Peripheral blood mononuclear cells are widely used as source material for anticancer immunotherapies. The conventional cryopreservation method for peripheral blood mononuclear cells is time-consuming and expansive, which involves controlled rate freezing followed by storage in liquid nitrogen. Instead, the convenient uncontrolled rate freezing cryopreservation method had been reported successfully in peripheral blood hematopoietic stem cells and peripheral blood progenitor cells. Therefore, we hypothesized that uncontrolled rate freezing cooling method maybe also applied to peripheral blood mononuclear cells cryopreservation. In this study, we evaluated the performance of uncontrolled rate freezing and controlled rate freezing cooling methods through cell recovery rate, viability, differentiation potential into cytokine-induced killer cells and the cellular properties of the cultured cytokine-induced killer cells. The results showed similar post-thaw viability and recovery rate in both controlled rate freezing and uncontrolled rate freezing cryopreserved peripheral blood mononuclear cells. Importantly, the uncontrolled rate freezing cryopreserved peripheral blood mononuclear cells exhibited higher growth ratio and earlier cell clustering during ex-vivo cytokine-induced killer cell culture than the controlled rate freezing ones. These two groups of expanded cytokine-induced killer cells also exhibited similar effector cell subset ratio and tumoricidal activity. In general, the performance of cryopreserved peripheral blood mononuclear cells using uncontrolled rate freezing cooling method, with the commercial cryoprotective agent CellBanker 2, was equal or better than the controlled rate freezing method. Our study implied that the combined use of cryoprotective agent CellBanker 2 and uncontrolled rate freezing could be a convenient cryopreservation method for peripheral blood mononuclear cells.

外周血单个核细胞被广泛用作抗癌免疫疗法的原料。外周血单个核细胞的常规冷冻保存方法既耗时又费力，涉及控制速度冷冻，然后储存在液氮中。取而代之的是，在外周血造血干细胞和外周血祖细胞中成功地报道了方便的不受控制的速率冷冻冷冻保存方法。因此，我们假设不受控速率冷冻冷却方法也可能应用于外周血单个核细胞的冷冻保存。在本研究中，我们通过细胞恢复率、存活率、分化为细胞因子诱导的杀伤细胞的潜能和培养的细胞因子诱导的杀伤细胞的细胞特性。结果显示，受控速率冷冻和非受控速率冷冻冷冻保存的外周血单核细胞的解冻后存活率和恢复率相似。重要的是，在离体细胞因子诱导的杀伤细胞培养过程中，与受控速率冷冻的外周血单核细胞相比，不受控制的速率冷冻冷冻保存的外周血单核细胞表现出更高的生长率和更早的细胞聚集。这两组扩增的细胞因子诱导的杀伤细胞也表现出相似的效应细胞亚群比率和杀肿瘤活性。一般而言，冷冻保存外周血单个核细胞的性能采用无控速冷冻冷却方法，使用商业冷冻保护剂 CellBanker 2，与控制速率冷冻方法相同或更好。我们的研究表明，冷冻保护剂 CellBanker 2 和不受控制的速率冷冻的联合使用可能是一种方便的外周血单核细胞冷冻保存方法。