Macromolecular cryoprotectants for the preservation of mammalian cell culture: lessons from crowding, overview and perspectives.

ABSTRACT

Cryopreservation is a process used for the storage of mammalian cells at a very low temperature, in a state of 'suspended animation.' Highly effective and safe macromolecular cryoprotectants (CPAs) have gained significant attention as they obviate the toxicity of conventional CPAs like dimethyl sulfoxide (DMSO) and reduce the risks involved in the storage of cultures at liquid nitrogen temperatures. These agents provide cryoprotection through multiple mechanisms, involving extracellular and intracellular macromolecular crowding, thereby impacting the biophysical and biochemical dynamics of the freezing medium and the cryopreserved cells. These CPAs vary in their structures and physicochemical properties, which influence their cryoprotective activities. Moreover, the introduction of polymeric crowders in the cryopreservation media enables serum-free storage at low-DMSO concentrations and high-temperature vitrification of frozen cultures (-80 °C). This review highlights the need for macromolecular CPAs and describes their mechanisms of cryopreservation, by elucidating the role of crowding effects. It also classifies the macromolecules based on their chemistry and their structure-activity relationships. Furthermore, this article provides perspectives on the factors that may influence the outcomes of the cell freezing process or may help in designing and evaluating prospective macromolecules. This manuscript also includes case studies about cellular investigations that have been conducted to demonstrate the cryoprotective potential of macromolecular CPAs. Ultimately, this review provides essential directives that will further improve the cell cryopreservation process and may encourage the use of macromolecular CPAs to fortify basic, applied, and translational research.