Some epidemiological studies are so large that it would be too expensive to isolate peripheral blood mononuclear cells (PBMCs) from every donor enrolled in them. Therefore, research studies, such as the Retrovirus Epidemiology Donors Study (REDS) have collected and aliquoted whole blood, added 10 percent v/v DMSO, and stored the specimens at-30 degree C for short-term followed by -80 degree C for long-term time period. During the cryopreservation process, transport and storage, the quality of these specimens is negatively affected by the temperature gradients and alterations to which they are exposed. Even though our understanding of the mechanisms involved in cryopreservation has improved to include a wide range of cell types, it has been suggested that oxidative stress is a key mediator of cold-induced apoptosis. This contest led the investigators to try characterizing, validating, and determining the efficacy of Ape-Arrest, a new apoptotic inhibitor in whole blood processing and cell preservation. Ape-Arrest is an anti-apoptotic (caspase) inhibitor (analog of Z-VAD FMK ) developed and patented by the Enzyme Systems Products, inc. In this proposal, the investigators describe their collaborative approach together with Enzyme Systems Products, Inc. to demonstrate the feasibility of characterizing and developing new applications for Ape-Arrest when added to a blood sample that is going to be processed or cryopreserved. The investigators predict this product will help prevent freezing-induced apoptosis in PBMCs isolated from whole blood, subjected to viable cryopreservation, and that may also experience temperature fluctuations during shipment or storage. The investigators believe there is an optimal concentration of Ape-Arrest that should be added to an anticoagulant mixture in whole blood to inhibit apoptosis induced in PBMCs. In addition, the investigators have decided to study the effect of Ape-Arrest on long-term whole blood storage conditions at room temperature using an apoptosis assay, cell viability, proliferation and other functional tests. The investigators plan to assess the dose response relationship of Ape- Arrest for the suppression of apoptosis induced by the cryopreservation of whole blood, while maintaining cell viability and proliferation. In addition, the investigators would like to answer the question of exactly when and for how long Ape-Arrest should be added to an anticoagulant solution to achieve the most effective inhibition of cold induced apoptosis. Their findings will greatly improve the process of viably preserving blood cells and possibly other cell types.
