Inhibition of Ferroptosis Enables Safe Rewarming of HEK293 Cells following Cooling in University of Wisconsin Cold Storage Solution.

The prolonged cooling of cells results in cell death, in which both apoptosis and ferroptosis have been implicated. Preservation solutions such as the University of Wisconsin Cold Storage Solution (UW) encompass approaches addressing both. The use of UW improves survival and thus extends preservation limits, yet it remains unclear how exactly organ preservation solutions exert their cold protection. Thus, we explored cooling effects on lipid peroxidation and adenosine triphosphate (ATP) levels and the actions of blockers of apoptosis and ferroptosis, and of compounds enhancing mitochondrial function. Cooling and rewarming experiments were performed in a cellular transplantation model using Human Embryonic Kidney (HEK) 293 cells. Cell viability was assessed by neutral red assay. Lipid peroxidation levels were measured by Western blot against 4-Hydroxy-Nonenal (4HNE) and the determination of Malondialdehyde (MDA). ATP was measured by luciferase assay. Cooling beyond 5 h in Dulbecco's Modified Eagle Medium (DMEM) induced complete cell death in HEK293, whereas cooling in UW preserved ~60% of the cells, with a gradual decline afterwards. Cooling-induced cell death was not precluded by inhibiting apoptosis. In contrast, the blocking of ferroptosis by Ferrostatin-1 or maintaining of mitochondrial function by the 6-chromanol SUL150 completely inhibited cell death both in DMEM- and UW-cooled cells. Cooling for 24 h in UW followed by rewarming for 15 min induced a ~50% increase in MDA, while concomitantly lowering ATP by >90%. Treatment with SUL150 of cooled and rewarmed HEK293 effectively precluded the increase in MDA and preserved normal ATP in both DMEM- and UW-cooled cells. Likewise, treatment with Ferrostatin-1 blocked the MDA increase and preserved the ATP of rewarmed UW HEK293 cells. Cooling-induced HEK293 cell death from hypothermia and/or rewarming was caused by ferroptosis rather than apoptosis. UW slowed down ferroptosis during hypothermia, but lipid peroxidation and ATP depletion rapidly ensued upon rewarming, ultimately resulting in complete cell death. Treatment throughout UW cooling with small-molecule Ferrostatin-1 or the 6-chromanol SUL150 effectively prevented ferroptosis, maintained ATP, and limited lipid peroxidation in UW-cooled cells. Counteracting ferroptosis during cooling in UW-based preservation solutions may provide a simple method to improve graft survival following cold static cooling.

Hibernator-Derived Cells Show Superior Protection and Survival in Hypothermia Compared to Non-Hibernator Cells.

Mitochondrial failure is recognized to play an important role in a variety of diseases. We previously showed hibernating species to have cell-autonomous protective mechanisms to resist cellular stress and sustain mitochondrial function. Here, we set out to detail these mitochondrial features of hibernators. We compared two hibernator-derived cell lines (HaK and DDT1MF2) with two non-hibernating cell lines (HEK293 and NRK) during hypothermia (4 °C) and rewarming (37 °C). Although all cell lines showed a strong decrease in oxygen consumption upon cooling, hibernator cells maintained functional mitochondria during hypothermia, without mitochondrial permeability transition pore (mPTP) opening, mitochondrial membrane potential decline or decreased adenosine triphosphate (ATP) levels, which were all observed in both non-hibernator cell lines. In addition, hibernator cells survived hypothermia in the absence of extracellular energy sources, suggesting their use of an endogenous substrate to maintain ATP levels. Moreover, hibernator-derived cells did not accumulate reactive oxygen species (ROS) damage and showed normal cell viability even after 48 h of cold-exposure. In contrast, non-hibernator cells accumulated ROS and showed extensive cell death through ferroptosis. Understanding the mechanisms that hibernators use to sustain mitochondrial activity and counteract damage in hypothermic circumstances may help to define novel preservation techniques with relevance to a variety of fields, such as organ transplantation and cardiac arrest.