Chapter 9
Use of Ice Recrystallization Inhibition Assays to Screen
for Compounds That Inhibit Ice Recrystallization
Anna A. Ampaw, August Sibthorpe, and Robert N. Ben
Abstract
Ice recrystallization inhibition assays are used to screen for compounds that possess the ability to inhibit ice
recrystallization. The most common of these assays are the splat cooling assay (SCA) and sucrose sandwich
assay (SSA). These two assays possess similarities; however, they vary in their sample size, cooling rate, and
the solution used to dissolve the analyte. In this chapter, both assay methods are described in detail, and we
perform a direct comparison of the assays by evaluating the IRI activity of an antifreeze protein (AFP I). IRI
activity is quantified by using ImageJ software to analyze ice crystals, and a quantitative value describing the
efficiency of the inhibitor is generated. This analysis emphasizes the importance of choosing the right assay
to measure IRI activity.
Key words Ice crystals, Ice recrystallization, Cryopreservation, Antifreeze protein
1 Introduction
1.1 General
Background
Ice recrystallization is a process that occurs during cryopreservation. During the freezing and thawing cycles of cryopreservation,
large ice crystals grow into larger ice crystals at the expense of
smaller ones resulting in an overall grain coarsening effect. This is
a thermodynamic phenomenon driven by an overall reduction in
free energy as ice crystals grow larger [1–4]. During cryopreservation, there are many mechanisms by which cellular injury occurs,
but the uncontrolled growth of ice (recrystallization) is one of the
main causes of cellular damage and death [5].
Most cryopreservation methods/protocols utilize a cryoprotective agent (CPA) such as glycerol or dimethyl sulfoxide (DMSO)
[6, 7]. These cryoprotective agents function by decreasing the
colligative freezing point of solutions and replace intracellular
water during cooling and dehydration; however, they do not prevent ice recrystallization and thus do not mitigate cellular injury
resulting from mechanical damage due to ice recrystallization
[8]. An effective solution to this problem lies with the discovery
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_9, © Springer Science+Business Media, LLC, part of Springer Nature 2021
271
Use of Ice Recrystallization Inhibition Assays to Screen
for Compounds That Inhibit Ice Recrystallization
Anna A. Ampaw, August Sibthorpe, and Robert N. Ben
Abstract
Ice recrystallization inhibition assays are used to screen for compounds that possess the ability to inhibit ice
recrystallization. The most common of these assays are the splat cooling assay (SCA) and sucrose sandwich
assay (SSA). These two assays possess similarities; however, they vary in their sample size, cooling rate, and
the solution used to dissolve the analyte. In this chapter, both assay methods are described in detail, and we
perform a direct comparison of the assays by evaluating the IRI activity of an antifreeze protein (AFP I). IRI
activity is quantified by using ImageJ software to analyze ice crystals, and a quantitative value describing the
efficiency of the inhibitor is generated. This analysis emphasizes the importance of choosing the right assay
to measure IRI activity.
Key words Ice crystals, Ice recrystallization, Cryopreservation, Antifreeze protein
1 Introduction
1.1 General
Background
Ice recrystallization is a process that occurs during cryopreservation. During the freezing and thawing cycles of cryopreservation,
large ice crystals grow into larger ice crystals at the expense of
smaller ones resulting in an overall grain coarsening effect. This is
a thermodynamic phenomenon driven by an overall reduction in
free energy as ice crystals grow larger [1–4]. During cryopreservation, there are many mechanisms by which cellular injury occurs,
but the uncontrolled growth of ice (recrystallization) is one of the
main causes of cellular damage and death [5].
Most cryopreservation methods/protocols utilize a cryoprotective agent (CPA) such as glycerol or dimethyl sulfoxide (DMSO)
[6, 7]. These cryoprotective agents function by decreasing the
colligative freezing point of solutions and replace intracellular
water during cooling and dehydration; however, they do not prevent ice recrystallization and thus do not mitigate cellular injury
resulting from mechanical damage due to ice recrystallization
[8]. An effective solution to this problem lies with the discovery
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_9, © Springer Science+Business Media, LLC, part of Springer Nature 2021
271