48. In vitro assessment of apoptosis and necrosis following cold storage in human airway cells

As advances in medical technology improve the ability and efficacy of cells and tissues to be transplanted, there remains a void in our knowledge of the specific molecular response of cells to low temperature storage. While much focus has been given to the formulation of solution to perfuse the tissues during storage to increase viability, investigations into the complex molecular changes that occur during cold exposure and their subsequent effect on outcome remain limited. An understanding of cellular stress on a molecular level is of fundamental importance as such information proves critical to tissue storage and transplantation as well as to the development of new technologies and processes in non-related areas of cell biology, bioprocessing, etc. The intent of this study was to begin to quantify the levels of cell death following hypothermic storage in a lung cell model in order to establish a foundation for future in depth molecular studies in support of improved lung transplantation. Normal human lung fibroblast (IMR-90) cells were stored statically for 1, 2 and 3 days at 4 °C in basal media, complete media and ViaSpan ® . Post-storage viability was assessed at 1, 2 and 3 days post-storage using the metabolic indicator alamarBlue. To determine the level of apoptotic and necrotic involvement, flow cytometry was performed and corroborated via visualization under fluorescent microscopy at 1, 4, 8, and 24 h post-storage using the Vybrant apoptosis assay. Sample analysis revealed that cells stored in ViaSpan ® were 68% (±4%) viable after 24 h of storage at 4C and repopulated to 103% (±6%) within 3 days. The other solutions resulted in complete cell loss after 24 h of hypothermic storage with no re-growth observed. Extension of the storage interval to 3 days resulted in complete cell loss in all conditions. Analysis of the apoptotic and necrotic populations in the ViaSpan ® stored samples revealed that 5% of the population was apoptotic at 8 h post-storage while around 20% of the same population was found to be necrotic. The data revealed a high level of sensitivity to cold storage for this cell system, resulting in a significant amount of cell death, through both apoptosis and necrosis. These data highlight the critical need for a more in depth understanding of the molecular changes that occur as a result of cold exposure in cells.