Preliminary Report: Evaluation of Storage Conditions and Cryococktails during Peripheral Blood Mononuclear Cell Cryopreservation

Abstract

The use of cryopreserved peripheral blood mononuclear cells (PBMCs) has proven critical in both clinical and biomedical applications. While utilized, in numerous settings, the functionality of frozen PBMCs is often limited, and a disconnect exists between measured viability and outcome. In this study we investigated parameters affecting outcome including storage protocol, freeze media, and assessment assay. PBMCs were isolated from seven healthy donors and cryopreserved in: (1) a media-based cocktail (SAIC), (2) CryoStor 7.5 (CS7.5), and (3) CryoStor 7.5 plus caspase inhibitors (CS7.5 ± Inh). All samples were stored in vapor phase liquid nitrogen (static) with replicates exposed to a thermal cycling regime, which mimicked typical sample handling (cycle). Viability was assessed immediately postthaw by trypan blue. Cryopreservation-induced delayed-onset cell death (CIDOCD) was evaluated postthaw using the Vybrant® Apoptosis Assay (VAA) that differentiated viability, apoptosis, and necrosis by fluorescent microscopy. Analysis of static stored PBMCs in CS7.5 demonstrated improved viability 1 h postthaw (82% –90%) compared to SAIC samples (60%–72%). Both cryococktails performed similarly after 24 h postthaw, yet fewer surviving PBMCs were evident, especially when assessed by VAA (SAIC: 31%; CS7.5: 36%). Replicate PBMC aliquots subjected to temperature cycling did not appear to have altered viability at 1 and 24 h postthaw (trypan blue). However, VAA showed that the SAIC cryococktail yielded improved viability at 1 h postthaw (61% vs. 51.8%, respectively) whereas by 24 h both the SAIC and CS7.5 samples were similar (∼20%). CIDOCD assessment revealed significant levels of both necrosis and apoptosis at 4 h and 8 h across all conditions. The observed decline in viability in the cycled samples was a result of increased apoptosis. Finally, the incorporation of caspase inhibitors did not improve static sample viability, but did significantly reduce the negative effect of temperature cycling. These preliminary investigations demonstrate that CIDOCD plays a significant role in PBMC cryopreservation failure, and that temperature fluctuations influence the overall level. Further, these studies demonstrate the importance of using multiple viability assessment techniques in evaluating PBMC cryococktail performance.