Impact of Nucleation Temperature and Hydroxyethyl Starch on Ice Crystal Growth: Implications for Cell Viability During Extreme Temperature Fluctuations

Extreme temperature fluctuations during routine handling and shipping of cryopreserved cell products significantly compromise product quality in ways that extend beyond the duration and peak temperature of the fluctuation. The type of cryoprotectant used and the initial ice nucleation temperature influence ice crystal growth during rewarming events, in turn impacting cell survival. Using a cryomicroscope together with temperature profiles recorded in cord-blood units, ice crystal growth was tracked through five transient-warming events (TWEs) that peaked at -30 °C, -20 °C, or -10 °C. Initial freezing conditions were modified either by adding 6 % (w/v) hydroxyethyl starch (HES) or by lowering the ice-nucleation temperature by 10 °C. Across five TWEs, ice-crystal area saw the greatest increase when the peak rewarming temperature was -10 °C. Although adding HES further accelerated this recrystallization, it still protected Jurkat cells after a single TWE. Lowering the nucleation temperature also improved viability in samples warmed to -20 °C, regardless of HES supplementation. These findings show that ice crystal growth is not the sole cause of injury during transient rewarming; other temperature-dependent stresses also play a role. Importantly, careful optimisation of cryoprotectant composition and nucleation temperature can bolster cellular resilience to temperature excursions, potentially reducing quality losses during the storage and transport of cryopreserved therapeutics.