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

Synovial mesenchymal stem cells (MSCs) are promising for cartilage regeneration, but freeze–thaw injury can reduce their proliferative capacity. This study examined whether cysteine preconditioning improves post-freeze–thaw proliferation and whether glutathione (GSH) responsiveness is associated with donor-dependent variability. Human synovial MSCs from six patients with osteoarthritis were cultured with or without 1 mM L-cysteine for one week before cryopreservation. After thawing, cell number, colony formation, and chondrogenic differentiation were assessed, and intracellular GSH levels were quantified. Group effects were evaluated using linear mixed-effects models. Compared with fresh MSCs, freeze–thawed MSCs without cysteine showed reduced cell numbers and colony formation in four and five donors, respectively. Cysteine preconditioning increased post-freeze–thaw cell numbers and colony formation in three of six donors, with no consistent adverse effect on chondrogenic differentiation. Mixed-effects analyses showed significantly higher cell and colony numbers with cysteine preconditioning than without it. Cysteine increased intracellular GSH levels in all six donors. Donor-matched analyses demonstrated positive correlations between GSH responsiveness and post-freeze–thaw changes in cell number (r = 0.95) and colony formation (r = 0.91). These findings suggest that cysteine preconditioning may partially mitigate freeze–thaw-associated proliferative impairment in synovial MSCs.