Wang Guangchuan, Yu Guang, Wang Dongmei, Guo Shengnan, Shan Fengping
Department of Immunology, School of Basic Medical Science, China Medical University, Shenyang, Liaoning 110122, P.R. China.
Department of Immunology, School of Basic Medical Science, Jinzhou Medical University, Jinzhou, Liaoning 121000, P.R. China.
Exp Ther Med. 2017 May;13(5):1875-1883. doi: 10.3892/etm.2017.4189. Epub 2017 Mar 7.
Natural killer (NK) cells are innate lymphocytes that aid in the protection of the host from infectious diseases and cancer. studies of NK cells have provided a foundation for developing clinical adoptive NK-cell transferred immunotherapy against human tumors. To elucidate the functions and mechanisms of NK cell populations, it is important to develop an optimal, highly reproducible and reliable isolation method. The present comparative study was performed with four different NK cell isolation kits of magnetic bead labeling made by Miltenyi and Stemcell companies, including positive selection kits [cluster of differentiation (CD)-49b, using the monoclonal antibody DX5) MicroBeads] and negative selection kits. In addition, the viability of NK cells isinterleukin-2 (IL-2)-dependent and thus the concentration of IL-2 is critical for maintaining longer cell viability of NK cells. NK cell purity and viability after culturing, for 24, 48 or 72 h, with or without IL-2 (0, 100, 300 or 500 U/ml) was investigated in the present study. Purity of NK cells varied depending on the purification kit used, despite the same method being applied. Furthermore, more granulocytes were present in purified NK cells using Miltenyi sorting kits, particularly when using the negative selection kit. The main disadvantage of DX5-positive selection using the Stemcell and Miltenyi kits was that a high percentage of CD3ε cells were mixed into the isolated NK cells. Additionally, a significant difference of NK cell purity (P=0.003) was observed while purification was performed using different surface markers. As a consequence, the use of the positive selection kit was modified and subsequently a significantly higher purity (P=0.002) and yield (P=0.004) of NK cells was obtained. Moreover, the purity of NK cells and viability with or without a range of concentrations of IL-2 was compared. Results indicated that with a higher IL-2 concentration, the NK cell purity and viability were significantly higher (P<0.05). To our knowledge, this is the first report that has compared the disadvantages of four commercial NK cell isolation kits from two well-known companies, and identified the effect of NK cell purity and viability, using different concentrations of IL-2. To conclude, the results of the present study are fundamental in aiding the further development of NK cell therapy protocols for murine models.
自然杀伤（NK）细胞是先天性淋巴细胞，有助于保护宿主免受传染病和癌症侵害。对NK细胞的研究为开发针对人类肿瘤的临床过继性NK细胞转移免疫疗法奠定了基础。为阐明NK细胞群体的功能和机制，开发一种优化的、高度可重复且可靠的分离方法很重要。本比较研究使用了Miltenyi公司和Stemcell公司生产的四种不同的磁珠标记NK细胞分离试剂盒，包括阳性选择试剂盒[分化簇（CD）-49b，使用单克隆抗体DX5微珠]和阴性选择试剂盒。此外，NK细胞的活力依赖于白细胞介素-2（IL-2），因此IL-2的浓度对于维持NK细胞更长的细胞活力至关重要。本研究调查了在有或无IL-2（0、100、300或500 U/ml）的情况下培养24、48或72小时后NK细胞的纯度和活力。尽管采用相同方法，但NK细胞的纯度因所用纯化试剂盒而异。此外，使用Miltenyi分选试剂盒纯化的NK细胞中存在更多粒细胞，特别是使用阴性选择试剂盒时。使用Stemcell和Miltenyi试剂盒进行DX5阳性选择的主要缺点是，高比例的CD3ε细胞混入分离的NK细胞中。此外，在使用不同表面标志物进行纯化时，观察到NK细胞纯度存在显著差异（P=0.003）。因此，对阳性选择试剂盒的使用进行了改进，随后获得了显著更高的NK细胞纯度（P=0.002）和产量（P=0.004）。此外，还比较了有或无一系列浓度IL-2时NK细胞的纯度和活力。结果表明，IL-2浓度越高，NK细胞的纯度和活力显著越高（P<0.05）。据我们所知，这是第一份比较两家知名公司的四种商用NK细胞分离试剂盒缺点，并确定不同浓度IL-2对NK细胞纯度和活力影响的报告。总之，本研究结果对于帮助进一步开发小鼠模型的NK细胞治疗方案至关重要。
