Laboratory Products
Characterisation of thermal excursions in cryogenically stored vials: implications for exceeding the glass transition temperature of water
Erica Waller, Azenta Life Sciences
Biological samples stored below −150°C in liquid nitrogen (LN₂) vapour-phase freezers rely on the assumption that biochemical and degradative processes cease below the glass transition temperature of water (Tg,H₂O ≈ −134°C). However, transient temperature excursions during handling can expose samples to warming rates suffi cient to exceed Tg,H₂O within seconds. This study characterises the warm-up behaviour of single H₂O-fi lled cryovials during typical handling conditions and quantifi es the risk of crossing Tg,H₂O. Results from experimental measurements and fi nite element simulations are used to inform best practices for safe cryogenic sample handling.
Introduction
Cryogenic storage is a cornerstone of long-term biological sample preservation. The critical threshold for sample stability is the water glass transition temperature (Tg,H₂O), below which molecular mobility and chemical activity are effectively halted. However, during handling - particularly transfers from LN₂ storage to ambient (RT) or −80°C dry ice environments - samples are susceptible to rapid thermal excursions. Understanding the dynamics of vial warm-up rates is crucial to mitigate degradation risk during such events.
Materials and Methods
Experimental Setup • Samples: FluidX™ 1.0 mL and 2.0 mL vials, and Wheaton™ 2.0 mL vials, fi lled with 100% or 25% of maximum working volume (MWV) with deionised H₂O.
• Storage: Vials equilibrated in a vapour-phase LN₂ freezer at −173°C.
• Measurement: Vials instrumented with three 32-gauge Type T thermocouples positioned at:
1. 2 mm below the water surface 2. Vial centre 3. Vial base
• Warm-Up Conditions: Vials warmed by suspension in ambient air or immersion in dry ice pellets; no user contact. Warm-up occurred under natural convection unless otherwise noted.
Simulation Setup
Finite element (FE) models were developed in ANSYS 15.0 and calibrated against empirical data. Ice properties were temperature-dependent; polypropylene vial properties were assumed at room temperature.
Results
Warm-Up Rates • Single Vials (−175°C to −120°C): Warm-up rates ranged from 55 to 255°C/min. • Inside Cryoboxes: Rates were reduced to 5.4 to 66.7°C/min. • Time to Exceed Tg,H₂O: o RT Environment: 24–40 s o Dry Ice: 9–45 s (faster due to enhanced conduction and CO₂ convection)
• Volume Effects: Smaller H₂O volumes warmed 15–35% faster, but volume sensitivity was modest compared to environmental impact.
• Thermal Uniformity: In both environments, spatial temperature variation in vial interiors was ≤4°C at any time, confi rming near-uniform thermal distribution.
Figure 1: Warm-up rate sensitivity to volume and exposure environment. Lower H₂O volumes provide decreased thermal mass at equivalent heat energy absorbed by the vial. This corresponds to slightly increased warm-up rates (15 – 30%), yet the effect is not signifi cant compared to a fast vial warm-up at T<-90°C. Single vials in dry ice warm above Tg,H₂O (-134°C) much faster than in an RT environment.
Table 1. Single tube warm-up rates1 from -175°C storage to lab bench (21°C) or dry ice temperature [°C/min]
H20 fi lled tubes RT, 100% MWV RT, 25% MWV
Dry ice, 100% MWV Dry ice, 25% MWV
FluidX 1.0 ml vial 94 – 101 101 – 112 171 – 213 195 – 255
FluidX 2.0 ml vial 55 – 62 67 – 72 92 – 120 106 – 141
1. Linearised warm-up rate in the -175°C to -120°C range.
Table 2. Cryobox stored tube warm-up rates1 from -175°C storage to lab bench (21°C) [°C/min] H20 fi lled tubes
FluidX 1.0 ml vial2
Full rack, cover on 6.8 – 22.3 Partial rack, cover on 18.2 – 26.2 Full rack, cover off 15.0 – 36.9 Partial rack, cover off 36.2 – 50.4
FluidX 2.0 ml vial3
5.9 – 12.5 16.8 – 21.6 13.0 – 19.8 28.5 – 66.7
Biocision 2.0 ml vial4
5.4 – 15.6 9.8 – 17.4 6.9 – 20.9 11.5 – 25.8
Wheaton 2.0 ml vial5
8.1 – 10.8 23.5 – 29.0 10.2 – 24.3 34.9 – 44.0
tube with screw cap fi lled with 1.8 ml H2 hinged cryobox with LN2
box, tubes fi lled with 1.8 ml H2
1. Linearised warm-up rate in the -175°C to -130°C range. 2. 96 format storage tube with screw cap fi lled with 0.73 ml H2
drain holes; tubes fi lled with 1.8 ml H2 0, perforated rack underside.
0; perforated rack underside. 3. 9x9 sample storage 0, no perforated rack underside. 4. 9x9 TruCool 0. 5. KeepIT-100 freezer
Wheaton 2.0 ml vial 57 – 62 70 – 76
100 – 143 164 – 192
INTERNATIONAL LABMATE - SEPTEMBER 2025
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