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  • Review Article
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Chemical approaches to cryopreservation

Nature Reviews Chemistry volume 6, pages 579–593 (2022)Cite this article

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Abstract

Cryopreservation of cells and biologics underpins all biomedical research from routine sample storage to emerging cell-based therapies, as well as ensuring cell banks provide authenticated, stable and consistent cell products. This field began with the discovery and wide adoption of glycerol and dimethyl sulfoxide as cryoprotectants over 60 years ago, but these tools do not work for all cells and are not ideal for all workflows. In this Review, we highlight and critically review the approaches to discover, and apply, new chemical tools for cryopreservation. We summarize the key (and complex) damage pathways during cellular cryopreservation and how each can be addressed. Bio-inspired approaches, such as those based on extremophiles, are also discussed. We describe both small-molecule-based and macromolecular-based strategies, including ice binders, ice nucleators, ice nucleation inhibitors and emerging materials whose exact mechanism has yet to be understood. Finally, looking towards the future of the field, the application of bottom-up molecular modelling, library-based discovery approaches and materials science tools, which are set to transform cryopreservation strategies, are also included.

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Fig. 1: Potential mechanisms of cellular damage during cryopreservation.
Fig. 2: Vitrification agents for cryopreservation.
Fig. 3: Ice recrystallization inhibitors.
Fig. 4: Macromolecular cryoprotectants for cryopreservation.
Fig. 5: Control of ice nucleation.
Fig. 6: Modulating biochemical pathways.

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Acknowledgements

K.A.M. thanks the Wellcome Trust and the University of Warwick for supporting a Translational Partnership Fellowship (WT-219429/Z/19/Z). This project (M.I.G.) has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement no. 866056) and the Royal Society for an Industry Fellowship (M.I.G., 191037) with Cytiva.

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  1. Department of Chemistry, University of Warwick, Coventry, UK

    Kathryn A. Murray & Matthew I. Gibson

  2. Division of Biomedical Sciences, Warwick Medical School, University of Warwick, Coventry, UK

    Matthew I. Gibson

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M.I.G. is co-founder and shareholder of Cryologyx Ltd, which is developing cryopreservation technology.

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Glossary

Phenotypic drift

A gradual change in the observable traits of a cell due to prolonged time in culture.

Cell therapies

Using a patient’s own cells, or donated cells, to elicit a medicinal response to disease.

Immunotherapies

Treatments that use a person’s immune system to fight diseases, such as cancer.

Cold chain

A low-temperature supply chain.

Cryoprotective agents

(CPAs). Compounds added to freezing solutions to provide a protective effect to the material being frozen.

Ice recrystallization

An increase in ice crystal size over time in an already frozen material.

Glass transition temperature

The temperature at which materials transition from rigid to flexible.

Super-flash freezing

Cooling at an incredibly high cooling rate, so the liquid freezes almost instantly.

Differential scanning calorimetry

(DSC). A thermal analysis technique used to study the heat flow of a sample.

Ice-binding proteins

Molecules found in a diverse range of organisms that bind to the interface between ice and water.

Thermal hysteresis

A difference in the melting and freezing temperatures.

Exopolysaccharides

Macromolecules secreted from microorganisms.

Extremophilic

A propensity for extreme environments, such as organisms that survive at very high or very low temperatures.

Heterogeneous ice nucleation

Ice crystallization induced by the presence of foreign particles.

Ice seeding

Introducing microscopic ice crystals to induce nucleation in a supercooled liquid.

Electrofreezing

Applying an electric current to induce ice nucleation in a supercooled liquid.

Shock cooling

Applying external force or rapid temperature changes to induce nucleation in a supercooled liquid.

Cryosurvival

The ability to survive exposure to cold temperatures, often referring to temperatures far below 0 °C.

Caspase

Protease enzyme involved in the activation and execution of apoptosis.

Calpain cascade

A signalling cascade driven by calcium-dependent cysteine proteases, known as calpains.

Lab-on-chip

A miniature microchip device used to integrate several processes that are typically performed in a laboratory, such as chemical or biological analyses.

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Murray, K.A., Gibson, M.I. Chemical approaches to cryopreservation. Nat Rev Chem 6, 579–593 (2022). https://doi.org/10.1038/s41570-022-00407-4

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