A Controlled Framework for Modeling Temperature Excursions in Cryopreserved Cells

The shipping and handling of cryopreserved biological materials require strict temperature control to preserve post-thaw viability and performance. Despite standard packaging and shipping controls, vials can experience warming events during transit, receipt, or transfers between storage systems. Because these excursions are unpredictable and difficult to reproduce, it can be difficult to compare excursion severity across cell types or evaluate mitigation approaches under consistent conditions.

We developed a programmable workflow to model controlled temperature excursions using a benchtop controlled-rate freezer (Strex CytoSAVER, FZ-2100). Due to the platform’s practical lower temperature limit of -80°C, we designed an excursion framework to probe dry ice relevant warming events that can occur during shipment and handling. A structured temperature-duration matrix spanning -80°C, -60°C, -40°C, -20°C, 0°C, and +20°C with dwell times from minutes to hours (5, 15, 30, 60, 120, and 240 minutes) was implemented. Cryopreserved vials were exposed to defined excursion conditions, returned to baseline storage temperature, and thawed using a standardized procedure. Post-thaw viability and cell concentration were quantified using the Vi-CELL BLU Cell Viability Analyzer.

The system was applied across THP-1, A-549, HepG2, U937, and SH-SY5Y cells to generate viability response surfaces and compare shared versus cell-specific susceptibility patterns. Excursion impact increased with both deviation temperature and exposure time, but sensitivity differed markedly by model. For example, following a -20°C excursion lasting 2 hours, THP-1 and SH-SY5Y lost >50% viability, whereas A-549 showed ~2% loss under the same condition.

This controlled excursion modeling framework enables benchmarking of biological robustness to cold-chain deviations and provides a reproducible testbed for evaluating mitigation strategies. The same matrix-based approach can be extended to newer biological models to support scale-up and logistics decisions for broader, more consistent use.