Abstract：
Amino acids are vital components of the serum-free medium that influence the expansion and function of NK cells. This study aimed to clarify the relationship between amino acid metabolism and expansion and cytotoxicity of NK cells. Based on analyzing the mino acid metabolism of NK-92 cells and Design of Experiments (DOE), we optimized the combinations and concentrations of amino acids in NK-92 cells culture medium. The results demonstrated that NK-92 cells showed a pronounced demand for glutamine, serine, leucine, and arginine, in which glutamine played a central role. Significantly, at a glutamine concentration of 13 mM, NK-92 cells expansion reached 161.9 folds, which was significantly higher than 55.5 folds at 2.5 mM. Additionally, under higher glutamine concentrations, NK-92 cells expressed elevated levels of cytotoxic molecules, the level of cytotoxic molecules expressed by NK-92 cells was increased and the cytotoxic rate was 68.42%, significantly higher than that of 58.08% under low concentration. In view of the close relationship between glutamine metabolism and intracellular redox state, we investigated the redox status within the cells. This study demonstrated that intracellular ROS levels in higher glutamine concentrations were significantly lower than those under lower concentration cultures with decreased intracellular GSH/GSSG ratio, NADPH/NADP+ ratio, and apoptosis rate. These findings indicate that NK-92 cells exhibit improved redox status when cultured at higher glutamine concentrations. Overall, our research provides valuable insights into the development of serum-free culture medium for ex vivo expansion of NK-92 cells.
摘要：
氨基酸是影响NK细胞扩增和功能的无血清培养基的重要成分。本研究旨在阐明氨基酸代谢和扩增与NK细胞毒性的关系。在分析NK-92细胞的氨基酸代谢和实验设计（DOE）的基础上，我们优化了NK-92细胞培养基中氨基酸的组合和浓度。结果表明，NK-92细胞对谷氨酰胺有明显的需求，丝氨酸,亮氨酸,还有精氨酸,其中谷氨酰胺发挥了核心作用。重要的是,在13mM的谷氨酰胺浓度下，NK-92细胞扩增达到161.9倍，显著高于2.5mM时的55.5倍。此外,在较高的谷氨酰胺浓度下，NK-92细胞表达水平升高的细胞毒性分子，NK-92细胞表达的细胞毒性分子水平升高，细胞毒性率为68.42%，显著高于低浓度下的58.08%。鉴于谷氨酰胺代谢与细胞内氧化还原状态的密切关系,我们研究了细胞内的氧化还原状态。这项研究表明，在较高的谷氨酰胺浓度下，细胞内ROS水平显着低于那些在较低浓度的培养物中，细胞内GSH/GSSG比率降低，NADPH/NADP+比值,和凋亡率。这些发现表明,当以较高谷氨酰胺浓度培养时,NK-92细胞表现出改善的氧化还原状态。总的来说,我们的研究为体外扩增NK-92细胞的无血清培养基的开发提供了有价值的见解。
