Temperature controlled dual hypoxic chamber design for in vitro ischemia experiments

In vitro ischemia models are designed to study various aspects of hypo-perfusion, focusing on the consequences of acute events under body temperature. Cold ischemia is less investigated even though the beneficial effects of cooling is expected. The aim of the present work was to develop a device modeling cold and warm ischemia in vitro. Oxygen-glucose deprivation was applied with continuous nitrogen flow and glucose-free cell culture media to mimic ischemia. The temperature in both chambers were independently set between 4 and 37 °C. Samples were placed inside for the ischemic period, followed by a reperfusion stage under standard cell culture conditions. We tested rat calvaria bone pieces undergoing 1, 7, 12 and 24 h of ischemia at 4 and 37 °C. After 24 h of reperfusion, cell number was measured with a tetrazolium cell viability assay. One hour of warm ischemia paradoxically increased the post-reperfusion cell count, while cold-ischemia had an opposite effect. After 7 h of warm ischemia the cells were already unable to recover, while under cold ischemia 60% of the cells were still functioning. After 12 h of cold ischemia 50% of the cells were still be able to recover, while at 24 h even the low temperature was unable to keep the cells alive. The markedly different effect of warm and cold ischemia suggests that this newly designed system is capable of reliable and reproducible modeling of ischemic conditions. Moreover, it also enables deeper investigations in the pathophysiology of cold ischemia at cellular and tissue level.