46: 1042-1046. Landa, V., 1982. Protection of non-cellular investments of rabbit morulae stored at - 196°C. Folia Biol., 28: 350-358. Landa, V. and Tepla, 0.. 1990. Cryopreservation of mouse 8-cell embryos in microdrops. Folia Biol., 36: 153-160. Leibo, S.P., 1983. A one-step in situ dilution method for frozen-thawed bovine embryos. Ctyo-Lett., 4: 387-400. Leibo, S.P. and Oda, K., 1993. High survival of mouse zygotes and embryos cooled rapidly or slowly in ethylene glycol plus polyvinylpyrrolidone. Cryo-Lett., 14: 133- 144. Liehman, P., Tepla, 0. and Fulka, J., 1990. Vitrification of mouse 8-cell embryos with glycerol as a cryoprotectant. Folia Biol., 36: 240-25 1. Mahmoudzadeh, A.R., Soom, A.V., Vlaenderen, I.V. and Kruif, A.D., 1993. A comparative study of the effect of one-step addition of different vitrification solutions on in vitro survival of vitrified bovine embryos. Theriogenology, 39: 1291-1302. Mazur. P. and Schneider, U., 1986. Osmotic responses of preimplantation mouse and bovine embryos and their cryobiological implications. Cell Biophys., 8: 259-284. Miyake, T., Kasai, M., Zhu, SE., Sakurai, T. and Machida, T., 1993. Vitrification of mouse oocytes and embryos at various stages of development in an ethylene glycol-based solution by a simple method. Theriogenology, 40: 121- 134. Nakagata, N., 1989. Survival of mouse embryos derived from in vitro fertilization after ultrarapid freezing and thawing. J. Mamm. Ova Res., 6: 23-26. (In Japanese.) Parks, J.E. and Ruffmg, N.A., 1992. Factors affecting low temperature survival of mammahan oocytes. Theriogenology, 37: 59-73.
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Rall, W.F., 1987. Factors affecting the survival of mouse embryos cryopreserved by vitrification. Cryobiology, 24: 387-402. Rall, W.F. and Fahy, G.M., 1985. Ice-free cryopreservation of mouse embryos at - 196°C by vitrification. Nature, 313: 573-575. Rall, W.F. and Meyer, T.K., 1989. Zona fracture damage and its avoidance during the cryopreservation of mammalian embryos. Thetiogenology, 3 1: 683-692. Rall, W.F., Reid, D.S. and Polge, C., 1984. Analysis of slow-warming injury of mouse embryos by cryomicroscopical and physiochemical methods. Cryobiology, 21: 106- 121. Saito, N., Imai, K. and Tomizawa, M., 1994. Effect of sugars-addition on the survival of vitrified bovine blastocysts produced in vitro. Theriogenology, 41: 1053- 1060. Scheffen, B., Van Der Zwalmen, P. and Massip, A., 1986. A simple and efficient procedure for preservation of mouse embryos by vitrification. Cryo-Lett., 7: 260-269. Schiewe, M.C., Rall, W.F., Stuart, L.D. and Wildt, D.E., 1991. Analysis of cryoprotectant, cooling rate and in situ dilution using conventional freezing or vitrification for cryopreserving sheep embryos. Theriogenology, 36: 279-293. Smorag, Z., Gajda, B., Wieczorek, B. and Jura, J., 1989. Stage-dependent viability of vitrified rabbit embryos. Theriogenology, 3 1: 1227- 1231. Tachikawa, S., Otoi, T., Kondo, S., Machida, T. and Kasai, M., 1993. Successful vitrification of bovine blastocysts derived by in vitro maturation and fertilization. Mol. Reprod. Dev., 34: 266-27 1. Tada, N., Sato, M., Amann, E. and Ogawa, S., 1993. A simple and rapid method for cryopreservation of mouse 2-cell embryos by vitrification: beneficial effect of sucrose and rafftnose on their cryosurvival rate. Theriogenology, 40: 333-344. Whittingham, D.G., Leibo, S.P. and Mazur, P., 1972. Survival of mouse embryos frozen to - 196°C and - 269°C. Science, 178: 41 l-414. Yoshino, J., Kojima, T., Shimizu, M. and Tomizuka, T., 1993. Cryopreservation of porcine blastocysts by vitrification. Cryobiology, 30: 413-422. Zhu, S.E., 1995. Studies on the cryopreservation of mammalian blastocysts by vitrification. Ph.D. Dissertation, Ehime University, Japan. (In Japanese.) Zhu, S.E., Kasai, M., Otoge, H., Sakurai, T. and Machida, T., 1993. Cryopreservation of expanded mouse blastocysts by vitrification in ethylene glycol-based solutions. J. Reprod. Fertil., 98: 139- 145. Zhu, SE., Sakurai, T., Edashige, K., Machida, T. and Kasai, M., 1994. Optimization of the procedures for the vitrification of expanded mouse blastocysts in glycerol-based solutions. J. Reprod. Dev., 40: 293-300.
Simple and efficient methods for vitrification of mammalian embryos Magosaburo Kasai
I
Laboratory of Animal Science, College of Agriculture, Kochi University, Nankoku, Kochi 783, Japan
Abstract greatly simplifies the cooling process for embryo cryopreservation, and eliminates caused by extracellular ice. However, embryos may be injured by various factors, toxicity of the solution, intracellular ice, fracture damage and osmotic swelling. By the effect of each of these factors, refined procedures for simple and efficient have been developed for mouse embryos.
Vitrification
any injuries such as the minimising vitrification
Keywords: Vitrification; Cryopreservation; Embryo; Mouse; Ethylene glycol
1. Introduction
If embryos can be cryopreserved efficiently, the technique can be applied to a wide range of mammalian species, i.e. farm animals, laboratory animals, wild animals and human. However, during the processing and cooling of embryos, or during recovery of stored embryos into a physiological solution, they are at risk of injury by various factors, including: (1) toxicity of cryoprotectants; (2) chilling injury; (3) physical injury by extracellular ice; (4) toxicity of concentrated electrolytes; (5) formation and growth of intracellular ice; (6) fracture damage; (7) osmotic swelling. By circumventing these obstacles, various mammalian embryos at various stages of preimplantation have been successfully cryopreserved using several methods. However, there is a need for improvement in the method because post-thawing survival is variable depending on the species, the strain, the developmental stage and the quality of the embryo. In addition, a simpler method would be preferable. Using the controlled slow freezing method established by Whittingham et al. (19721, embryos equilibrated in l-l.5 M cryoprotectant must be cooled slowly so that cellular ’Tel.: 8 I-888-5 194; fax: 8 I-888-64-5200. 0378-4320/%/$15.00
6 1996 Elsevier Science B.V. All rights reserved.
PII SO378-4320(96)01536-9
68
M. Kasai/Animal Reproduction Science 42 (1996) 67-75
contents are concentrated by gradual dehydration caused by the growth of extracellular ice. When the sample is cooled in liquid nitrogen, the cytoplasm of the embryos, together with the extracellular concentrated fraction, will turn to glassy solids without ice formation by an extreme elevation in viscosity, i.e. they will vitrify. The disadvantage of this slow freezing method is that controlled cooling requires time and elaborate equipment. If embryos are suspended, at 0°C or above, in a concentrated solution which will vitrify in liquid nitrogen, they are expected to survive direct cooling in liquid nitrogen, with both extracellular solution and cytoplasm being vitrified. Based on this idea, Rall and Fahy (1985) devised a vitrification method for mouse embryos. This method not only greatly simplifies the cooling process but also eliminates all the physical and chemical injuries caused by extracellular ice. In addition, it may even lessen the chilling injury, which is observed in embryos of some species, by passing through critical temperatures very rapidly. However, embryos can still be injured by toxicity, intracellular ice formation, fracture and osmosis. By minimising the chances of damage to the embryos by each of these mechanisms, simple and efficient vitrification methods can be developed. The primary obstacle in vitrification is toxicity of the cryoprotectant.
2. Low toxicity vitrification solutions consisting of three categories of agents 2.1. Permeating cryoprotectants The essential component of a vitrification solution is the permeating agent. Rall and Fahy (1985) used a solution containing 6.5 M (in total) of dimethyl sulphoxide (DMSO), acetamide and propylene glycol as permeating agents. Since their pioneering report, however, numerous vitrification solutions have been devised using various cryoprotectants in combination or as a single permeating component (Table 1). Because the permeating agent is responsible for the toxicity (Kasai et al., 1992a), we have tested the relative toxicity of five such agents on mouse morulae by suspending them in phosphate-buffered saline (PBS) containing 30% (v/v> cryoprotectant for 20 min at 20°C. Survival rates assessed by the developmental potential in vitro showed that ethylene glycol is the least toxic (98%), followed by glycerol (88%) and then DMSO (68%). Propylene glycol (16%) and acetamide (0%) are very toxic (Kasai, 1994). Another important characteristic for evaluating the cryoprotectant is its permeating property. In general, rapidly permeating agents are favoured, because the exposure time before rapid cooling can be shortened, and because they are more likely to diffuse out of the cell rapidly, thus preventing osmotic injury. To compare the permeating speed of cryoprotectants, we observed the volume change of mouse morulae during 5 min of suspension in five cryoprotectant solutions. As shown in Fig. l(a), embryos in ethylene glycol shrank the least and regained their original volume most quickly, indicating that of the five agents, ethylene glycol permeates the most rapidly. When toxicity and permeating property of the cryoprotectants are considered, ethylene glycol was the best candidate tested for the permeating component of a vitrification solution and glycerol may be the second choice, at least for mouse morulae.
M. Kasai/Animal Reproduction Science 42 (1996) 67-75
69
Table I Vitrification solutions used for the cryopmservation of mammalian embryos Reference
Cryoprotective additives
Rall and Fahy, 1985 Rail and Fahy, 1985 Scheffen et al., 1986 Rall, 1987 Rail, 1987 Nakagata, 1989 Smorag et al., 1989 Liehman et al., 1990 Landa and Tepla, 1990 Kasai et al., 1990 Schiewe et al., 1991 Ishimori et al., 1992 Leibo and &la, 1993 Tachikawa et al., 1993 Yoshino et al., 1993 Zhu et al., 1993 Tada et al., 1993 Ah and Shelton, 1993 Saito et al., 1994
Permeating agents
Non-permeating agents
(%, v/v)
Macromolecules a (%, w/v)
18.6D+13.4A+9.6P 16.7 D+ 12 A+8.7 P 25G+25P 40.3 P 47.4 G 14.2D+5.1 A+22P 35G+35P 50 G 30G 4OE 47.4 G 25E+25D 44E 4QG 40E 30E 19.5 D+ 20.2 P 31 E 20E+20G
6 PEG 5.4 PEG
Saccharides (MI
6 PEG 6 PEG -
18 Ficoll 6 BSA 7.5 PVP 18 Ficoll 18 Ficoll 21 Ficoll
1.o sue 0.3 sue
0.3 sue 0.3 Treha 0.35 sue 1.o sue 1.o sue 0.75 (Sue + Glc)
a Serum and O.l-0.3% BSA am not shown. D, dimethyl sulphoxide; A, acetamide; P, propylene glycol; G. glycerol; E, ethylene glycol; PEG, polyethylene glycol; BSA, bovine serum albumin; Sue, sucrose; Treha, trehalose; Glc, glucose.
a) YORUlA
“V
I--
b) ZYGOTE
EG
-AA -
DMSO
-
GL
iu _I
or2345012345 Exposure time (min)
Fig. 1. The volume change of mouse (a) morulae and (b) one-cell zygotes in various cryoprotectant solutions at 20°C. Bach embryo was held by a holding pipette connected to a micromanipulator and intrcduced into a cryoprotectant solution with the aid of a covering pipette. The volume change was calculated from the images recorded by a time-lapse video tape recorder. EG, 10% (v/v) ethylene glycol; AA, 1.5 M acetamide; DMSO, 10% (v/v) dimethyl sulphoxide; PG, 10% (v/v) propylene glycol; GL, 10% (v/v) glycerol (Pedro et al., unpublished data).
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2.2. Macromolecules
It is known that incorporation of a macromolecule promotes vitrification of a solution (Fahy et al., 1984). Although macromolecules are generally less toxic, we have found in a toxicity test that Ficoll 70 was less toxic than polyethylene glycol when it was mixed with 40% (v/v) ethylene glycol (Kasai, 1994). Of. other macromolecules, polyvinyl pyrrolidone (Fahy et al., 1984; Leibo and Oda, 1993) and bovine serum albumin (BSA) (Schiewe et al., 1991) have been used. 2.3. Small saccharides As a non-permeating agent, sucrose exerts a considerable osmotic effect. In a vitrification solution containing 40% (v/v> ethylene glycol and 18% (w/v) Ficoll, it was found that incorporation of sucrose reduces the toxicity, probably because the amount of intracellular cryoprotectant is reduced (Kasai et al., 1990). The beneficial effect is not specific to sucrose, with other saccharides, such as trehalose (Yoshino et al., 1993) also being effective. It was shown that sucrose and other saccharides such as glucose, fructose and galactose are virtually non-toxic (Kasai, 1986). 2.4. Composition of vitrijkation solutions After considering the different properties of agents in each category, we developed a vitrification solution, designated EFS40, containing 40% (v/v> ethylene glycol, 18% (w/v) Ficoll and 0.3 M sucrose (Kasai et al., 1990). A precise protocol for preparing this solution has been described elsewhere (Kasai, 1995). A similar solution, CFS40, in which 40% ethylene glycol was replaced by 40% glycerol, has also been developed (Tachikawa et al., 1993; Zhu et al., 1994). A lower toxicity solution, EFS30, with a reduced proportion (30%) of ethylene glycol and with constant molalities of Ficoll and sucrose has also been described (Zhu et al., 1993). However, this solution may not be called a true vitrification solution because it devitrifies during warming.
3. Toxicity of the solution %mdintracellular ice formation 3.1. Time of exposure A strategy to avoid toxicity of a vitrification solution will be to shorten the exposure time of embryos to the solution. If the exposure is too short, however, permeation of the cryoprotectant will not be sufficient, and intracellular ice can be formed even if extracellular ice is absent. Therefore, optimal exposure time for successful vitrification must be a compromise between preventing toxic injury and preventing intracellular ice formation: We vitrified mouse morulae after exposure to EFS40 at 2O”C, and quite high survival was obtained with a wide range of exposure periods (30 s-5 min) (Fig. 2(a); Kasai, 1994). This contrasts with the results obtained with a high toxicity solution containing
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71
ii~lyq~~i 0 0
30
60
90
120
0
30
60
90
120
0
30
60
90
120
Exposure time (set)
Fig. 2. Percentage survival of mouse morulae vitrified in (a) EFS40, (b) EFS30 and (c) GFS40 after exposure for various times at 20°C (closed symbols) and 25°C (opensymbols)(Pedro et al., unpublished data).
DMSO, acetamide and propylene glycol, in which embryos can survive only about 10 s of exposure (Nakagata, 1989). Our results show that the cytoplasm of mouse morulae can be concentrated rapidly and that these cells are less sensitive to the toxicity of EFS4O. On the other hand, when expanded mouse blastocysts were vitrified using the same procedure, the maximal survival rate was still 57% (Miyake et al., 19931, suggesting that they are injured by the toxicity before enough ethylene glycol permeates, possibly into the blastocoel. In this case, a two step procedure, in which embryos were first equilibrated in a dilute ethylene glycol solution before a brief exposure to EFS40, improved survival (Zhu et al., 1993). Thus, the optimal time and procedure of exposure differ depending on the stage of development at which vitrification is carried out. Further evidence for the importance of the developmental stage was obtained from experiments on the permeating property of various cryoprotectants used on mouse one-cell zygotes. As shown in Fig. I(b), the permeating speed of most agents was much slower than when used on morulae; furthermore, the permeating pattern was quite different from that observed in morulae, suggesting that even adequate cryoprotectants can behave differently depending on the stage of development. The optimal conditions for vitrifying embryos will also differ between species, because embryo characteristics, such as size, shape, membrane properties and sensitivity to cryoprotectant toxicity, will differ. The efficacy of EFS40 has essentially been confirmed for mouse embryos at the one-cell stage to expanded blastocysts (Miyake et al., 1993; Zhu et al., 1993), rabbit morulae (Kasai et al., 1992b), in vitro derived bovine blastocysts (Tachikawa et al., 1993; Mahmoudzadeh et al., 1993) and horse blastocysts (Hochi et al., 1994). 3.2. Temperature of exposure Permeation and toxicity of a cryoprotectant is largely influenced by temperature. One strategy to reduce toxicity is to lower the temperature (Rall and Fahy, 1985). Optimal exposure time in EFS40 for vitrifying mouse morulae was extended at 5°C (Kasai et al., 1992a); however, in expanded mouse blastocysts survival was not improved by lowering the temperature of exposure, probably because sufficient permeation of ethylene glycol was not attained (Zhu et al., 1993). In this case, a rather brief exposure at an elevated
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temperature seems preferable, because 93% (108/116) of blastocysts survived after vitrification with 30 s of exposure at 3O”C, although survival decreased sharply with the extension of exposure time (Zhu, 1995). 3.3. Vitri~cation solutions other than EFS40 When mouse morulae were vitrified in EFS30 after exposure for 2 min at 2O“C,they survived as well as in EFS40. As expected, EFS30 has lower toxicity than EFS4O but longer exposure times or higher temperatures were needed to attain high survival (Fig. 2(b)). In GFS40, which was based on a less permeating cryoprotectant than EFS40, results similar to those using EFS30 were observed (Fig. 2(c)). 4. Fracture damage
After cryopreservation, cracked embryos are occasionally found. This physical injury is thought to be caused by non-uniform volume change of the solution during phase change, and is called fracture damage (Rail and Meyer, 1989). In conventional freezing, more than 50% of embryos can be damaged physically, and efforts have been made to reduce this (Rail and Meyer, 1989; Landa, 1982). In vitrification, the incidence of physical damage is considerably lower than with conventional freezing despite extremely rapid cooling and warming, probably because of the absence of ice. In our vitrification experiments using EFS40, incidences of zona damage were 1.6% in mouse morulae and 3.6% in rabbit morulae (Kasai et al., 1992b). In order to study fracture damage during vitrification, mouse blastocysts were subjected to 10 cycles of rapid vitrification and warming. As a result, 75% of the embryos were found to have an injured zona pellucida. Because rapid phase change is caused by rapid cooling and warming, samples in the next experiment were passed somewhat more slowly through the temperature zone at which the phase change occurs (- 110 to - 130°C) (Rall et al., 1984; Rall, 1987) by suspending samples in liquid nitrogen gas (3 min) or room temperature air (15 s). With slow cooling followed by rapid warming or with rapid cooling followed by slow warming, the respective percentages of embryos with an injured zona after ten vitrification-warming cycles were 41% and 16%, suggesting that the fracture damage occurs during both processes but more damage occurs during warming. Finally, when both cooling and warming were performed slowly, 100% of the embryos had intact zona even after ten cycles of vitrification and warming. Therefore, the fracture damage can be completely prevented by buffering the cooling and warming velocities during passage through the critical temperature zone. This method will be especially effective for rabbit embryos, where an intact zona is essential for in vivo development. 5. Osmotic injury Because of the toxicity of the solution, quick dilution of the vitrification solution after warming is necessary. When embryos permeated by a cryoprotectant are directly recovered into an isotonic solution, they are threatened by injury from osmotic swelling,
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because water permeates far more rapidly than the cryoprotectant diffuses out (Jackowski et al., 1980). To counteract excess water inflow, embryos are often suspended in a sucrose solution (Kasai et al., 1980; Leibo, 1983). However, the molality of sucrose has to be high enough to prevent any swelling upon suspension. An effective method to prevent this is to make embryos shrink before they are transferred to a sucrose solution by suspending them in a solution containing the cryoprotectant plus sucrose (Kasai et al., 1980). In the vitrification using EFS solutions, sucrose contained in the solution promoted shrinkage and reduced the intracellular amount of ethylene glycol before dilution. The rapid permeating property of ethylene glycol allows its rapid diffusion out of the cell. Therefore, embryos would be effectively protected from swelling upon direct dilution in 0.5 M sucrose solution. Mazur and Schneider (1986) showed that fresh mouse and bovine embryos can survive swelling to 200% of their volume in isotonic solution. We have also observed that more than 80% of fresh mouse blastocysts can survive 30 min of exposure in a 0.3 X isotonic solution at 25°C. When vitrified mouse blastocysts after successful recovery in an isotonic solution were suspended in hypotonic solution, only 12% of them survived. The results show that cryopreserved embryos are more sensitive to osmotic swelling than fresh embryos. The embryos of some species have not been cryopreserved efficiently at some stages of development, and oocytes are more sensitive to cryopreservation than embryos (Parks and Ruffing, 1992). Osmotic injury would presumably contribute to the difficulties in successful vitrification of these embryos and oocytes, because apparently normal cells, just after recovery in isotonic solution, are sometimes observed to lose their clear outline and degenerate. 6. Conclusions Low toxicity vitrification solutions, designated EFS, comprise representatives of three different categories of agents, that is, ethylene glycol as a rapidly permeating low toxic agent, Ficoll as a macromolecule, and sucrose as a disaccharide. The solutions are efficient for vitrifying various mammalian embryos after brief exposure at room temperature. However, the optimal time of exposure depends on (1) the concentration of ethylene glycol, (2) the temperature, and (3) the developmental stage and species of embryo. If embryos are likely to be injured before sufficient permeation of the cryoprotectant, a two-step method will improve the survival. Fracture damage can be prevented completely by slow cooling and warming. In many embryos, permeated ethylene glycol can be removed efficiently in a sucrose solution, because sucrose contained in the EFS solution promotes shrinkage. In some embryos or oocytes, however, osmotic damage may still be an obstacle, because cryopreserved cells are more sensitive to osmotic swelling than fresh cells. Acknowledgements
I thank Dr. A. Bartke, Southern Illinois University, USA, for critical reading of the manuscript.
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