Effects of DMSO concentration on cryopreservation-induced stress in keratinocytes

Le résumé fourni par la source

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.

Ce résumé expose les affirmations des auteurs. BNTIC ne l’interprète pas comme une validation indépendante des résultats.