Sabanai Rinko, Suzuki Yoshifumi, Mizushige Takafumi, Uehara Nobuo, Inagawa Arinori
School of Engineering, Utsunomiya University, 7-1-2, Yoto, Utsunomiya, Tochigi, 321-8585, Japan.
School of Agriculture, Utsunomiya University, 350, Minemachi, Utsunomiya, Tochigi, 321-8585, Japan.
Anal Sci. 2025 Sep;41(9):1555-1563. doi: 10.1007/s44211-025-00815-8. Epub 2025 Jul 4.
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.
我们研究了用于细胞冷冻保存的冷冻二甲基亚砜（DMSO）水溶液中形成的冷冻浓缩溶液（FCS）的形态特征。具体而言，我们研究了冷却速率和初始DMSO浓度对FCS形态的影响。在1℃/min的冷却速率下，由于细胞外冰晶的结晶形成了相对较大的FCS通道，能够有效地容纳细胞。相反，快速冷却速率导致形成细小的冰晶，从而形成较窄的FCS通道。缓慢冷却促进了细胞容纳，这归因于冰晶的重新定向和更大的FCS尺寸，强调了FCS宽度分布对冷冻过程中细胞聚集的影响。还通过改变冷冻速率研究了C2C12成肌细胞的复苏情况。C2C12成肌细胞的细胞复苏率表明，缓慢冷却可提高复苏率，而快速冷却则会降低复苏率。中等冷却速率下的复苏表现出更大的变异性，表明冷冻保存结果的一致性降低。虽然在冷冻过程中有许多因素决定细胞活力，但我们的研究结果突出了FCS形态是设计有效冷冻保存方案的关键决定因素。
