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

Abstract

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.