生物学杂志

生物学杂志 ›› 2026, Vol. 43 ›› Issue (3): 31-.

• 研究报告 • 上一篇    下一篇

利用磁性微球构建feeder-free体系扩增PBMC来源的NK细胞

舒　迅1,2,3， 张钦汉1,2,3， 李雨桐1,2,3， 李洋阳1,2,3， 马　婕1,2,3， 方　芳1,2,3， 肖卫华1,2,3
  

  1. 1. 中国科学技术大学 生命科学与医学部 免疫应答与免疫治疗全国重点实验室， 合肥 230027；
    2. 中国科学技术大学附属第一医院， 合肥 230027；
    3. 安徽省重组蛋白质药物工程技术研究中心， 合肥 230027
  • 出版日期:2026-06-18 发布日期:2026-06-16
  • 通讯作者: 肖卫华，教授，研究方向为免疫治疗技术和产品的研究开发和应用，E-mail：xiaow@ustc.edu.cn
  • 作者简介:舒迅，硕士，研究方向为细胞生物学，E-mail：seanshu98@mail.ustc.edu.cn
  • 基金资助:
    国家重点研发计划项目（2023YFC2506400）； 中国科学院先导专项B（XDB29030202）

Development of a feeder-free system for amplifying NK cells derived from PBMC using magnetic microspheres

SHU Xun1,2,3, ZHANG Qinhan1,2,3, LI Yutong1,2,3, LI Yangyang1,2,3, MA Jie1,2,3,FANG Fang1,2,3, XIAO Weihua1,2,3   

  1. 1. State Key Laboratory of Immune Response and Immunotherapy, Division of Life Sciences and Medicine,
    University of Science and Technology of China, Hefei 230027, China; 2. The First Affiliated Hospital of USTC, Hefei
    230027, China; 3. Anhui Provincial Engineering Research Center for Recombinant Protein Drugs, Hefei 230027, China
  • Online:2026-06-18 Published:2026-06-16

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。

关键词: 自然杀伤细胞, 肿瘤免疫治疗, 细胞免疫治疗, 磁性微球体外扩

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

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