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, 18 (4), e31
eCollection

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

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

Bokyung Min et al. Immune Netw.

Abstract

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.

Keywords: 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.

Figures

Figure 1
Figure 1. Characterization of NK cells expanded for 21 days in large-scale. CD3-depleted cells were expanded by stimulating irradiated PBMCs in the presence of IL-2 and OKT3 on 0 days and 7 days. (A) The fold expansion of NK cells was assessed after 21 days of culture (n=18). (B) The viability of expanded cells was measured by an automatic cell counter using propidium iodide (n=18). (C) The percentages of NK cells, T cells, monocytes, and B cells were analyzed by CD3CD56+, CD3+, CD14+ and CD19+ phenotype, respectively (n=18). (D) Direct cytotoxicity was assessed by Calcein-AM release assay. Expanded NK cells were co-cultured with K562 cells for 4 h at E:T) ratios of 10:1 to 0.3:1 (n=18). (E) Intracellular cytokines (IFN-γ and TNF-α) and CD107a expression were assessed by flow cytometry. Expanded cells were co-cultured with K562 cells for 4 h at the E:T ratio of 1:1 (n=16). Mean and standard error are presented.
Figure 2
Figure 2. Comparison of freshly expanded and cryopreserved NK cells. (A) The cell recovery was expressed as the number of total nucleated cells per bag before and after cryopreservation (n=9). (B) The viability was measured by an automatic cell counter using propidium iodide before and after cryopreservation (n=9). (C) Killing activity of NK cells was assessed by co-culture with various tumor cells for 4 h at the E:T ratio of 10:1. (D) Intracellular cytokines and CD107a expression of NK cells were assessed by co-culture with various tumors for 4 h at the E:T ratio of 1:1. Cryopreserved NK cells were analyzed immediately after thawing without further culture or cytokine stimulation. Mean and standard error are presented (n=6).
***p<0.001.
Figure 3
Figure 3. Phenotypic analysis of NK cells before expansion (D0), after expansion (D21), and after cryopreservation. Expression of activating receptors (A), inhibitory receptors (B), and chemokine receptors (C) of NK cells was analyzed by flow cytometry. Cryopreserved NK cells were analyzed immediately after thawing without further culture or cytokine stimulation.
**p<0.01; ***p<0.001.
Figure 4
Figure 4. Anti-tumor activity of freshly expanded and cryopreserved NK cells in SNU354 xenograft model. SNU354 cells were subcutaneously transplanted into nude mice. NK cells were administered into a caudal vein of nude mice 2 h after transplantation of SNU354 cells, and NK cells were administered once a week (total 4 times). Cryopreserved NK cells were used immediately after thawing without further culture or cytokine stimulation. Doxorubicin (positive control) was administered total 13 times at 2-day intervals. (A) Tumor size was measured 9 times from day 9. (B) Tumors were isolated and weighed at the final day. (C) Body weight changes of mice compared to the body weight at day 0 (100%) were assessed. Mean and standard deviation are presented (n=10).
*p<0.01.
Figure 5
Figure 5. The ADCC activity of cryopreserved NK cells in combination with rituximab in Raji lymphoma model. (A) In vitro ADCC activity of expanded NK cells with or without cryopreservation was assessed against Raji cells. Cryopreserved NK cells were used immediately after thawing without further culture or cytokine stimulation. Calcein-AM labeled Raji cells were co-cultured with NK cells for 4 h at E:T ratios of 30:1 to 0.3:1 in the presence of rituximab or huIgG. The percentage of specific lysis of Raji cells is expressed as the mean±standard error (n=6). (B, C) To determine in vivo ADCC activity of cryopreserved NK cells, Raji cells were intravenously transplanted into SCID mice and cryopreserved NK cells were administered 5 times at 2 to 3-day intervals. Cryopreserved NK cells were used immediately after thawing without further culture or cytokine stimulation. Rituximab was administered with NK cells together on day 1. Paralysis (%) (B) and survival rate (%) (C) were assessed.
huIgG, human IgG. *p<0.05; **p<0.01; ***p<0.001.

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