Winter is coming: the future of cryopreservation

Abstract

The preservative effects of low temperature on biological materials have been long recognised, and cryopreservation is now widely used in biomedicine, including in organ transplantation, regenerative medicine and drug discovery. The lack of organs for transplantation constitutes a major medical challenge, stemming largely from the inability to preserve donated organs until a suitable recipient is found. Here, we review the latest cryopreservation methods and applications. We describe the main challenges-scaling up to large volumes and complex tissues, preventing ice formation and mitigating cryoprotectant toxicity-discuss advantages and disadvantages of current methods and outline prospects for the future of the field.

Keywords: Biostasis; Cryobiology; Freezing; Organ banking; Vitrification.

Fig. 1

An overview of the mechanisms of cryopreservation and CPAs. Cryopreservation utilises either slow cooling, in which the sample is frozen at a controlled rate to allow water to flow out of the cell and prevent intracellular ice formation, or vitrification in which a high freezing rate and/or high CPA concentration prevents ice formation in the sample. Ice growth can be further controlled by ice nucleation inhibitors, controlled ice nucleation, ice growth inhibitors or ice recrystallization inhibitors. Devitrification occurs when a vitrified sample is warmed too slowly, resulting in ice formation. Apoptosis inhibitors may also be utilised to prevent cells from dying after cryopreservation from stress-induced apoptosis

Fig. 2

How cryoprotective agents (CPAs) work. A When a sample is cooled, ice first forms in the extracellular space. Ice excludes solutes, so the extracellular solute concentration increases as extracellular water becomes part of the ice crystals. Intracellular water is then drawn out of the cells via osmosis. B In unprotected cells, the intracellular solute concentration increases and causes damage. C Penetrating CPAs permeate the cell membrane and increase the intracellular solute concentration which prevents water loss and dilutes other solutes inside the cell which can cause damage at high concentrations. Ice blockers bind to ice crystals, preventing them from growing, or to nucleators, which prevents heterogenous ice nucleation. D1, D2 Penetrating CPAs interfere with homogenous ice nucleation by colligative interference, which depresses the freezing point of the solution. D1 Water forms a regular ice lattice. D2 A CPA molecule disrupts the hydrogen bonding between water molecules

Fig. 3

A vitrified substance (left) is formed when liquid transitions into a highly viscous, glass-like state that prevents translational molecular motion. In vitrification, molecules remain in the position they were in when the substance was vitrified. This is different to freezing (right), in which ice crystals progressively grow as the temperature is decreased, excluding solutes and thus causing the solute concentration to increase

Fig. 4

In directional freezing, cold is applied to one side of the sample causing a temperature gradient. Uniform ice lamellae form along this gradient with heat being conducted through the unfrozen part of the sample