Certain bone marrow transplantation (BMT) methods require prior cryopreservation of hematopoietic stem cells (HSCs). Properly preserving these cells proves critical to ensure product quality and, consequently, bone marrow engraftment outcome. Cooling the cells to temperatures below 0 ºC reduces their metabolism but fails to completely stop it, leading to a gradual deterioration of the cells. Evidence demonstrates that storage at -196 °C in liquid nitrogen slows this deterioration process by interrupting intracellular metabolic pathways (1-5). However, storing in liquid nitrogen presents a significant challenge for many transplant programs as they expand and accumulate products, causing a consistent decrease in available space.

This study aimed to evaluate the cell viability of HSCs maintained at short—and long-term storage in -80°C, the relationship between storage time and cell viability, and the impact of cell concentration (total nucleated cells—TNC) on cell viability after thawing. Finally, analyze the impact of the number of CD34+ cells viable on engraftment outcome.

For this study, we prepared twenty-nine aliquots of HSCs from 21 patients with multiple myeloma and 8 patients with lymphoma. These were cryopreserved in cryovials with a solution of 5% dimethyl sulfoxide, 6% hydroxyethylamide, and 4% albumin in a -80 C mechanical freezer. We evaluated the TNC concentrations in all samples using the XN-350 Sysmex Hematology Analyzer, and the viability of CD34+ cells using 7AAD (flow cytometry; ISHAGE protocol; FACS Canto II and FACS Lyric, BD Biosciences). The samples were then categorized into three groups: T0 (before cryopreservation), T1 (HSCs cryopreserved for up to 6 months), and T2 (HCSs cryopreserved for 4-6 years).

There was no difference in the cell viability of groups T0, T1, in cell viability of groups T0, T1, and T2 (pre-cryopreservation, until 6 months and 4 to 6 years, respectively). In the paired analysis, when comparing the T1 and T2 groups, the cell viability was not impacted by storage time (p < 0.05). When evaluating, together or separately, the correlations between viability storage time and cell concentration, there was no significant association. Finally, there was no association between engraftment time and cell viability, engraftment time, and CD34+cells/Kg, even when adjusted for viability and recovery.

Findings indicate that cryopreservation at -80°C for up to six years does not impact cell viability. This finding is significant for safely storing cryopreserved cells without compromising their quality when nitrogen storage remains unfeasible. Despite this encouraging result, careful monitoring of the clonogenic assay should be followed to ensure the functional capacity of the HSCs.