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Immune Netw. 2018 Aug 21;18(4):e31. doi: 10.4110/in.2018.18.e31. eCollection 2018 Aug.

Optimization of Large-Scale Expansion and Cryopreservation of Human Natural Killer Cells for Anti-Tumor Therapy.

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BioMedical Science and Engineering Interdisciplinary Program, KAIST, Daejeon 34141, Korea.
Cell Therapy Research Center, GC LabCell, Yongin 16924, Korea.
GC Pharma, GC LabCell, Yongin 16924, Korea.
Laboratory of Immunology and Infectious Diseases, Graduate School of Medical Science and Engineering, KAIST, Daejeon 34141, Korea.


Allogeneic natural killer (NK) cell therapy is a potential therapeutic approach for a variety of solid tumors. We established an expansion method for large-scale production of highly purified and functionally active NK cells, as well as a freezing medium for the expanded NK cells. In the present study, we assessed the effect of cryopreservation on the expanded NK cells in regards to viability, phenotype, and anti-tumor activity. NK cells were enormously expanded (about 15,000-fold expansion) with high viability and purity by stimulating CD3+ T cell-depleted peripheral blood mononuclear cells (PBMCs) with irradiated autologous PBMCs in the presence of IL-2 and OKT3 for 3 weeks. Cell viability was slightly reduced after freezing and thawing, but cytotoxicity and cytokine secretion were not significantly different. In a xenograft mouse model of hepatocellular carcinoma cells, cryopreserved NK cells had slightly lower anti-tumor efficacy than freshly expanded NK cells, but this was overcome by a 2-fold increased dose of cryopreserved NK cells. In vivo antibody-dependent cell cytotoxicity (ADCC) activity of cryopreserved NK cells was also demonstrated in a SCID mouse model injected with Raji cells with rituximab co-administration. Therefore, we demonstrated that expanded/frozen NK cells maintain viability, phenotype, and anti-tumor activity immediately after thawing, indicating that expanded/frozen NK cells can provide 'ready-to-use' cell therapy for cancer patients.


Allogeneic NK therapy; Cancer; Cryopreservation; NK cell expansion; Natural killer cells

Conflict of interest statement

Conflict of Interest: The authors declare no potential conflicts of interest.

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