Raman spectroscopic analysis of intracellular ice-induced degradation of mesenchymal stromal cells

Cryopreservation enables the long-term maintenance of cellular characteristics, thereby serving as a foundation for establishing a stable supply chain for cells. However, during the freezing process, which is part of cryopreservation, phase-transition-associated biophysical events, such as the formation of intracellular ice crystals (IICs), have the potential to induce cellular degradation. During freezing, the cooling rate influences cellular dehydration and acts as a balancing factor between IIC formation and cellular shrinkage. In particular, IIC formation has been regarded as a lethal event, and various studies have focused on elucidating the underlying mechanisms. However, studies investigating the mechanisms of cellular degradation caused by IIC formation remain limited. The objective of this study is to investigate the degradation mechanisms of mesenchymal stromal cells induced by IIC formation during the freezing process at different cooling rates (1 and 5 K/min). Cell viability assessments at multiple time points after thawing suggested that the 5 K/min group exhibited an increase in the population of cells that lost membrane integrity. To investigate the mechanisms underlying membrane integrity loss, Raman spectroscopic microscopy was employed to analyze changes in cellular size and IIC formation during the freezing process. The analyses suggested that, compared to the 1 K/min group, the 5 K/min group exhibited increased IIC formation and a more uniform spatial distribution, as well as the presence of a specific subpopulation characterized by increased cellular size. These biophysical events may be associated with the loss of membrane integrity after thawing. In this study, cellular degradation during freezing at different cooling rates was analyzed from both biological and physical perspectives. Our findings advance understanding of the mechanisms of cellular degradation induced by IICs and are expected to offer valuable insights for optimizing cell freezing processes.