Cysteine preconditioning improves post–freeze–thaw proliferative capacity of human synovial mesenchymal stem cells

Background Synovial mesenchymal stem cells (MSCs) are promising for cartilage regeneration; however, freeze–thaw injury during cryopreservation impairs their proliferative capacity and may limit therapeutic efficacy. Glutathione, a key intracellular antioxidant, maintains redox homeostasis during cellular stress. Although redox modulation has been shown to improve cryopreservation outcomes in other stem cell types, its effectiveness in synovial MSCs remains unclear. This study aimed to determine whether cysteine preconditioning improves the proliferative capacity of synovial MSCs after freeze-thawing and to investigate the association between cysteine-induced glutathione responsiveness and donor-dependent variability in cryopreservation outcomes. Methods Human synovial MSCs were isolated from synovial tissue obtained from six patients with osteoarthritis undergoing total knee arthroplasty. MSCs were cultured with or without 1 mM L-cysteine for one week prior to cryopreservation. After freeze–thawing, proliferative capacity, colony-forming ability, and chondrogenic differentiation potential were assessed using multiple samples (six samples) from each donor. Intracellular glutathione (GSH) levels were quantified using fluorescence-based assays, and donor-matched analyses were performed to examine correlations between cysteine-induced GSH responsiveness and post–freeze–thaw changes in cell number and colony formation. Results Freeze–thaw without cysteine supplementation consistently reduced cell numbers and colony formation compared with fresh MSCs across all donors. In contrast, cysteine preconditioning before cryopreservation increased post–freeze–thaw cell numbers and colony counts in all donors, although the magnitude of recovery varied among individuals. Chondrogenic differentiation capacity after freeze–thaw was largely preserved, and cysteine preconditioning did not adversely affect chondrogenic outcomes. Cysteine preconditioning significantly increased intracellular glutathione (GSH) levels in all six donors. Donor-matched analyses demonstrated strong positive correlations between cysteine-induced GSH responsiveness and post–freeze–thaw changes in cell numbers (r = 0.95) and colony counts (r = 0.91). Conclusions Cysteine preconditioning improved the proliferative capacity of synovial MSCs after freeze-thawing without impairing chondrogenic differentiation. Strong associations between cysteine-induced glutathione responsiveness and recovery of cell number and colony formation after freeze-thawing indicate that donor-dependent differences contribute to variability in cryopreservation outcomes. These findings suggest that targeting glutathione-related redox pathways may represent a useful strategy for optimizing the cryopreservation of synovial MSCs.