Elsevier

Cryobiology

Volume 54, Issue 1, February 2007, Pages 77-86
Cryobiology

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

https://doi.org/10.1016/j.cryobiol.2006.11.008Get rights and content

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.

Section snippets

Animals

Male Wistar rats (250–300 g) were obtained from the Zentrales Tierlaboratorium (Universitätsklinikum Essen). Animals were kept under standard conditions with free access to food and water. All animals received humane care in compliance with the institutional guidelines.

Chemicals

Leibovitz L-15 medium, 2,2′-dipyridyl, choline chloride, choline bicarbonate, choline free base and lactobionic acid were obtained from Sigma–Aldrich (Taufkirchen, Germany). Deferoxamine mesylate was purchased from Novartis Pharma

Effect of iron chelators on cold-induced injury to rat hepatocytes during/after cold incubation in cell culture medium

When incubated for 24 h at 4 °C in cell culture medium (Leibovitz L-15) and rewarmed thereafter, cultured rat hepatocytes showed major cold-induced injury (Fig. 1). Part of this injury occurred during the cold incubation itself, part during rewarming. When cold incubation was performed in the presence of the iron chelator 2,2′-dipyridyl, cells were almost completely protected during cold incubation, but a major part of the protection was lost during rewarming. Practically identical results as

Discussion

We here further characterized cold-induced cell injury/apoptosis to rat hepatocytes and showed that the iron-independent component of cold-induced hepatocyte injury is a chloride-dependent component, predominantly occurs during rewarming, is associated with marked cell detachment and does not occur in organ preservation solutions.

Acknowledgments

We thank Mrs. E. Hillen and Ms. B. Lammers for their excellent technical assistance.

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    This study was supported by the Deutsche Forschungsgemeinschaft (RA 960/1-2 and RA 960/1-3).

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    U.R. and H.d.G. are consultants of Dr. Franz Köhler Chemie GmbH, Alsbach-Hähnlein, Germany.

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