Evaluation of hematopoietic stem cell cryopreservation: A comparative study of liquid nitrogen versus storage at -80°C

Abstract Hematopoietic stem cell (HSC) cryopreservation is a critical component of cellular therapy that directly influences 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 viability of HSC grafts preserved via LN₂ cryopreservation with those preserved via mechanical freezing at − 80°C. 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: (1) validation of a controlled-rate freezing protocol, (2) assessment of postthaw recovery in low-cell-density samples, and (3–4) a direct comparison between LN₂ and − 80°C cryopreservation using paired aliquots. Postthaw CD34⁺ cell counts were analyzed via flow cytometry, and freezing curves were recorded to assess the consistency of the decrease in temperature.Both methods demonstrated comparable short-term CD34⁺ cell viability, with similar postthaw cell loss rates (~ 2 × 10⁶ cells/kg). However, LN₂ cryopreservation provided superior thermal control, as evidenced by strict adherence to the programmed freezing curve. Moreover, the LN₂ method reduced the reliance on scarce cryoprotectants such as Voluven®, enhancing reproducibility and long-term storage potential. No instances of bag leakage or contamination were observed with sterile packaging.While − 80°C cryopreservation remains a viable short-term option, LN₂ cryopreservation offers distinct advantages in terms of thermal stability, cryoprotectant flexibility, and long-term cell viability. Our validated LN₂ protocol meets clinical standards for safety, sustainability, and quality, confirming 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.