Abstract: To construct a fully closed and automatedin vitroexpansion 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.

Key words: natural killer cells, tumor immunotherapy, cellular immunotherapy, magnetic microsphere-basedin vitroexpansion system