Impact of cold storage protocols on passive arterial wall mechanical behavior

This study systematically evaluated how common cold storage protocols alter passive arterial wall mechanics before ex vivo testing. A total of 177 mechanically analyzed specimens were prepared from 12 porcine descending thoracic aortas and assigned to fresh testing or one of six storage protocols: refrigeration at 4 °C for 1 week, slow freezing in PBS at -80 °C, slow freezing in PBS with 10% DMSO, snap freezing without medium, snap freezing with DMSO, or snap freezing in pre-cooled isopentane. The authors used displacement-driven multidirectional biaxial testing to measure passive elastic and viscous behaviour, then used linear mixed-effects models to account for animal-to-animal and anatomical-location variability. Across all groups, arterial samples showed nonlinear strain-stiffening and higher circumferential than longitudinal stress, consistent with anisotropic arterial wall mechanics. Cold storage affected elastic behaviour more than viscous behaviour: refrigeration significantly decreased strain energy density, indicating softening, while snap freezing and cryoprotectant use generally increased strain energy density, especially under circumferential loading. Slow freezing in PBS to -80 °C best preserved fresh-tissue mechanical behaviour among the protocols tested. Variance analysis showed that animal identity and anatomical location explained a larger proportion of mechanical variability than storage protocol, emphasizing the importance of paired or mixed-effects study designs when comparing arterial biomechanics data.