A High-Throughput Platform for Measuring and Predicting Vitrification Behavior in Multicomponent Aqueous Solutions

Area of Science:

• Cryobiology
• Biophysics
• Materials Science

Background:

• Cryopreservation relies on cryoprotective agents (CPAs) to prevent ice formation.
• Limited data exist on the critical CPA concentration for vitrification (Cv).

Purpose of the Study:

• To develop a high-throughput platform for rapid Cv determination.
• To investigate factors influencing vitrification behavior.
• To create a predictive model for CPA formulations.

Main Methods:

• A 384-well automated liquid handling platform with randomized layouts.
• A binary-search strategy for efficient Cv determination.
• Analysis of ~200 CPA compositions and environmental boundary conditions.

Main Results:

• The platform achieved a ~50-fold increase in throughput, reducing measurement time from a year to a week.
• Sealed plates demonstrated lower Cv than open plates, indicating improved vitrification.
• Cv decreased with increasing CPA molecular weight, suggesting larger molecules are more effective ice suppressors.
• A predictive mixture model accurately estimated Cv for complex CPA mixtures (R² ≥ 0.93).

Conclusions:

• The developed platform enables rapid and efficient determination of CPA concentrations for vitrification.
• Environmental conditions and CPA molecular properties significantly impact vitrification.
• A predictive model facilitates the rational design of low-toxicity CPA formulations near their vitrification threshold.