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 (ϳ20%). 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. 189

Postthaw characterization of umbilical cord blood: markers of storage lesion

“…These studies demonstrated that damage to the mitochondria resulted in release of caspases and ultimately, to the loss of the cell due to apoptosis. A similar outcome was observed by Cosentino and colleagues who demonstrated that improper storage (specifically, cycling of temperature in storage) resulted in higher levels of postthaw apoptosis for peripheral blood mononuclear cells (PBMNCs) stored on liquid nitrogen. Similarly, a study of UCB in long‐term storage suggests that HSCs expressing signs of apoptosis (annexin V staining) did not engraft when transplanted into an animal transplant model .…”

“…Our hypothesis was that apoptosis could be used as a potential early marker for degradation of UCB units during long‐term storage. Cosentino and colleagues demonstrated improper temperature storage conditions resulted in increased apoptosis for PBMNCs and Shim and colleagues demonstrated that frozen and thawed CD34+ cells isolated from thawed UCB units expressing annexin V did not engraft in animals. To test whether apoptosis could be used as a biomarker for long‐term damage, UCB units were moved from liquid nitrogen to −80°C storage to impose a well‐characterized temperature lesion.…”

“…Theoretically, storage of cryopreserved cells in liquid nitrogen should result in a shelf life of thousands of years . In practice, temperature excursions commonly experienced in liquid nitrogen storage units when the tank is accessed to insert or remove samples can lead to degradation of affected units …”

Cryopreservation

“…Media including Viaspan, CryoStor, Unisol, Adesta, Celsior and others, to name a few, when combined with CPAs for have been reported to improve cell survival to varying degrees. Improvements have been observed in systems including hepatocytes, 58,103 cord blood stem cells, 104 PBMCs, 67,105 fibroblasts, 11 keratinocytes, 69 blood vessels 101 and engineered tissues. 56 In these studies, evaluation of the cryopreservation media was conducted and correlated with improvements in cell survival, function and growth.…”

Characterization and modulation of human mesenchymal stem cell stress pathway response following hypothermic storage

“…Numerous studies have now established that bioprocessing techniques are associated with the activation of molecular-based stress responses which contribute to cell loss during and following processing leading to failure[3,4,5,6,12,13,15,30,35]. These molecular responses can manifest as apoptosis or programmed cell death signaling.…”