Chapter 1
Principles Underlying Cryopreservation and Freeze-Drying
of Cells and Tissues
Willem F. Wolkers and Harrie ¨ tte Oldenhof
Abstract
Cryopreservation and freeze-drying can be used to preserve cells or tissues for prolonged periods. Vitrification, or ice-free cryopreservation, is an alternative to cryopreservation that enables cooling cells to
cryogenic temperatures in the absence of ice. The processing pathways involved in (ice-free) cryopreservation and freeze-drying of cells and tissues, however, can be very damaging. In this chapter, we describe the
principles underlying preservation of cells for which freezing and drying are normally lethal processes as well
as for cells that are able to survive in a reversible state of suspended animation. Freezing results in solution
effects injury and/or intracellular ice formation, whereas drying results in removal of (non-freezable) water
normally bound to biomolecules, which is generally more damaging. Cryopreservation and freeze-drying
require different types of protective agents. Different mechanistic modes of action of cryoprotective and
lyoprotective agents are described including minimizing ice formation, preferential exclusion, water
replacement, and vitrification. Furthermore, it is discussed how protective agents can be introduced into
cells avoiding damage due to too large cell volume excursions, and how knowledge of cell-specific
membrane permeability properties in various temperature regimes can be used to rationally design
(ice-free) cryopreservation and freeze-drying protocols.
Key words Cryopreservation, Vitrification, Cryoprotectants, Lyoprotectants, Anhydrobiosis, Preferential exclusion theory, Water replacement theory, Membrane phase behavior, Membrane permeability
1 Nature’s Way to Preserve Life
Living systems are dependent on the presence of water. Removal of
water from tissues, cells, and biomolecules by drying or freezing
typically is not without consequences. It may result in osmotic
stress and conformational changes that affect cellular and biomolecular structure and function (see Fig. 1d). In addition, ice formation may lead to mechanical stress. One possibility to preserve life in
the frozen or dried state, for systems that normally will not survive
exposure to extreme conditions, is to develop “bioinspired” protective formulations and strategies.
In nature, there are spectacular examples of preservation of life
in a reversible state of suspended animation. Anhydrobiosis refers
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_1, © Springer Science+Business Media, LLC, part of Springer Nature 2021
3
Principles Underlying Cryopreservation and Freeze-Drying
of Cells and Tissues
Willem F. Wolkers and Harrie ¨ tte Oldenhof
Abstract
Cryopreservation and freeze-drying can be used to preserve cells or tissues for prolonged periods. Vitrification, or ice-free cryopreservation, is an alternative to cryopreservation that enables cooling cells to
cryogenic temperatures in the absence of ice. The processing pathways involved in (ice-free) cryopreservation and freeze-drying of cells and tissues, however, can be very damaging. In this chapter, we describe the
principles underlying preservation of cells for which freezing and drying are normally lethal processes as well
as for cells that are able to survive in a reversible state of suspended animation. Freezing results in solution
effects injury and/or intracellular ice formation, whereas drying results in removal of (non-freezable) water
normally bound to biomolecules, which is generally more damaging. Cryopreservation and freeze-drying
require different types of protective agents. Different mechanistic modes of action of cryoprotective and
lyoprotective agents are described including minimizing ice formation, preferential exclusion, water
replacement, and vitrification. Furthermore, it is discussed how protective agents can be introduced into
cells avoiding damage due to too large cell volume excursions, and how knowledge of cell-specific
membrane permeability properties in various temperature regimes can be used to rationally design
(ice-free) cryopreservation and freeze-drying protocols.
Key words Cryopreservation, Vitrification, Cryoprotectants, Lyoprotectants, Anhydrobiosis, Preferential exclusion theory, Water replacement theory, Membrane phase behavior, Membrane permeability
1 Nature’s Way to Preserve Life
Living systems are dependent on the presence of water. Removal of
water from tissues, cells, and biomolecules by drying or freezing
typically is not without consequences. It may result in osmotic
stress and conformational changes that affect cellular and biomolecular structure and function (see Fig. 1d). In addition, ice formation may lead to mechanical stress. One possibility to preserve life in
the frozen or dried state, for systems that normally will not survive
exposure to extreme conditions, is to develop “bioinspired” protective formulations and strategies.
In nature, there are spectacular examples of preservation of life
in a reversible state of suspended animation. Anhydrobiosis refers
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_1, © Springer Science+Business Media, LLC, part of Springer Nature 2021
3