BACKGROUND This study examined the freezing responses of peripheral blood mononuclear cells (PBMCs) and specific white blood cell subsets contained therein when cryopreserved in three combinations of osmolytes composed of sugars, sugar alcohols and amino acids. METHODS A differential evolution algorithm with multiple objectives was used to optimize cryoprotectant composition and thus the post-thaw recoveries for both helper and cytotoxicity T cells simultaneously. RESULTS The screening of various formulations using a differential evolution algorithm showed post-thaw recoveries greater than 80% for the two subsets of T cells. The phenotypes and viabilities of PBMC subsets were characterized using flow cytometry. Significant differences between the post-thaw recovery for helper T cells and cytotoxic T cells were observed. Statistical models were used to analyze the importance of individual osmolytes and interactions between post-thaw recoveries of three subsets of T cell including helper T cells, cytotoxic T cells and natural killer T cells. The statistical model indicated that the preferred concentration levels of osmolytes and interaction modes were distinct between the three subsets studied. PBMCs were cultured for 72 h post-thaw to determine the stability of the cells. Because post-thaw apoptosis is a significant concern for lymphocytes, apoptosis of helper T cell and cytotoxic T cells frozen in a DMSO-free cryoprotectant was analyzed immediately post-thaw and 24 h post-thaw. Both cell types showed a decrease in cell viability 24 h post-thaw compared with immediately post-thaw. Helper T cell viability dropped 17%, and cytotoxic T cells had a 10% drop in viability. Immediately post-thaw, both cell types had >30% of cells in early apoptosis, but after 24 h the number of cells in early apoptosis decreased to below 20%. CONCLUSION This study helped us identify the freezing responses of different human PBMC subsets using combinations of osmolytes.

中文翻译：

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使用无 DSMO 冷冻保护剂了解 T 细胞和其他人外周血单核细胞亚群的冷冻反应

背景这项研究检查了外周血单核细胞 (PBMC) 和其中包含的特定白细胞亚群在冷冻保存在由糖、糖醇和氨基酸组成的三种渗透剂组合中时的冷冻反应。方法 使用具有多个目标的差异进化算法来优化冷冻保护剂的组成，从而同时优化辅助 T 细胞和细胞毒性 T 细胞的解冻后恢复。结果 使用差异进化算法对各种配方进行筛选显示，两个 T 细胞亚群的解冻后回收率大于 80%。使用流式细胞术表征 PBMC 亚群的表型和活力。观察到辅助 T 细胞和细胞毒性 T 细胞的解冻后恢复之间的显着差异。统计模型用于分析单个渗透剂的重要性以及三个 T 细胞亚群（包括辅助 T 细胞、细胞毒性 T 细胞和自然杀伤 T 细胞）的解冻后恢复之间的相互作用。统计模型表明，所研究的三个子集之间的渗透物和相互作用模式的首选浓度水平是不同的。PBMCs 在解冻后培养 72 小时以确定细胞的稳定性。由于解冻后细胞凋亡是淋巴细胞的一个重要问题，因此在解冻后立即和解冻后 24 小时分析冷冻在不含 DMSO 的冷冻保护剂中的辅助 T 细胞和细胞毒性 T 细胞的凋亡。与解冻后立即相比，两种细胞类型在解冻后 24 小时均显示细胞活力降低。辅助 T 细胞活力下降 17%，和细胞毒性 T 细胞的活力下降了 10%。解冻后，两种细胞类型均具有>30% 的早期凋亡细胞，但 24 小时后早期凋亡的细胞数量降至 20% 以下。结论 本研究帮助我们确定了使用渗透剂组合的不同人类 PBMC 亚群的冷冻反应。