Identification of Cell Death Markers in Keratinocytes Due to Cryopreservation with Various DMSO Concentrations

Cryopreservation of cells is a critical challenge in contemporary cryobiology, particularly in the burgeoning field of tissue engineering. The method of cryopreservation significantly affects the quality of cells post-thaw, as cells are sensitive to the stress induced by freezing and thawing, leading to physical damage, loss of functionality, or cell death. It is essential to develop protocols that ensure good physiological and adherent conditions post-thaw. This study investigates the effect of cryopreservation on human keratinocytes using the intracellular cryoprotective agent dimethyl sulfoxide (DMSO), known for mitigating cell damage during freezing and thawing, although its toxicity remains debated. We evaluated the cryopreservation of human keratinocytes with low (1.8% and 2.2% v/v) and standard (5% and 10% v/v) concentrations of DMSO during short-term storage (4 days) at -80°C. Post-thaw, we examined the impact of the cryopreservation process on cell viability, plasma membrane fluidity, and identified signs of cell death depending on the concentration of DMSO used in the freezing medium. This study is the first to systematically examine the impact of various DMSO concentrations on cell viability and plasma membrane fluidity of keratinocytes in the context of 30% FBS concentration in the freezing medium, slow cooling, and − 80°C temperature, highlighting the originality and significance of our research. This research demonstrated that lower DMSO concentrations (1.8% and 2.2%) significantly reduce keratinocyte viability due to increased apoptotic activity and cellular stress. In contrast, higher concentrations (5% and 10%) provide better protection and maintain higher cell viability. The study revealed that membrane fluidity increases with higher DMSO concentrations, which may facilitate the clustering of death receptors and the formation of apoptotic signaling complexes, thereby increasing the sensitivity of keratinocytes to apoptotic stimuli. Morphological analysis showed that lower DMSO concentrations lead to significant morphological changes and apoptosis, while higher concentrations result in cell enlargement and shape alteration. Ultrastructural analysis provided detailed insights into the internal structure of cells, revealing changes in the nucleus, mitochondria, and the presence of vesicles around the plasma membrane at lower DMSO concentrations, whereas higher DMSO concentration led to significant nuclear damage. These findings have implications for cryopreservation to improve cell viability and functionality post-thaw and may enhance the success of cell preservation in biomedical applications.