Viable and Functional: Long-Term -80 °C Cryopreservation Sustains CD34⁺ Integrity and Transplant Success

Background: Cryopreservation of hematopoietic stem cells (HSCs) at -80 °C using uncontrolled-rate freezing is frequently employed in resource-constrained settings, yet concerns remain regarding long-term viability and clinical efficacy. Reliable post-thaw assessment is essential to ensure graft quality and engraftment success. Methods: This single-center, retrospective study evaluated 72 cryopreserved stem cell products from 25 patients stored at -80 °C for a median of 868 days. Viability was assessed using both acridine orange (AO) staining and 7-AAD (7-aminoactinomycin D) flow cytometry at three time points: collection (T0), pre-infusion (T1), and delayed post-thaw evaluation (T2). Associations between viability loss, storage duration, and clinical engraftment outcomes were analyzed. Results: Median post-thaw viability remained high (94.8%) despite a moderate time-dependent decline (~1.02% per 100 days; R² = 0.283, p < 0.001). Mean viability loss at T2 was 9.2% (AO) and 6.6% (flow cytometry). AO demonstrated greater sensitivity to delayed degradation, with a significant difference between methods (p < 0.001). Engraftment kinetics were preserved in most patients, with neutrophil and platelet recovery primarily influenced by disease type rather than product integrity. Notably, storage duration and donor age were not significantly associated with engraftment outcomes or CD34⁺ cell dose. Conclusions: Long-term cryopreservation at -80 °C maintains HSC viability sufficient for durable engraftment, despite gradual decline. While transplant outcomes are primarily dictated by disease biology and remission status, AO staining provides enhanced sensitivity for detecting delayed cellular damage. Notably, our viability-loss model offers a practical framework for predicting product quality, potentially supporting graft selection and clinical decision-making in real-world, resource-constrained transplant settings.