Cryoprotectants: A review of the actions and applications of cryoprotective solutes that modulate cell recovery from ultra-low temperatures

Abstract

Cryopreservation has become a central technology in many areas of clinical medicine, biotechnology, and species conservation within both plant and animal biology. Cryoprotective agents (CPAs) invariably play key roles in allowing cells to be processed for storage at deep cryogenic temperatures and to be recovered with high levels of appropriate functionality. As such, these CPA solutes possess a wide range of metabolic and biophysical effects that are both necessary for their modes of action, and potentially complicating for cell biological function. Early successes with cryopreservation were achieved by empirical methodology for choosing and applying CPAs. In recent decades, it has been possible to assemble objective information about CPA modes of action and to optimize their application to living systems, but there still remain significant gaps in our understanding. This review sets out the current status on the biological and chemical knowledge surrounding CPAs, and the conflicting effects of protection versus toxicity resulting from the use of these solutes, which are often required in molar concentrations, far exceeding levels found in normal metabolism. The biophysical properties of CPAs that allow them to facilitate different approaches to cryogenic storage, including vitrification, are highlighted. The topics are discussed with reference to the historical background of applying CPAs, and the relevance of cryoprotective solutes in natural freeze tolerant organisms. Improved cryopreservation success will be an essential step in many future areas such as regenerative medicine, seed banking, or stem cell technology. To achieve this, we will need to further improve our understanding of cryobiology, where better and safer CPAs will be key requirements.

Mechanisms underlying insect freeze tolerance

“…4; Lee, 2010;Zachariassen, 1985). When ice melts, the decreased osmotic pressure could also cause damage if cell rehydration is too rapid ('osmotic shock', as reviewed by Elliott, Wang & Fuller, 2017). Thus, both dehydration and/or osmotic stress could damage cells in frozen and thawed insects (Yi & Lee, 2003;Worland et al, 2004;Pegg, 2010).…”

Serum- and albumin-free cryopreservation of endothelial monolayers with a new solution

“…For cryopreservation, in contrast to cold storage, 19 this protection by the chloride-poor solutions could in part be caused by the presence of the fairly high concentration (95 mM) of the large impermeable anion lactobionate rather than by the mere absence of chloride. Lactobionate is a disaccharide derivative, and other disaccharides are known to act as cryoprotective agents in various cell types 53 ; the presence of the disaccharide sucrose in combination with DMSO has also been shown to improve functionality of cryopreserved vessels. 54 Lactobionate might stabilize the plasma membrane during cryopreservation as known for other disaccharides and/or the low concentration of chloride might avoid chloride influx via a destabilized plasma membrane/fluid-to-gel phase transition during freezing/thawing.…”

Designing the next generation of cryoprotectants – From proteins to small molecules

“…Cells and tissues can be preserved at low temperatures using vitrification, anhydrous preservation, or cryopreservation . Vitrification is a process in which high cooling rates are employed making a solution extremely viscous during freezing, ultimately resulting in a “glass like” state.…”