Departments of Mechanical Engineering, Institute for Therapeutics Discovery and Development, University of Minnesota, Minneapolis, Minnesota, USA.
Departments of Biomedical Engineering, Institute for Therapeutics Discovery and Development, University of Minnesota, Minneapolis, Minnesota, USA.
Cytotherapy. 2020 May;22(5):291-300. doi: 10.1016/j.jcyt.2020.01.013. Epub 2020 Mar 25.
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.
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.
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%.
This study helped us identify the freezing responses of different human PBMC subsets using combinations of osmolytes.
本研究考察了三种渗透压调节剂组合冻存时外周血单个核细胞（PBMC）及其所含特定白细胞亚群的冷冻反应。
使用具有多个目标的差分进化算法来优化冷冻保护剂组成，从而同时提高辅助性和细胞毒性 T 细胞的冻后回收率。
使用差分进化算法筛选各种配方显示，两种 T 细胞亚群的冻后回收率均大于 80%。使用流式细胞术对 PBMC 亚群的表型和活力进行了特征分析。观察到辅助性 T 细胞和细胞毒性 T 细胞的冻后回收率之间存在显著差异。使用统计模型分析了个体渗透物的重要性以及三种 T 细胞亚群（辅助性 T 细胞、细胞毒性 T 细胞和自然杀伤 T 细胞）的冻后回收率之间的相互作用。统计模型表明，研究的三种亚群之间优选的渗透物浓度水平和相互作用模式是不同的。PBMC 在冻后 72 小时进行培养，以确定细胞的稳定性。由于冻后细胞凋亡是淋巴细胞的一个重要问题，因此分析了在无 DMSO 冷冻保护剂中冷冻的辅助性 T 细胞和细胞毒性 T 细胞的冻后即刻和 24 小时的细胞凋亡情况。与冻后即刻相比，两种细胞类型在 24 小时后细胞活力均下降。辅助性 T 细胞活力下降 17%，细胞毒性 T 细胞活力下降 10%。冻后即刻，两种细胞类型均有>30%的细胞处于早期凋亡，但 24 小时后，早期凋亡的细胞数量减少到 20%以下。
本研究帮助我们确定了不同人 PBMC 亚群在使用渗透压调节剂组合时的冷冻反应。
