Development of Novel Low-DMSO Cryoprotectant for Peripheral Blood Stem Cell Preservation

Peripheral blood hematopoietic stem cell (PBHSC) cryopreservation is critical for autologous stem cell transplantation (ASCT), but traditional cryoprotective agents (TCPAs) containing 10% dimethyl sulfoxide (DMSO) raise safety concerns due to toxicity risk. This study aimed to validate a novel low-DMSO cryoprotective agent (CPA, 2% DMSO) for PBHSC preservation at -80 °C, eliminating the need for liquid nitrogen storage. PBHSCs from six donors were divided into CPA and TCPA groups. The CPA was mixed with PBHSCs (1:1 vol/vol) and directly stored at -80 °C. TCPA (10% DMSO + 5% human albumin) underwent gradual cooling (1 °C/min) and liquid nitrogen storage. After 1 month, both groups were thawed in a 37 °C water bath. Cell viability, cytoskeletal integrity (microfilaments/microtubules), mitochondrial activity, and colony-forming capacity were compared. After thawing, PBHSC survival was comparable between CPA (91.29%) and TCPA (90.07%). However, CPA outperformed TCPA in cell viability assays (CPA: 89.38% versus TCPA: 79.55%; p < 0.05). Cytoskeletal analysis revealed intact microfilaments and microtubules in CPA-preserved cells, with structural clarity exceeding TCPA. Mitochondrial activity in CPA-treated cells mirrored fresh PBHSCs, exhibiting 8.5% higher activity than TCPA (p < 0.05) and increased mitochondrial complex numbers. Colony-forming assays further confirmed CPA's superiority, with higher colony counts post-induction. CPA enables safe, convenient PBHSC cryopreservation at -80 °C using ultralow DMSO (2%), eliminating liquid nitrogen reliance. Its enhanced cell viability and mitochondrial preservation suggest clinical advantages by reducing infusion toxicity risks. This protocol offers a transformative strategy for ASCT, optimizing safety and operational efficiency.