Zixuan Zhang, Qiang Xu, Xinli Zhou
BACKGROUND: Jurkat cells, a human T lymphocyte line widely used in immunological studies, require reliable cryostorage to maintain post-thaw quality. Dimethyl sulfoxide (Me 2 SO) is the standard cryoprotectant, but its cytotoxicity may impair cell performance after thawing. OBJECTIVE: To develop a Me 2 SO-free multicomponent cryoprotectant agent (CPA) for Jurkat cell cryopreservation. MATERIALS AND METHODS: A Me 2 SO-free CPA composed of ethylene glycol (EG), disaccharides (trehalose, sucrose, glucose), and amino acids (histidine, isoleucine, tetrahydropyrimidine) was formulated. The effects of CPA composition and concentration on Jurkat cell cryopreservation were evaluated. Post-thaw viability and proliferation capacity were measured to identify optimal formulations. Cryomicroscopy was used to observe ice crystal growth and recrystallization under different CPA conditions. RESULTS: Disaccharides at 0.1 M improved post-thaw viability, whereas higher concentrations caused a marked decline in viability, indicating concentration-dependent cryoinjury. Among the disaccharides tested, trehalose showed the most consistent protection. Adding 20 mM histidine to 12.5% EG + 0.1 M trehalose increased viability from 87.8% to 93.8% (p < 0.05). Reducing EG to 10% while maintaining trehalose and histidine preserved viability above 91%, comparable to 10% Me 2 SO. Proliferation assays confirmed sustained recovery, with 40 mM histidine producing the highest proliferation. Cryomicroscopy showed that trehalose and histidine suppressed ice crystal growth and recrystallization. CONCLUSION: The optimized Me 2 SO-free, EG-based multicomponent CPA provides effective cryoprotection for Jurkat cells with reduced cytotoxicity and represents a practical alternative to Me 2 SO-based protocols.