Vitrification: Methods Contributing to Successful Cryopreservation Outcomes
The only method of stable and long-term preservation of perishable biological materials is to keep them in the glassy (vitreous) state. Embryo and gamete storage has come far since the discovery of glycerol, to the successful cryopreservation of mouse emb
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Vitrification: Methods Contributing to Successful Cryopreservation Outcomes James J. Stachecki 54.1
Fundamentals of Vitrification – 666
54.1.1 54.1.2 54.1.3 54.1.4 54.1.5 54.1.6
itrification Studies: Animal Research – 667 V Human Research – 668 Current Vitrification Technologies: DMSO Methods – 668 Non-DMSO Methods – 669 Vitrification Outcomes – 669 Emerging Concepts – 670
54.2
Beyond Vitrification: Other Factors Affecting Outcomes – 670
54.2.1 54.2.2
E mbryo Culture – 670 Patient Preparation and Embryo Transfer – 671
54.3
Conclusion – 671
Review Questions – 672 References – 672
© Springer Nature Switzerland AG 2019 Z. P. Nagy et al. (eds.), In Vitro Fertilization, https://doi.org/10.1007/978-3-319-43011-9_54
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666
J. J. Stachecki
Learning Objectives 55 To provide a basic understanding of key cryopreservation/vitrification concepts 55 To review past research leading to current methodologies 55 To discuss concepts important to frozen embryo transfer success
54.1 Fundamentals of Vitrification
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The only method of stable and long-term (practically infinite) preservation and storage of any perishable biological materials, particularly cells, is to keep them in the glassy (vitreous) state. This was apparent to Father Luyet when he titled his pioneering work “The vitrification of organic colloids and of protoplasm” and “Revival of frog’s spermatozoa vitrified in liquid air” [1, 2]. He and other pioneers of the cryobiological frontiers including Lovelock, Meryman, Mazur, Polge, Smith, Levitt, Farrant, and Willadsen clearly understood some 40–70 years ago that only a glassy state would insure stable and non lethal preservation of cells [3–6]. With time, we saw the development of a variety of biopreservation methods, such as slow-cooling, ultra rapid cooling, and kinetic vitrification [7, 8]. It was Luyet’s work that would make cryopreservation a science. From the outset, he recognized that ice damage must be avoided and vitrification could be a method for long-term preservation of cell viability [2]. In order to understand rapid-cooling or “modern” vitrification techniques, let us compare them to the slow-cooling method. During slow-cooling, embryos are exposed to relatively low concentrations of cryoprotectants (1.5 M PrOH and usually some sucrose, around 0.2 M), equilibrated for 10–25 min at room temperature, loaded into a straw or vial, sealed, and placed into a controlled rate freezer. Ice formation is induced extracellularly by seeding at a temperature, whereby ice can perpetuate (around −5.5 °C or lower), and, as a result of the solute gradient created, freezable water flows out of the cells, minimizing the chance of intracellular ice formation during cooling. As the temperature is gradually lowered, the concentration of cryoprotectant in the liquid phase, which includes the intracellular fluid, increases correspondingly until a level is reached at which additional formation and growth of ice crystals, although possible, are unlikely, even if the temperature drops further
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