Optimizing NK cell expansion with K562-mbIL-18/-21 feeder cells and ensuring feeder cell-free NK products — ASN Events
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Optimizing NK cell expansion with K562-mbIL-18/-21 feeder cells and ensuring feeder cell-free NK products (#145)

Hantae Jo 1 , Yujung Jo 2 , Seung Kwon Koh 2 , Jinho Kim 1 , SoonHo Kweon 3 , Jeehun Park 4 5 , Sang-ki Kim 6 , Hyun-Young Kim 1 , Duck Cho 1 2 7 8
  1. Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, South Korea
  2. Department of Health Sciences and Technology, Samsung Advanced Institute for Health Sciences and Technology, Sungkyunkwan University, Seoul, South Korea
  3. Department of Medicine, Weill Cornell Medicine, New York, NY, USA
  4. Department of Molecular Bioscience, College of Biomedical Science, Kangwon National University, Chuncheon-si, Gangwon-do, South Korea
  5. Multidimensional Genomics Research Center, Kangwon National University, Chuncheon-si, Gangwon-do, South Korea
  6. Department of Companion & Laboratory Animal Science, Kongju National University, Yesan, Korea
  7. Cell and Gene Therapy Institute, Samsung Medical Center, Seoul, South Korea
  8. Department of Biopharmaceutical Convergence, Sungkyunkwan University, Suwon, South Korea

Efficient ex vivo natural killer (NK) cell expansion is essential for NK cell-based immunotherapies. While cancer cell line-derived feeder cells enhance NK cell proliferation, concerns exist about residual feeder cells in the final product despite irradiation. Although clinical-grade feeder-free media have been developed to address these safety concerns, they typically show lower expansion efficiency. This study compared NK cell expansion rates with and without irradiated genetically engineered K562-mbIL-18/-21 (GE-K562) feeder cells using clinical-grade media (CTS™ NK-Xpander™ Medium), optimized a feeder-based protocol, and confirmed the absence of residual feeder cells in the final NK products.

We first compared NK cell fold expansion between feeder and feeder-free conditions. To optimize feeder-based NK cell expansion, we tested various PBMC-to-feeder ratios (2:1, 4:1, 6:1) and different re-stimulation frequencies over 21 days, evaluating their effects on NK cell fold expansion. Flow cytometry and BCR::ABL1 quantitative reverse transcription-PCR analyses were performed on the final NK cell product to ensure complete removal of feeder cells.

Our results showed that while Xpander is designed for feeder-free systems, the addition of feeder cells significantly enhanced NK cell expansion. NK cell fold expansion increased proportionally with higher feeder cell ratios and more frequent re-stimulation. The optimal expansion was achieved at a 2:1 PBMC-to-feeder ratio with weekly re-stimulation over 21 days. Although a large number of feeder cells were introduced during the culture period, GE-K562 feeder cells were effectively eliminated through irradiation and NK cell-mediated killing, with no residual feeder cells detected in the final product.

These findings demonstrate that a feeder-based NK cell expansion system combined with clinical-grade medium provides an efficient and scalable approach for clinical applications. This method ensures robust NK cell fold expansion while addressing safety concerns regarding residual feeder cells, making it suitable for large-scale NK cell manufacturing.

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