Chapter 2
Principles of Ice-Free Cryopreservation by Vitrification
Gregory M. Fahy and Brian Wowk
Abstract
Vitrification is an alternative to cryopreservation by freezing that enables hydrated living cells to be cooled
to cryogenic temperatures in the absence of ice. Vitrification simplifies and frequently improves cryopreservation because it eliminates mechanical injury from ice, eliminates the need to find optimal cooling and
warming rates, eliminates the importance of differing optimal cooling and warming rates for cells in mixed
cell type populations, eliminates the need to find a frequently imperfect compromise between solution
effects injury and intracellular ice formation, and can enable chilling injury to be “outrun” by using rapid
cooling without a risk of intracellular ice formation. On the other hand, vitrification requires much higher
concentrations of cryoprotectants than cryopreservation by freezing, which introduces greater risks of both
osmotic damage and cryoprotectant toxicity. Fortunately, a large number of remedies for the latter problem
have been discovered over the past 35 years, and osmotic damage can in most cases be eliminated or
adequately controlled by paying careful attention to cryoprotectant introduction and washout techniques.
Vitrification therefore has the potential to enable the superior and convenient cryopreservation of a wide
range of biological systems (including molecules, cells, tissues, organs, and even some whole organisms),
and it is also increasingly recognized as a successful strategy for surviving harsh environmental conditions in
nature. But the potential of vitrification is sometimes limited by an insufficient understanding of the
complex physical and biological principles involved, and therefore a better understanding may not only
help to improve present outcomes but may also point the way to new strategies that may be yet more
successful in the future. This chapter accordingly describes the basic principles of vitrification and indicates
the broad potential biological relevance of this alternative method of cryopreservation.
Key words Vitrification, Freezing, Cryopreservation, Intracellular ice formation, Devitrification,
Recrystallization, Chilling injury, Cryoprotective agents, Cryoprotectant toxicity, Osmotic limits,
Protein denaturation, Biobanking, Glass transition, Glassy state, Optimal cooling rate, Organ
preservation
1 Introduction and General Orientation
1.1 Overview
Vitrification is the solidification of a liquid into a non-crystalline or
amorphous (meaning, literally, “without structure”) solid known as
a glass [1, 2]. The industrial significance of glass is well understood
and long-standing. The first artificial glass artifacts date to 3100 BC
[3], while obsidian, which is a vitrified form of lava [4], was used to
make artifacts such as arrowheads as long ago as the Stone Age
Willem F. Wolkers and Harrie ¨ tte Oldenhof (eds.), Cryopreservation and Freeze-Drying Protocols, Methods in Molecular Biology,
vol. 2180, https://doi.org/10.1007/978-1-0716-0783-1_2, © Springer Science+Business Media, LLC, part of Springer Nature 2021
27
Principles of Ice-Free Cryopreservation by Vitrification
Gregory M. Fahy and Brian Wowk
Abstract
Vitrification is an alternative to cryopreservation by freezing that enables hydrated living cells to be cooled
to cryogenic temperatures in the absence of ice. Vitrification simplifies and frequently improves cryopreservation because it eliminates mechanical injury from ice, eliminates the need to find optimal cooling and
warming rates, eliminates the importance of differing optimal cooling and warming rates for cells in mixed
cell type populations, eliminates the need to find a frequently imperfect compromise between solution
effects injury and intracellular ice formation, and can enable chilling injury to be “outrun” by using rapid
cooling without a risk of intracellular ice formation. On the other hand, vitrification requires much higher
concentrations of cryoprotectants than cryopreservation by freezing, which introduces greater risks of both
osmotic damage and cryoprotectant toxicity. Fortunately, a large number of remedies for the latter problem
have been discovered over the past 35 years, and osmotic damage can in most cases be eliminated or
adequately controlled by paying careful attention to cryoprotectant introduction and washout techniques.
Vitrification therefore has the potential to enable the superior and convenient cryopreservation of a wide
range of biological systems (including molecules, cells, tissues, organs, and even some whole organisms),
and it is also increasingly recognized as a successful strategy for surviving harsh environmental conditions in
nature. But the potential of vitrification is sometimes limited by an insufficient understanding of the
complex physical and biological principles involved, and therefore a better understanding may not only
help to improve present outcomes but may also point the way to new strategies that may be yet more
successful in the future. This chapter accordingly describes the basic principles of vitrification and indicates
the broad potential biological relevance of this alternative method of cryopreservation.
Key words Vitrification, Freezing, Cryopreservation, Intracellular ice formation, Devitrification,
Recrystallization, Chilling injury, Cryoprotective agents, Cryoprotectant toxicity, Osmotic limits,
Protein denaturation, Biobanking, Glass transition, Glassy state, Optimal cooling rate, Organ
preservation
1 Introduction and General Orientation
1.1 Overview
Vitrification is the solidification of a liquid into a non-crystalline or
amorphous (meaning, literally, “without structure”) solid known as
a glass [1, 2]. The industrial significance of glass is well understood
and long-standing. The first artificial glass artifacts date to 3100 BC
[3], while obsidian, which is a vitrified form of lava [4], was used to
make artifacts such as arrowheads as long ago as the Stone Age
Willem F. Wolkers and Harrie ¨ tte Oldenhof (eds.), Cryopreservation and Freeze-Drying Protocols, Methods in Molecular Biology,
vol. 2180, https://doi.org/10.1007/978-1-0716-0783-1_2, © Springer Science+Business Media, LLC, part of Springer Nature 2021
27