Iron-dependent vs. iron- independent cold-induced injury to cultured rat hepatocytes: A comparative study in physiological media and organ preservation solutions

Abstract

We previously described the entity of cold-induced apoptosis to rat hepatocytes and characterized its major, iron-dependent pathway. However, after cold incubation in some solutions, e.g. cell culture medium, hepatocytes show an additional, yet uncharacterized component of cold-induced injury. We here assessed the effects of organ preservation solutions on both components of cold-induced injury and tried to further characterize the iron- independent component. None of the preservation solutions (University of Wisconsin, histidine–tryptophan–ketoglutarate, Euro-Collins, histidine–lactobionate, sodium–lactobionate–sucrose and Celsior solutions) provided significant protection against cold-induced cell injury (LDH release after 24-h cold incubation/3 h rewarming >65% for all solutions); three solutions even enhanced cold-induced injury. However, when the predominant iron-dependent mechanism was eliminated by the addition of iron chelators, all preservation solutions yielded hepatocyte protection that was clearly superior to the one obtainable in cell culture medium or Krebs–Henseleit buffer with iron chelators (LDH release after 24-h cold incubation/3 h rewarming ⩽35% in all preservation solutions and 65 ± 10% in culture medium). The iron-dependent and the weaker iron- independent component of cold-induced injury showed a different temperature dependence, and in experiments with modified Krebs–Henseleit buffer the principle of the preservation solutions that inhibits the iron- independent component was identified as the low chloride concentration of these solutions (LDH release after cold incubation/rewarming in the presence of iron chelators: 66 ± 6% in regular and 22 ± 8% in chloride-poor Krebs–Henseleit buffer). Taken together, these results suggest that solutions for cold storage of hepatocytes should be chloride-poor and contain an iron chelator.