【摘要】 为构建全封闭、自动化的自然杀伤(NK)细胞体外扩增体系，通过对比羧基，氨基磁性微球，并优化磁性微球偶联4-1BBL和IL-21蛋白的条件，包括温度、pH、蛋白偶联比例，初步构建了基于培养瓶的NK细胞磁性微球扩增方案。随后，基于此方案进一步优化了磁性微球与细胞的比例以及磁性微球的添加时间，并最终将培养容器更换为培养袋，使体系能够成为全封闭系统。最后，扩增后的NK细胞有着较高的纯度和数量，对多种肿瘤细胞的杀伤效果较好。通过流式细胞术检测扩增后NK细胞的特性分子表达水平，证实其杀伤特性分子表达较高。本研究所构建的扩增体系能够得到高纯度、高活性的NK细胞，同时能够满足完全封闭式的NK细胞扩增培养需求，适合工艺放大和满足细胞制剂的GMP要求，为建设全封闭、自动化的NK细胞生产工艺打下基础。更多还原

【Abstract】 To construct a fully closed and automated in vitro expansion system for natural killer(NK) cells, carboxyl and amino magnetic microspheres were compared, and the conditions such as temperature, pH, and protein conjugation ratios for conjugating 4-1BBL and IL-21 proteins onto the magnetic microspheres were optimized. This preliminary step allowed the construction of a magnetic microsphere-based NK cell expansion protocol in culture flasks. Subsequently, this protocol was further refined by optimizing the ratio of magnetic microspheres to cells and the timing of microsphere addition. The culture vessel was ultimately replaced with a culture bag to achieve a fully closed system. The expanded NK cells exhibited high purity and yield, along with strong cytotoxic effects against various tumor cell lines. Flow cytometry analysis confirmed elevated expression levels of cytotoxicity-related markers in the expanded NK cells. The expansion system developed in this study enabled the production of NK cells with high purity and activity while meeting the requirements for fully closed expansion culture, making it suitable for process scale-up and compliance with GMP standards for cell-based therapeutics. These findings lay a solid foundation for establishing a fully closed and automated NK cell manufacturing process.更多还原