Endothelial Cell Preservation at 10°C Minimizes Catalytic Iron, Oxidative Stress, and Cold-Induced Injury

Abstract

There is growing evidence that oxidative stress plays an important role in mediating the injury induced by hypothermia during the preservation of cells and tissues for clinical or research use. In cardiovascular allografts, endothelial cell loss or injury may lead to impaired control of vascular permeability and tone, thrombosis, and inflammation. We hypothesized that hypothermia-induced damage to the endothelium is linked to increases in intracellular catalytic iron pools and oxidative stress. In this study, bovine aortic endothelial cells and cell culture methods were used to model the response of the endothelium of cardiovascular tissues to hypothermia. Confluent cells were stored at 0°C to 25°C and cell damage was measured by lipid peroxidation (LPO) and lactate dehydrogenase release. Varying the bleomycin-detectible iron (BDI) in cells modulated cold-induced LPO and cell injury. In untreated cells, injury was highest at 0°C and a minimum at 10°C. A similar temperature-dependent trend was found in BDI levels and cell plating efficiencies. Arrhenius plots of cell killing and iron accumulation rates showed biphasic temperature dependence, with minima at 10°C and matching activation energies above and below 10°C. These findings imply that the mechanisms underlying the hypothermic increase in catalytic iron, oxidative stress, and cell killing are the same and that preservation of the endothelium may be optimized at temperatures above those routinely used.

Effect of Storage Temperature on Cultured Epidermal Cell Sheets Stored in Xenobiotic-Free Medium

“…Low cell viability (∼20%) seen at 4°C is in agreement with other studies that have shown cell viability ranging from ∼20% to ∼33% after storage at this temperature [5], [12]. Rewarming after cold-storage has been shown to induce mitochondrial swelling and initiate apoptosis, which may account for the high cell death seen at 4°C [23], [29]. Evidence of injury to epidermal cells associated with cold and re-warming seen at 4°C is particularly clinically relevant as refrigeration is the most common method of split skin graft storage and low cell viability may influence skin graft healing [5].…”

“…The favorable conditions at 10°C were explained by minimal generation of catalytically available intracellular iron and reduced oxidative stress. Similarly, high proliferative capability was retained [23]. Higher cell viability, at approximately 60% or above, was shown in the present study at several temperatures (12°C, 24°C, 28°C, 32°C and 37°C), which is comparable to that reported in cultured epithelial autograft cryopreservation studies [27], [32].…”

“…A smaller peak in cell viability was seen at 12°C (∼60%) which coincided with highest PCNA expression. A temperature study on storage of lung endothelial cells also reported similar results at 10°C [23]. The favorable conditions at 10°C were explained by minimal generation of catalytically available intracellular iron and reduced oxidative stress.…”

“…Cells were stored for 14 days in tightly sealed air-tight culture wells by means of Nunclon adhesive sheets. Following storage, MEM storage medium was replaced with KM, and cells were allowed to equilibrate in the 37°C incubator for 3 hours before all analyses in order to assess any potential damage incurred upon rewarming [23]. An extended storage period of 14 days was chosen to accentuate any temperature associated differences.…”

The analysis of viability for mammalian cells treated at different temperatures and its application in cell shipment

“…The preservation of endothelial cells at 10°C can reduce oxidative stress and cold-induced injury [23]. In our work mammalian cells were able to maintain a considerably high level of viability and excellent recovery when stored at 16°C or 22°C for 4 days (Figs 1D and 2B–2D).…”