■外周血和腹水中的单核细胞是翻译和基础研究中常用的重要临床资源。然而,不同的冷冻保存时间和额外的冻融循环对单核细胞数量和功能的影响尚不清楚。
■从健康志愿者和卵巢癌患者收集外周血样品(n=21)和腹水样品(n=8)。分离单核细胞,冷冻,在6个月和12个月时解冻。冷冻保存对细胞活力的影响,表型,流式细胞术分析T细胞的活化和增殖情况。应用单细胞测序研究其潜在机制。
■冷冻保存后,外周血和腹水中单个核细胞的细胞数量和活力显着降低。T淋巴细胞,尤其是CD4+T细胞,受影响最大。相比之下,单核细胞,自然杀伤(NK)细胞,自然杀伤T(NKT)细胞,B细胞的耐受性更强。同时,长期冷冻保存后,T细胞增殖和IL-2分泌受到显着影响。机械上,高活性氧(ROS)诱导的细胞死亡与冷冻保存后CD4+T细胞的减少有关。
■我们的数据表明,冷冻保存后,单核细胞的不同亚型表现出不同的耐受能力。因此,我们的研究可以为使用冷冻的临床患者来源的单核细胞进行实验的个体提供证据和支持,用于基础研究或临床试验。此外,当研究人员在不同冷冻保存条件下获得的数据集之间比较外周血或腹水的免疫细胞功能时,值得格外小心.
UNASSIGNED: Mononuclear cells in peripheral blood and ascites are important clinical resources commonly used in translational and basic research. However, the impact of different cryopreservation durations and extra freeze-thaw cycles on the number and function of mononuclear cells is unknown.
UNASSIGNED: Peripheral blood samples (n = 21) and ascites samples (n = 8) were collected from healthy volunteers and ovarian cancer patients. Mononuclear cells were isolated, frozen, and thawed at 6 and 12 months. The impact of cryopreservation on cell viability, the phenotype, and the activation and proliferation of T cells were analyzed by flow cytometry. Single-cell sequencing was applied to investigate the underlying mechanism.
UNASSIGNED: The cell number and viability of mononuclear cells in peripheral blood and ascites were significantly decreased after cryopreservation. The T lymphocytes, especially CD4+ T cells, were affected the most significantly. By contrast, monocytes, natural killer (NK) cells, natural killer T (NKT) cells, and B cells were more tolerant. Meanwhile, T cell proliferation and IL-2 secretion are significantly affected after long-term cryopreservation. Mechanistically, the cell death induced by elevated reactive oxygen species (ROS) was involved in the reduction of CD4+ T cells after cryopreservation.
UNASSIGNED: Our data indicates that different subtypes of mononuclear cells exhibit different tolerance capacities upon cryopreservation. Thus, our research can provide evidence and support for individuals who are conducting experiments using frozen clinical patient-derived mononuclear cells, for basic research or clinical trials. In addition, extra caution is worthwhile when researchers compare immune cell functionality from peripheral blood or ascites across datasets obtained in different cryopreservation conditions.