Theriogenology 81 (2014) 96–102
Contents lists available at ScienceDirect
Theriogenology journal homepage: www.theriojournal.com
40th Anniversary Special Issue
Cryopreservation of oocytes and embryos A. Arav* FertileSafe, Shlomzion Hamalca, Tel Aviv, Israel 62266
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 July 2013 Received in revised form 11 September 2013 Accepted 11 September 2013
Two hundred years have passed since the first description of supercooled water by GeyLussac to the recently high survival rates of embryo and oocytes after vitrification. This review discusses important milestones that have made vitrification the method of choice for oocytes and embryos cryopreservation. We will go through the first cells ever to survive low temperature exposure in the beginning of the last century, the finding of glycerol in the late 1940s and the first mouse and bovine embryos freezing in the 1970s. During the 1980s, embryo vitrification began and the time since is a tribute to the development of oocytes vitrification. Standardization and an automatic vitrification procedure are currently under development. The next evolutionary step in oocyte and embryo cryopreservation will be preserving them in the dry state at room temperature, allowing home storage for future use a reality. Ó 2014 Elsevier Inc. All rights reserved.
Keywords: Freezing Vitrification Drying Oocytes Embryos Cryopreservation
1. Two hundred years of vitrification Going back to the book “Life and Death at Low Temperatures” by Basile J. Luyet and Marie Pierre Gehenio, published at 1940 [1], I found out that the small volume vitrification (the minimum drop size technique) that is generally attributed to my work was already thought at the beginning of the 19th century. It was done by the great French chemist and physicist Joseph Louis Gay-Lussac. He is known mostly for his two laws of gases and for his work on alcohol–water mixtures. Gay Lussac found that water can be cooled to 12 C without freezing, finding with this discovery the basis of vitrification [2]. In 1804, Gay-Lussac was ascended in a hot air balloon (Fig. 1) and noticed that the drops in the clouds are not frozen despite the subzero temperatures. He published later the discovery of the effect of small volume of water droplets on supercooling. The size of water drops in clouds is around 8 to 10 mm, which maintains them at a liquid state at a subfreezing temperature of 5 C. Luyet described it in his book: “Some of the oldest investigations on subcooling were made by Gay-Lussac * Corresponding author. Tel.: þ972 523 638022. E-mail address: [email protected]. 0093-691X/$ – see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2013.09.011
(1836) who observed that water can be subcooled to 12 C when it is enclosed in small tubes” [1,3]. Already in 1858, Johann Rudolf Albert Mousson sprayed droplets of water less than 0.5 mm in diameter on a dry surface and observed that the smaller the drops the longer they stayed subcooled [4]. Not only was volume important to achieve supercooling, among other factors that might have an influence in inducing crystallization, as mentioned by Luyet, are cooling velocity and concentration of the supercooled or supersaturated solutions. Luyet wrote, “To avoid freezing, the temperature should drop at a rate of some hundred degrees per second, within the objects themselves,” and “The only method of vitrifying a substance is to take it in the liquid or gas state and cool it rapidly so as to skip over the zone of crystallization temperatures in less time than is necessary for the material to freeze.” Luyet further wrote, “It is evident that when crystals grow faster one must traverse the crystallization zone more rapidly if one wants to avoid crystallization” [1].
2. Basic principles The velocity of cooling depends on the thermal mass of the sample and on its surface area. To achieve rapid cooling,
A. Arav / Theriogenology 81 (2014) 96–102
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Later attempts at vitrifying pure water have been made by a few investigators; Hawkes [8] published an experiment in which a drop of solid amorphous water was obtained, by chance, during rapid cooling. Burton and Oliver [9] obtained from steam some solid water in which x-ray analysis did not reveal any crystalline structure. As we can see, these achievements were mainly owing to the samples’ small volume and not the velocity of cooling. A review on supercooling of water can be found also in “Cryobiology” by Meryman published in1966 [10]. 3. The first cells survival after vitrification The most important year for cryobiology was 1938; Basile J. Luyet and Eugene L. Hodapp [11] published the first successful vitrification of sperm. Luyet began his research with colloids (gelatin, milk, or agar) and found that their water content determines the possibility or impossibility of vitrification. In general, with 50% gelatin solutions, they had vitrified layers of 0.3 mm thick (by the method of immersion in liquid nitrogen [LN]), whereas with solutions containing 90% water, they could vitrify only smears of few microns thick [3]. They were the first to demonstrate successful cryopreservation of frog sperm by vitrification using 2 mol/L sucrose and small drops.
Fig. 1. .Joseph Louis Gay Lussac (1778–1850) and portrait and an illustration of from 1804 of Gay Lussac and Jean Baptiste Biot in the hot air balloon at 4000 m.
we should use material with the lowest heat mass and maximum surface to volume ratio. Indeed, Gregory M. Fahy and William F. Rall published in 2007 the critical cooling rates needed to vitrify aqueous solutions that contain different concentrations of cryoprotectants (CPs) [5]. It was extrapolated that for pure water over 100 106 C/min are needed to form a glass state without crystallization. Also, it is interesting to note that for 15% (v/v) of most CPs almost 1 106 C/min is needed, which is difficult to achieve. We have shown that 15% (v/v) of CPs can vitrify at relative slow cooling rates when the volume of the drop is 0.07 mL [6]. Therefore, it is much more feasible to achieve vitrification by lowering the volume than by increasing the cooling rate. James H. Walton and Roy C. Judd measured the velocity of ice crystal growth and found that it is in the range of 65 mm/s [7]. This means that if we want to avoid crystallization in a drop placed on a cold metal surface and that has a diameter of 0.1 mm, we need a velocity of 1/6500 mm/s, which is 0.0001 second. If we cool from room temperature to 180 C, this means we need to reduce 200 C at a rate of 0.1 ms or at 78 106 C/min. This is actually the cooling rate that was estimated by Balds and by Bruggeller [1,5]. However, because this cooling rate is impossible to achieve, the ability to reach vitrification of pure water in a small drop can be achieved in relatively slow cooling rates, which indicate that the small volume has an independent effect on the probability of vitrification.
4. The beginning of slow freezing In 1949, Christopher Polge, Audrey Smith, and Alan Parkes [12], when trying to duplicate Luyet’s results, discovered by mistake the cryoprotective property of glycerol and so opened the field of slow freezing. Currently, there are two methods for gametes cryopreservation: slow freezing and vitrification. Slow freezing has the advantage of using low concentrations of CPs, which are associated with chemical toxicity and osmotic shock. Vitrification is a rapid method, which reduces chilling sensitivity and crystallization damage caused to cells. Sherman and Lin [13] showed that mouse oocytes need 8 to 10 minutes for equilibration in a freezing solution containing 5% glycerol at 37 C. In addition, he demonstrated that mouse oocytes will survive supercooling to 20 C after slow cooling at 0.6 C/min, however, oocytes that were cooled faster or to lower temperatures were damaged owing to intracellular crystallization. In the early 1970s, two groups were competing on achieving the first success of slow freezing of embryos. One group included Whittingham, Leibo, and Mazur, and the other Wilmut and Polge. Whittingham had partially succeeded in freezing embryos to 79 C for 30 minutes using polyvinylpyrrolidone; however, these experiment could not be duplicated [14–16]. Both groups published in 1972 the first survival of mouse embryos after slow freezing [15,16] and live offspring [15]. The technique included cooling at a slow rate in the presence of 1 mol/L DMSO, which most likely was the ingredient that enabled this. In 1976, sheep embryos were slow frozen by Willadsen using 1.5 mol/L DMSO and a cooling rate of 0.3 C/min. [17]. However, the first farm animal to be born after transplantation of frozen and thawed embryos was a calf. This was published by Wilmut and Rowson at 1973 [18]. Since
98
A. Arav / Theriogenology 81 (2014) 96–102
then, dozens of species have been successfully cryopreserved by slow freezing (for a review, see [19]).
5. The comeback of vitrification For many years, slow freezing and not vitrification was the method of choice for embryo cryopreservation. This was because vitrification was not achieved easily owing to the need of high CPs concentrations and relatively high volume samples. In 1985, the first successful vitrification of mouse embryos using a relatively large volume sample was done [20]. Rall and Fahy vitrified mouse embryos with mixture of DMSO, acetamide, and polyethylene glycol and in a relatively large volume inside a 0.25-mL straw plunged into LN. At that time, I was a veterinary student at the University of Bologna, Italy, and I met Bill Rall, who told me about the exciting work he did on mouse embryos. Two years later, I started to work on cryomicoscopy of oocytes and embryos. As in our laboratory, we used to prepare oocytes for histology evaluation by fixing them with a small drop over a microscopic slide. I had the idea of using the same technique for vitrification in a small drop, which I later named as the “minimum drop size” [6,21–24]. As noted, the probability of vitrification increases as the volume of the sample decreases. Pure water is vitrified only in very small droplets obtain from aerosols. Vitrification of thin layers (
Contents lists available at ScienceDirect
Theriogenology journal homepage: www.theriojournal.com
40th Anniversary Special Issue
Cryopreservation of oocytes and embryos A. Arav* FertileSafe, Shlomzion Hamalca, Tel Aviv, Israel 62266
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 July 2013 Received in revised form 11 September 2013 Accepted 11 September 2013
Two hundred years have passed since the first description of supercooled water by GeyLussac to the recently high survival rates of embryo and oocytes after vitrification. This review discusses important milestones that have made vitrification the method of choice for oocytes and embryos cryopreservation. We will go through the first cells ever to survive low temperature exposure in the beginning of the last century, the finding of glycerol in the late 1940s and the first mouse and bovine embryos freezing in the 1970s. During the 1980s, embryo vitrification began and the time since is a tribute to the development of oocytes vitrification. Standardization and an automatic vitrification procedure are currently under development. The next evolutionary step in oocyte and embryo cryopreservation will be preserving them in the dry state at room temperature, allowing home storage for future use a reality. Ó 2014 Elsevier Inc. All rights reserved.
Keywords: Freezing Vitrification Drying Oocytes Embryos Cryopreservation
1. Two hundred years of vitrification Going back to the book “Life and Death at Low Temperatures” by Basile J. Luyet and Marie Pierre Gehenio, published at 1940 [1], I found out that the small volume vitrification (the minimum drop size technique) that is generally attributed to my work was already thought at the beginning of the 19th century. It was done by the great French chemist and physicist Joseph Louis Gay-Lussac. He is known mostly for his two laws of gases and for his work on alcohol–water mixtures. Gay Lussac found that water can be cooled to 12 C without freezing, finding with this discovery the basis of vitrification [2]. In 1804, Gay-Lussac was ascended in a hot air balloon (Fig. 1) and noticed that the drops in the clouds are not frozen despite the subzero temperatures. He published later the discovery of the effect of small volume of water droplets on supercooling. The size of water drops in clouds is around 8 to 10 mm, which maintains them at a liquid state at a subfreezing temperature of 5 C. Luyet described it in his book: “Some of the oldest investigations on subcooling were made by Gay-Lussac * Corresponding author. Tel.: þ972 523 638022. E-mail address: [email protected]. 0093-691X/$ – see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2013.09.011
(1836) who observed that water can be subcooled to 12 C when it is enclosed in small tubes” [1,3]. Already in 1858, Johann Rudolf Albert Mousson sprayed droplets of water less than 0.5 mm in diameter on a dry surface and observed that the smaller the drops the longer they stayed subcooled [4]. Not only was volume important to achieve supercooling, among other factors that might have an influence in inducing crystallization, as mentioned by Luyet, are cooling velocity and concentration of the supercooled or supersaturated solutions. Luyet wrote, “To avoid freezing, the temperature should drop at a rate of some hundred degrees per second, within the objects themselves,” and “The only method of vitrifying a substance is to take it in the liquid or gas state and cool it rapidly so as to skip over the zone of crystallization temperatures in less time than is necessary for the material to freeze.” Luyet further wrote, “It is evident that when crystals grow faster one must traverse the crystallization zone more rapidly if one wants to avoid crystallization” [1].
2. Basic principles The velocity of cooling depends on the thermal mass of the sample and on its surface area. To achieve rapid cooling,
A. Arav / Theriogenology 81 (2014) 96–102
97
Later attempts at vitrifying pure water have been made by a few investigators; Hawkes [8] published an experiment in which a drop of solid amorphous water was obtained, by chance, during rapid cooling. Burton and Oliver [9] obtained from steam some solid water in which x-ray analysis did not reveal any crystalline structure. As we can see, these achievements were mainly owing to the samples’ small volume and not the velocity of cooling. A review on supercooling of water can be found also in “Cryobiology” by Meryman published in1966 [10]. 3. The first cells survival after vitrification The most important year for cryobiology was 1938; Basile J. Luyet and Eugene L. Hodapp [11] published the first successful vitrification of sperm. Luyet began his research with colloids (gelatin, milk, or agar) and found that their water content determines the possibility or impossibility of vitrification. In general, with 50% gelatin solutions, they had vitrified layers of 0.3 mm thick (by the method of immersion in liquid nitrogen [LN]), whereas with solutions containing 90% water, they could vitrify only smears of few microns thick [3]. They were the first to demonstrate successful cryopreservation of frog sperm by vitrification using 2 mol/L sucrose and small drops.
Fig. 1. .Joseph Louis Gay Lussac (1778–1850) and portrait and an illustration of from 1804 of Gay Lussac and Jean Baptiste Biot in the hot air balloon at 4000 m.
we should use material with the lowest heat mass and maximum surface to volume ratio. Indeed, Gregory M. Fahy and William F. Rall published in 2007 the critical cooling rates needed to vitrify aqueous solutions that contain different concentrations of cryoprotectants (CPs) [5]. It was extrapolated that for pure water over 100 106 C/min are needed to form a glass state without crystallization. Also, it is interesting to note that for 15% (v/v) of most CPs almost 1 106 C/min is needed, which is difficult to achieve. We have shown that 15% (v/v) of CPs can vitrify at relative slow cooling rates when the volume of the drop is 0.07 mL [6]. Therefore, it is much more feasible to achieve vitrification by lowering the volume than by increasing the cooling rate. James H. Walton and Roy C. Judd measured the velocity of ice crystal growth and found that it is in the range of 65 mm/s [7]. This means that if we want to avoid crystallization in a drop placed on a cold metal surface and that has a diameter of 0.1 mm, we need a velocity of 1/6500 mm/s, which is 0.0001 second. If we cool from room temperature to 180 C, this means we need to reduce 200 C at a rate of 0.1 ms or at 78 106 C/min. This is actually the cooling rate that was estimated by Balds and by Bruggeller [1,5]. However, because this cooling rate is impossible to achieve, the ability to reach vitrification of pure water in a small drop can be achieved in relatively slow cooling rates, which indicate that the small volume has an independent effect on the probability of vitrification.
4. The beginning of slow freezing In 1949, Christopher Polge, Audrey Smith, and Alan Parkes [12], when trying to duplicate Luyet’s results, discovered by mistake the cryoprotective property of glycerol and so opened the field of slow freezing. Currently, there are two methods for gametes cryopreservation: slow freezing and vitrification. Slow freezing has the advantage of using low concentrations of CPs, which are associated with chemical toxicity and osmotic shock. Vitrification is a rapid method, which reduces chilling sensitivity and crystallization damage caused to cells. Sherman and Lin [13] showed that mouse oocytes need 8 to 10 minutes for equilibration in a freezing solution containing 5% glycerol at 37 C. In addition, he demonstrated that mouse oocytes will survive supercooling to 20 C after slow cooling at 0.6 C/min, however, oocytes that were cooled faster or to lower temperatures were damaged owing to intracellular crystallization. In the early 1970s, two groups were competing on achieving the first success of slow freezing of embryos. One group included Whittingham, Leibo, and Mazur, and the other Wilmut and Polge. Whittingham had partially succeeded in freezing embryos to 79 C for 30 minutes using polyvinylpyrrolidone; however, these experiment could not be duplicated [14–16]. Both groups published in 1972 the first survival of mouse embryos after slow freezing [15,16] and live offspring [15]. The technique included cooling at a slow rate in the presence of 1 mol/L DMSO, which most likely was the ingredient that enabled this. In 1976, sheep embryos were slow frozen by Willadsen using 1.5 mol/L DMSO and a cooling rate of 0.3 C/min. [17]. However, the first farm animal to be born after transplantation of frozen and thawed embryos was a calf. This was published by Wilmut and Rowson at 1973 [18]. Since
98
A. Arav / Theriogenology 81 (2014) 96–102
then, dozens of species have been successfully cryopreserved by slow freezing (for a review, see [19]).
5. The comeback of vitrification For many years, slow freezing and not vitrification was the method of choice for embryo cryopreservation. This was because vitrification was not achieved easily owing to the need of high CPs concentrations and relatively high volume samples. In 1985, the first successful vitrification of mouse embryos using a relatively large volume sample was done [20]. Rall and Fahy vitrified mouse embryos with mixture of DMSO, acetamide, and polyethylene glycol and in a relatively large volume inside a 0.25-mL straw plunged into LN. At that time, I was a veterinary student at the University of Bologna, Italy, and I met Bill Rall, who told me about the exciting work he did on mouse embryos. Two years later, I started to work on cryomicoscopy of oocytes and embryos. As in our laboratory, we used to prepare oocytes for histology evaluation by fixing them with a small drop over a microscopic slide. I had the idea of using the same technique for vitrification in a small drop, which I later named as the “minimum drop size” [6,21–24]. As noted, the probability of vitrification increases as the volume of the sample decreases. Pure water is vitrified only in very small droplets obtain from aerosols. Vitrification of thin layers (
