外周淋巴细胞的冻存在肿瘤免疫治疗中的应用研究

Application of Cryopreservation of Peripheral Lymphocytes in Tumor Immunotherapy

目的通过建立体外分离高活性和高复苏率的淋巴细胞冻存方法,用于抗肿瘤自体免疫细胞回输治疗。方法按照人体回输细胞的临床应用要求,改良Ficoll梯度密度离心法,从外周血分离淋巴细胞,测定淋巴细胞活力和回收率。采用二甲亚砜作为细胞防冻剂,细胞冻存液分为含胎牛血清组和自体血浆组,纯化的淋巴细胞保存于-150℃气态液氮中,在1、3、6、12、24个月时复苏免疫细胞并评价效果。采用SPSS13.0统计学软件分析数据。结果改良的Ficoll梯度密度离心法获得的淋巴细胞活力为(99.6±0.6)%,淋巴细胞回收率(62.4±13.6)%,日处理血量500ml。在冻存了1、3、6、12、24个月后两种细胞冻存液均使淋巴细胞活率保持在95%以上,质量控制体系经过2年的运行,稳定可靠。结论通过体外人工分离淋巴细胞的方法可以获得足量的高活性淋巴细胞,经长时间冷冻保存后仍可维持高活性,有利于临床抗肿瘤免疫细胞回输疗法的实施。

Objective To establish a method to isolate high viability of peripheral lymphocyte in vitro and to store cryopreserved lymphocyte for autologous immune cells reinfusion in tumor immunotherapy. Methods According to the clinical application of human cell reinfusion requires, peripheral lymphocytes were isolated by the Ficoll density gradient technique with a newly developed method. The viability and recovery oflymphocytes were determined. Dimethyl sulfoxide （DMSO） as cryoprotectant, containing fetal bovine medium group and autologous plasma medium group were tested to cryopreserve the lymphocyte. Purified lymphocytes were stored in -150 ℃ vapor liquid nitrogen freezer. After thawing, the cell viability was assessed at months 1, 3, 6, 12, and 24. The statistical analysis was performed by using sottware SPSS 13.0. Results After separating, the cell viability was （99.6 ± 0.6） %, and the cell recovery of lymphocyte was （62.4 ± 13.6） %, with daily process of 500 ml whole blood. After thawing, the cell viability was over 95% by using two different cell cryopreservation media at months 1, 3, 6,12, and 24. The quality assurance program was reliable in two years of running. Conclusion Manual separation of lymphocyte in vitro can get enough high-quality lymphocytes. Cryopreserved peripheral lymphocytes still maintain high viability for long-term. It would be advantageous to clinical autologous immune cells reinfusion in tumor immunotherapy.