Pneumococcal Research, Murdoch Children's Research Institute, Melbourne, Melbourne, VIC 3052, Australia.
Pneumococcal Research, Murdoch Children's Research Institute, Melbourne, Melbourne, VIC 3052, Australia; Department of Paediatrics, University of Melbourne, Australia.
J Immunol Methods. 2019 Feb;465:61-66. doi: 10.1016/j.jim.2018.11.006. Epub 2018 Nov 14.
Cryopreservation of blood-derived immune cells is commonly used in clinical trials to examine immunological responses. However, studies elucidating the effects of cryopreservation on peripheral blood mononuclear cell (PBMC) responses have shown inconsistent results making it difficult to draw meaningful conclusions. Therefore we sought to address this issue by comparing key innate and adaptive immune parameters between freshly-isolated and cryopreserved PBMCs from healthy adults. We examined the effect of cryopreservation on the expression of key markers on innate and adaptive immune cell populations (i.e. CD4+ and CD8+ [T cells], CD14+ [monocytes], CD19+ [B cells], CD56+ [NK cells] or CD19 + CD27+ [memory B cells]), on cytokine secretion (TNF-α, INF-γ, IL-1β, IL-10, IL-6, MCP-1 and RANTES) in cultured PBMC supernatants following stimulation with a range of Toll-like receptor (TLR) agonists, as well as on antigen-specific memory B cell enumeration by ELISpot. We found that cryopreservation had no effect on the expression of immune markers on innate and adaptive immune cells as well on the number of antigen-specific memory B cells. However, the response to TLR ligands such as FLA-ST, CpG and LPS was variable with increased cytokine production by cryopreserved PBMCs observed compared to freshly-isolated PBMCs. Our results suggest that the effect of cryopreservation on the biological response of immune cell populations needs to be carefully considered, particularly in the context of clinical studies that rely on these immune outcomes.
血液来源的免疫细胞的冷冻保存通常用于临床试验中以检查免疫反应。然而，阐明冷冻保存对外周血单核细胞（PBMC）反应影响的研究结果不一致，使得难以得出有意义的结论。因此，我们试图通过比较健康成年人新鲜分离和冷冻保存的 PBMC 之间的关键固有和适应性免疫参数来解决这个问题。我们研究了冷冻保存对固有和适应性免疫细胞群（即 CD4+和 CD8+[T 细胞]、CD14+[单核细胞]、CD19+[B 细胞]、CD56+[NK 细胞]或 CD19+CD27+[记忆 B 细胞]）上关键标志物表达的影响、培养 PBMC 上清液中细胞因子（TNF-α、INF-γ、IL-1β、IL-10、IL-6、MCP-1 和 RANTES）的分泌以及 ELISpot 检测抗原特异性记忆 B 细胞计数。我们发现，冷冻保存对固有和适应性免疫细胞上的免疫标志物表达以及抗原特异性记忆 B 细胞数量没有影响。然而，对 TLR 配体（如 FLA-ST、CpG 和 LPS）的反应是可变的，与新鲜分离的 PBMC 相比，冷冻保存的 PBMC 中观察到细胞因子产生增加。我们的结果表明，冷冻保存对免疫细胞群体生物学反应的影响需要仔细考虑，特别是在依赖这些免疫结果的临床研究中。
