Ahmadkhani, N.; Sugden, C.; Brown, D.; Drummond, N.; Snyder, A.; Uden, M.; Higgins, A. Z.

Cryopreservation depends critically on suppression of ice formation by cryoprotective agents (CPAs), but limited data is available on the CPA concentration required for vitrification (Cv). Here, we introduce a high-throughput 384-well platform that integrates automated liquid handling, randomized plate layouts, and a binary-search strategy to rapidly determine Cv across hundreds of formulations. Relative to conventional methods, this approach increases throughput by ~50-fold, compressing a year of measurements into one week, while markedly reducing manual labor. Across ~200 CPA compositions, we demonstrate that environmental boundary conditions strongly influence vitrification behavior: plates sealed with silicone mats exhibited lower Cv than open plates, indicating that sealed configurations promote vitrification. Further, the data reveal a decrease in Cv with increasing CPA molecular weight, consistent with enhanced ice suppression by larger molecules. We also present a simple mixture model that accurately predicts Cv for a broad range of CPA formulations, including mixtures containing up to seven CPAs (R2 > 0.94), and use this model to evaluate published CPA toxicity data to identify formulations that operate near their vitrification threshold while maintaining relatively low toxicity. Together, these results establish a framework for rapid Cv determination, predictive modeling of vitrification behavior, and rational design of CPA formulations.