The need for novel cryoprotectants and cryopreservation protocols: Insights into the importance of biophysical investigation and cell permeability

Background: Cryopreservation is a key method of preservation of biological material for both medical treatments and conservation of endangered species. In order to avoid cellular damage, cryopreservation relies on the addition of a suitable cryoprotective agent (CPA). However, the toxicity of CPAs is a serious concern and often requires rapid removal on thawing which is time consuming and expensive. Scope of review: The principles of Cryopreservation are reviewed and recent advances in cryopreservation methods and new CPAs are described. The importance of understanding key biophysical properties to assess the cryoprotective potential of new non-toxic compounds is discussed. Major conclusions: Knowing the biophysical properties of a particular cell type is crucial for developing new cryopreservation protocols. Similarly, understanding how potential CPAs interact with cells is key for optimising protocols. For example, cells with a large osmotically inactive volume may require slower addition of CPAs. Similarly, a cell with low permeability may require a longer incubation time with the CPA to allow adequate penetration. Measuring these properties allows efficient optimisation of cryopreservation protocols. General significance: Understanding the interplay between cells and biophysical properties is important not just for developing new, and better optimised, cryopreservation protocols, but also for broader research into topics such as dehydration and desiccation tolerance, chilling and heat stress, as well as membrane structure and function.