Martí F, Miralles A, Peiró M, Amill B, de Dalmases C, Piñol G, Rueda F, García J
Department of Cryobiology and Cell Therapy, Hospital Duran i Reynals, Barcelona, Spain.
Transfusion. 1993 Aug;33(8):651-5. doi: 10.1046/j.1537-2995.1993.33893342746.x.
The use of lymphokine-activated killer (LAK) cell therapy in delayed treatment requires the use of cryopreserved effector cells. The purpose of this study was to determine the optimal cryopreservation protocol for the maintenance of cytotoxic activity in mononuclear cells (MNCs). MNCs were cryopreserved with dimethyl sulfoxide or 1,2-propanediol before and after 3 days of culture with recombinant interleukin 2. The effects of cryopreservation on cell recovery, LAK cell and natural killer (NK) cell cytotoxic activities, and surface antigen markers were studied. Recovery of nonactivated MNCs was higher with 1,2-propanediol than with dimethyl sulfoxide (p < 0.05). Cytotoxic activities, measured with a 51Cr release assay, significantly decreased after thawing, on both activated cells (76.3%; range, 35.8-92.2%) and fresh cells (54.6%; range, 17.5-75.4%). A 6-day kinetic test was used to compare the cytotoxic activity of cryopreserved and fresh cells. The results showed different patterns for NK cells (cryopreserved cells had lower levels of activity than fresh cells) and LAK cells (cryopreserved cells had higher levels of activity than fresh cells). Phenotype changes of effector cells in culture, with and without cryopreservation, were monitored by flow cytometry using monoclonal antibodies. These results were compared with changes in the cytotoxicity of cells with and without cryopreservation. After thawing, there was a decrease in MNCs expressing CD14 and CD56. Recovery of the CD56 marker correlates with increased cytotoxic activity. Despite some loss of NK cell activity, it is concluded that MNCs may be successfully cryopreserved before their use in immunotherapeutic treatment.
在延迟治疗中使用淋巴因子激活的杀伤细胞（LAK）疗法需要使用冷冻保存的效应细胞。本研究的目的是确定用于维持单核细胞（MNC）细胞毒性活性的最佳冷冻保存方案。在用重组白细胞介素2培养3天之前和之后，用二甲基亚砜或1,2 - 丙二醇对MNC进行冷冻保存。研究了冷冻保存对细胞回收率、LAK细胞和自然杀伤（NK）细胞细胞毒性活性以及表面抗原标志物的影响。未激活的MNC用1,2 - 丙二醇的回收率高于用二甲基亚砜（p < 0.05）。用51Cr释放试验测量的细胞毒性活性在解冻后显著降低，无论是激活细胞（76.3%；范围35.8 - 92.2%）还是新鲜细胞（54.6%；范围17.5 - 75.4%）。使用6天动力学试验比较冷冻保存细胞和新鲜细胞的细胞毒性活性。结果显示NK细胞（冷冻保存细胞的活性水平低于新鲜细胞）和LAK细胞（冷冻保存细胞的活性水平高于新鲜细胞）呈现不同模式。使用单克隆抗体通过流式细胞术监测有或没有冷冻保存的培养效应细胞的表型变化。将这些结果与有或没有冷冻保存的细胞的细胞毒性变化进行比较。解冻后，表达CD14和CD56的MNC减少。CD56标志物的回收率与细胞毒性活性增加相关。尽管NK细胞活性有所损失，但得出结论，MNC在用于免疫治疗之前可以成功冷冻保存。
