The impact of initial cooling rates on cell preservation in frozen water-dimethyl sulfoxide media: a morphological study

We investigated the morphological features of a freeze-concentrated solution (FCS) formed in frozen aqueous dimethyl sulfoxide (DMSO) solutions for cell cryopreservation. Specifically, we investigated the effects of cooling rates and initial DMSO concentrations on FCS morphology. At a cooling rate of 1 ℃/min, relatively large FCS channels formed owing to the crystallization of the extracellular ice crystals, accommodating cells effectively. Conversely, rapid cooling rates resulted in fine ice crystals, leading to the formation of narrower FCS channels. Slow cooling promoted cell accommodation, attributed to ice crystal reorientation and larger FCS sizes, underscoring the impact of FCS width profiles on cell accumulation during freezing. The recovery of C2C12 myoblasts was also studied by altering the freezing rate. The cell recovery rate of C2C12 myoblasts demonstrated that slow cooling improved recovery, whereas rapid cooling decreased it. Medium cooling rates showed greater variability in recovery, suggesting reduced consistency in cryopreservation outcomes. While numerous factors determine cell viability during freezing processes, our findings highlight FCS morphology as a critical determinant in designing effective cryopreservation protocols.