Abstract
Cryopreservation of whole organs and specific tissues is an important and continually expanding field of medicine. The protocols currently used for organ preservation do not ensure survivability and functionality; the protocols for ovarian tissue lead to acceptable outcomes, but these are still capable of further improvement. In general, cryopreservation protocols need to be optimized. One important approach to improving cryopreservation protocols in general involves reducing exposure to cytotoxic cryoprotective agents prior to freezing. This study, therefore, evaluated the real-time tissue penetration of dimethyl sulfoxide, a cryoprotective agent that is widely used in cryopreservation. Dimethyl sulfoxide penetration in rat hearts perfused with a 15% (v/v) dimethyl sulfoxide solution was examined in real-time using dynamic contrast-enhanced micro-computed tomography imaging. Viability of cardiomyocytes was not significantly affected by the dimethyl sulfoxide perfusion procedure. Two different perfusion rates were evaluated and compared with perfusion using a common iodine-based contrast agent (iomeprol). The dynamic contrast-enhanced micro-computed tomography imaging data showed that dimethyl sulfoxide flushes both the extracellular and intracellular spaces in rat heart tissue to 95% equilibration after ≈ 35 s via perfusion. Subsequent wash-out via perfusion is completed to 95% within ≈ 49 s. The equilibration duration routinely used in dimethyl sulfoxide-based protocols for cryopreservation should therefore be questioned. Shorter incubation duration would perhaps be sufficient, as well as being beneficial in relation to cell survivability. It would be helpful to have techniques for non-invasive real-time monitoring of the penetration of cryoprotective agents and such techniques should be used to revise cryopreservation protocols. Switching to perfusion-based equilibration procedures might be beneficial, if feasible.