Large-scale ex vivo expansion and characterization of natural killer cells for clinical applications.
Large-scale ex vivo expansion and characterization of natural killer cells for clinical applications.
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DOI：
10.3109/14653249.2012.700767
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发表时间：
2012-10
期刊：
影响因子：
4.5
通讯作者：
Rooney CM
中科院分区：
文献类型：
--
作者：
Lapteva N;Durett AG;Sun J;Rollins LA;Huye LL;Fang J;Dandekar V;Mei Z;Jackson K;Vera J;Ando J;Ngo MC;Coustan-Smith E;Campana D;Szmania S;Garg T;Moreno-Bost A;Vanrhee F;Gee AP;Rooney CM
由于大量临床级细胞的纯化和扩增的新方案已经可用，因此对基于自然杀伤（NK）细胞的免疫疗法的兴趣已经恢复。我们已经成功地采用了先前描述的NK扩增方法，该方法使用表达白细胞介素（IL）-15和4-1 BB配体（BBL）的K562细胞（K562-mb 15 - 4 - 1 BBL）在新型透气静态细胞培养瓶中（G-Rex）培养NK细胞。使用该系统，我们在培养8-10天内从未分离的单采产物中产生了高达19 × 109个功能性NK细胞，从15 × 107个CD 3 − CD 56 + NK细胞开始。G-Rex产生了比传统透气袋更高倍数的NK细胞扩增，并且在培养期间不需要细胞操作或喂养。我们还表明，K562-mb 15 - 41 BBL细胞上调表面HLA I类抗原表达后，刺激与NK培养物的上清液和刺激同种异体反应性的CD 8 + T细胞内的NK培养物。然而，这些CD 3 + T细胞可以使用CliniMACS系统成功去除。我们描述了我们优化的NK细胞冷冻保存方法，并表明NK细胞即使在冷冻保存12个月后也是可行的和功能性的。我们已经成功地开发了一种静态培养方案，用于在良好生产规范（GMP）条件下在透气G-Rex系统中大规模扩增NK细胞。这种策略目前被用于生产用于癌症免疫治疗的NK细胞。
Interest in natural killer (NK) cell-based immunotherapy has resurged since new protocols for the purification and expansion of large numbers of clinical-grade cells have become available. We have successfully adapted a previously described NK expansion method that uses K562 cells expressing interleukin (IL)-15 and 4-1 BB Ligand (BBL) (K562-mb15-41BBL) to grow NK cells in novel gas-permeable static cell culture flasks (G-Rex). Using this system we produced up to 19 × 109 functional NK cells from unseparated apheresis products, starting with 15 × 107 CD3− CD56+ NK cells, within 8–10 days of culture. The G-Rex yielded a higher fold expansion of NK cells than conventional gas-permeable bags and required no cell manipulation or feeding during the culture period. We also showed that K562-mb15-41BBL cells up-regulated surface HLA class I antigen expression upon stimulation with the supernatants from NK cultures and stimulated alloreactive CD8+ T cells within the NK cultures. However, these CD3+ T cells could be removed successfully using the CliniMACS system. We describe our optimized NK cell cryopreservation method and show that the NK cells are viable and functional even after 12 months of cryopreservation. We have successfully developed a static culture protocol for large-scale expansion of NK cells in the gas permeable G-Rex system under good manufacturing practice (GMP) conditions. This strategy is currently being used to produce NK cells for cancer immunotherapy.