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

Area of Science:

• Cryobiology
• Biomaterials Science
• Cellular Biology

Background:

• Vitrification offers ice-free cryopreservation for tissues and organs.
• High concentrations of cryoprotective agents (CPAs) required for vitrification cause toxicity, limiting its application.
• Minimizing CPA toxicity is crucial for advancing long-term tissue and organ storage.

Purpose of the Study:

• To characterize the toxicity kinetics of common CPAs and their mixtures in bovine pulmonary artery endothelial cells (BPAEC).
• To develop faster and more accurate experimental methods for determining CPA toxicity kinetics.
• To expand the existing mathematical model for CPA toxicity to include multi-CPA mixtures.

Main Methods:

• Utilized automated liquid handling to test toxicity kinetics of five CPAs (glycerol, DMSO, propylene glycol, ethylene glycol, formamide) and their binary/ternary mixtures.
• Employed a previously developed mathematical approach based on toxicity cost function minimization.
• Designed and implemented novel experimental protocols for rapid and precise toxicity assessment.

Main Results:

• Identified common CPA toxicity trends, such as increased toxicity with higher concentrations and relatively low toxicity of ethylene glycol.
• Discovered a potential toxicity neutralization effect between glycerol and formamide in mixtures.
• Generated a comprehensive dataset on CPA toxicity kinetics for BPAEC.

Conclusions:

• The developed methods enable quicker and more accurate toxicity characterization of CPAs and their mixtures.
• Findings suggest strategies for reducing CPA toxicity, including specific CPA combinations.
• The expanded dataset will enhance mathematical modeling for optimizing vitrification solutions with multiple CPAs.