Rapid quantification of multi-cryoprotectant toxicity using an automated liquid handling method

Cryopreservation in a vitrified state has vast potential for long-term storage of tissues and organs that may be damaged by ice formation. However, the toxicity imparted by the high concentration of cryoprotectants (CPAs) required to vitrify these specimens remains a hurdle. To address this challenge, we previously developed a mathematical approach to design less toxic CPA equilibration methods based on the minimization of a toxicity cost function. This approach was used to design improved methods for equilibration of bovine pulmonary artery endothelial cells (BPAEC) with glycerol. To fully capitalize on the toxicity cost function approach, it is critical to describe the toxicity kinetics of additional CPAs, including multi-CPA mixtures that are commonly used for vitrification. In this work, we used automated liquid handling to characterize the toxicity kinetics of five of the most common CPAs (glycerol, dimethyl sulfoxide (DMSO), propylene glycol, ethylene glycol, and formamide), along with their binary and ternary mixtures for BPAEC. In doing so, we developed experimental methods that can be used to determine toxicity kinetics more quickly and accurately. Our results highlight some common CPA toxicity trends, including the relatively low toxicity of ethylene glycol and a general increase in toxicity as the CPA concentration increases. Our results also suggest potential new approaches to reduce toxicity, including a surprising toxicity neutralization effect of glycerol on formamide. In the future, this dataset will serve as the basis to expand our CPA toxicity model, enabling application of the toxicity cost function approach to vitrification solutions containing multiple CPAs.