Screening for cryoprotective agent toxicity and toxicity reduction in mixtures at subambient temperatures

Organ transplantation faces major challenges in preserving and transporting organs due to the limitations of existing cold storage methods. Cryopreservation offers a promising alternative for extending preservation time, but it remains a challenge to avoid toxicity from the high concentrations of cryoprotective agents (CPAs) required to prevent ice formation. In this study, we expanded a previously reported high-throughput CPA toxicity screening platform by retrofitting an automated liquid handling system with subambient cooling capabilities. This enabled systematic assessment of CPA toxicity at 4 °C, a temperature commonly used for CPA equilibration in tissue and organ cryopreservation. Overall, we screened 22 individual CPAs and a wide range of binary mixtures at concentrations up to 12 mol/kg, allowing us to identify CPA combinations that reduce toxicity. Our findings revealed that at 4 °C, CPA toxicity was significantly reduced compared to room temperature. Several CPA combinations resulted in significantly lower toxicity than their constituent CPAs at the same concentration, including 12 CPA mixtures at 6 mol/kg and 8 CPA mixtures at 12 mol/kg. Toxicity neutralization was also observed in 9 cases, especially in combinations involving formamide, acetamide, dimethyl sulfoxide, and glycerol. For example, exposure to 6 mol/kg formamide alone resulted in 20% viability, but the addition of 6 mol/kg glycerol to create a mixture with a total concentration of 12 mol/kg eliminated this toxicity, resulting in a viability of 97%. These findings support the rationale for using multi-CPA cocktails and underscore the potential of rational mixture design to reduce toxicity.