Effects of DMSO concentration on cryopreservation-induced stress in keratinocytes

BACKGROUND: Despite the widespread use of DMSO for cryopreservation, its concentration dependent effects on epithelial cells remain insufficiently understood, particularly regarding sublethal stress responses. OBJECTIVES: To investigate how different DMSO concentrations affect cryopreservation-induced stress in human keratinocytes at both structural and functional levels. METHODS: Keratinocytes were cryopreserved using 1.8%, 2.2%, 5%, and 10% DMSO and stored at −80°C. Post‐thaw viability, proliferation, membrane fluidity, and transmembrane potential were assessed alongside ultrastructural analysis using SEM and TEM. RESULTS: Unexpectedly, 5% DMSO preserved optimal ultrastructure and membrane integrity, yet induced persistent hyperpolarization, suggesting latent mitochondrial stress. Although 10% DMSO yielded the highest initial viability (97.9%), it triggered depolarization, nuclear lobulation, and mitochondrial swelling, indicating early onset of oxidative stress. Low concentrations (1.8%, 2.2%) failed to maintain membrane stability, resulting in apoptosis‐like morphology and a 50% drop in proliferation. A novel negative correlation (r = −0.84) between DMSO concentration and membrane fluidity was identified, redefining the assumption that higher DMSO always rigidifies membranes. CONCLUSIONS: This study provides the first evidence that cryopreservation‐induced membrane potential shifts occur in a nonlinear, concentration-dependent manner, decoupled from immediate cell survival. The finding that 5% DMSO induces functional stress despite ideal morphology suggests that structural preservation does not equate to cellular homeostasis. These insights challenge current cryopreservation standards and offer a new perspective on balancing cytoprotection with post-thaw functionality in cells. The results highlight the need for next generation cryoprotective formulations targeting mitochondrial resilience and membrane dynamics.