The efficacy of ice recrystallization inhibitors in rat lung cryopreservation using a low cost technique for ex vivo subnormothermic lung perfusion

Abstract

Although lung transplant remains the only option for patients with end-stage lung failure, short preservation times result in an inability to meet patient demand. Successful cryopreservation may ameliorate this problem; however, very little research has been performed on lung cryopreservation due to the inability to prevent ice nucleation or growth. Therefore, this research sought to characterize the efficacy of a small-molecule ice recrystallization inhibitor (IRI) for lung cryopreservation given its well-documented ability to control ice growth.Sprague-Dawley heart-lung blocks were perfused at room temperature using a syringe-pump. Cytotoxicity of the IRI was assessed through the subsequent perfusion with 0.4% (w/v) trypan blue followed by formalin-fixation. Ice control was assessed by freezing at a chamber rate of −5 °C/min to −20 °C and cryofixation using a low-temperature fixative. Post-thaw cell survival was determined by freezing at a chamber rate of −5 °C/min to −20 °C and thawing in a 37 °C water bath before formalin-fixation. In all cases, samples were paraffin-embedded, sliced, and stained with eosin.The IRI studied was found to be non-toxic, as cell membrane integrity following perfusion was not significantly different than controls (p = 0.9292). Alveolar ice grain size was significantly reduced by the addition of this IRI (p = 0.0096), and the addition of the IRI to DMSO significantly improved post-thaw cell membrane integrity when compared to controls treated with DMSO alone (p = 0.0034).The techniques described here provide a low-cost solution for rat ex vivo lung perfusion which demonstrated that the ice control and improved post-thaw cell survival afforded by IRI-use warrants further study.