Comparative Evaluation of Hematopoietic Stem Cell Cryopreservation Using Liquid Nitrogen Versus -80°C Storage

Hematopoietic stem cell (HSC) cryopreservation is a critical component of cellular therapy, directly influencing transplant outcomes. Although mechanical freezing at -80°C is widely used in resource-limited settings, liquid nitrogen (LN₂) cryopreservation with controlled-rate freezing remains the gold standard for long-term storage. However, comparative data on the efficacy and operational feasibility of these methods remain scarce. This study aimed to compare the efficacy, cellular integrity, and post-thaw CD34⁺ cell recovery of HSC grafts preserved using LN₂ cryopreservation versus -80°C mechanical freezing. Additionally, we sought to validate a controlled-rate freezing protocol and evaluate its clinical applicability within a cell therapy unit. We conducted four experimental trials: (i) validation of a controlled-rate freezing protocol, (ii) assessment of post-thaw recovery in low-cell-density samples, and (iii-iv) a direct comparison between LN₂ and -80°C cryopreservation using paired aliquots. Post-thaw CD34⁺ cell counts were analyzed by flow cytometry using a BD FACSCanto™ II flow cytometer (Becton, Dickinson and Company, Franklin Lakes, NJ, USA), and freezing curves were recorded using a Consarctic® controlled-rate freezer (Consarctic GmbH, Schöllkrippen, Bavaria, Germany) to assess temperature descent consistency. Both cryopreservation methods demonstrated measurable short-term post-thaw CD34⁺ cell recovery. In the comparative experiment, the absolute post-thaw CD34⁺ cell loss was similar (approximately 2 × 10⁶ cells/kg) between the two preservation methods. However, because baseline CD34⁺ cell contents differed between grafts, percentage recovery also differed, and recovery should therefore be interpreted cautiously. Liquid nitrogen cryopreservation provided superior thermal control, as demonstrated by strict adherence to the programmed freezing curve, and eliminated reliance on hydroxyethyl starch (HES) (Voluven®; Fresenius Kabi AG, Bad Homburg, Germany) as a cryoprotective component. No cryobag leakage or contamination was observed during the study. Given the exploratory pilot design and limited sample size, these findings should be considered preliminary and require confirmation in larger studies incorporating direct viability assays and functional assessments. While -80°C cryopreservation remains a viable short-term option, LN₂ cryopreservation offers distinct advantages in thermal stability, cryoprotectant flexibility, and long-term cell viability. Our validated LN₂ protocol meets clinical standards for safety, sustainability, and quality, solidifying its role as the preferred method for high-quality HSC biobanking and transplantation. Further studies incorporating functional assays and extended follow-up are needed to assess long-term engraftment potential.