Technique / Cell Biology / Cell culture / Cell preservation

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 degrees C to 25 degrees 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 degrees C and a minimum at 10 degrees 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 degrees C and matching activation energies above and below 10 degrees 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.

The expanding complexity of biologics banked for therapeutic applications necessitates the development of improved preservation technologies for support of the emerging fields of reparative and regenerative medicine. Currently, a number of media or "solutions" are utilized for the preservation of biologics. Given the diversity of cell systems utilized in the regenerative medicine arena, we hypothesized that the development of unique (individualized) preservation solutions designed to meet the distinct molecular biological requirements of individual systems would provide for enhanced and extended preservation. To evaluate this hypothesis, coronary artery smooth muscle cells (CASMCs), coronary artery endothelial cells (CAECs), hepatic cells (C3A), and skeletal muscle cells (SKMCs) were hypothermically preserved for 2 to 7 days at 4 degrees C in either cell culture medium, University of Wisconsin Solution (UW or ViaSpan), or HypoThermosol (HTS) variants. Cells were then assayed for viability, using the alamarBlue assay as well as calcein-AM, subsequent to their return to normothermic (37 degrees C) temperatures for up to 5 days. CASMC viability was best maintained when preserved in HTS plus Trolox/EDTA, CAEC viability was highest when preserved in HTS plus Trolox, SKMCs stored in HTS plus Trolox/RGD demonstrated enhanced viability, and C3A cells were best preserved in HTS plus FK041. The data suggest that solution compositions that address the differences in cell death mechanisms limiting preservation efficacy can result in targeted improvement matched to specific cell types. These observations support the custom solution hypothesis of cell and tissue preservation.