Blood Res.  2014 Sep;49(3):154-161. 10.5045/br.2014.49.3.154.

Development of NK cell expansion methods using feeder cells from human myelogenous leukemia cell line

• Bae DS
• Lee JK

• KMID: 2172786
• DOI: http://doi.org/10.5045/br.2014.49.3.154

Abstract

BACKGROUND
Natural killer (NK) cells constantly survey surrounding tissues and remove newly generated cancer cells, independent of cancer antigen recognition. Although there have been a number of attempts to apply NK cells for cancer therapy, clinical application has been somewhat limited because of the difficulty in preparing a sufficient number of NK cells. Therefore, ex vivo NK cell expansion is one of the important steps for developing NK cell therapeutics.
METHODS
CD3+ depleted lymphocytes were cocultured with IL-2 and with feeder cells (peripheral blood mononuclear cells [PBMCs], K562, and Jurkat) for 15 days. Expanded NK cells were tested for cytotoxicity against cancer cell lines.
RESULTS
We compared feeder activities of three different cells-PBMC, K562, and Jurkat. K562 expanded NK cells by almost 20 fold and also showed powerful cytotoxic activity against cancer cells. K562-NK cells remarkably expressed the NK cell activation receptors, NKG2D, and DNAM-1. K562-NK cells exhibited more than two-fold production of cytotoxic granules compared with Jurkat-NK cells, producing more perforin and granzyme B than naive NK cells.
CONCLUSION
Our findings suggest that K562 are more efficient feeder cells than Jurkat or PBMCs. K562 feeder cells expanded NK cells by almost 20 fold and showed powerful cytotoxic activity against cancer cells. We herein propose an intriguing approach for a design of NK cell expansion.

Keyword

Natural killer cells; Feeder cell; K562; Cytotoxic activity; DNAM-1; NKG2D

MeSH Terms

Cell Line*
Feeder Cells*
Granzymes
Humans
Interleukin-2
Killer Cells, Natural*
Leukemia, Myeloid*
Lymphocytes
Perforin
Granzymes
Interleukin-2
Perforin

Figure

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  Fig. 1 Comparison of total PBMC expansion with and without feeder cells. CD3dep PBMC cells were cocultured with IL-2 and with feeder cells (PBMC, K562, and Jurkat) for 15 days. The total numbers of cells were counted on Days 0, 3, 5, 7, 9, 11, 13, and 15. The data from four independent experiments performed in triplicate are expressed as mean±SD.

• 

  Fig. 2 Optimization of NK cell expansion. CD3dep PBMCs were cultured as described in Fig. 1 and the percentages of CD56+/CD16+ double-positive NK cells. (A) and CD3+ T cells. (B) were determined. The number of CD56+/CD16+ double-positive NK cells was counted using flow cytometry. (C) The data from three independent experiments performed in triplicate are expressed as mean±SD.

• 

  Fig. 3 Comparison of cytotoxic properties of NK cells expanded with PBMC, K562, and Jurkat feeder cells. Expanded NK cells were tested for cytotoxicity against K562, Jurkat, Hep3B, Raji, MCF-7, and Ramos. (A) Cytotoxic activity of K562-NK cells was measured according to culture duration. (B) Cancer cell lines (6×104) and NK cells (1.8×105) were added on to the plates and incubated for 4 hours at 37℃. NK cell cytotoxicity towards cancer cell lines was measured using LDH release assay. The data from four independent experiments performed in triplicate are expressed as mean±SD.

• 

  Fig. 4 Comparison of cytotoxic properties of naïve NK cells and K562-feeder expanded NK cells. Cytotoxic activities of naïve and K562-NK cells were compared using LDH assay. Cancer cell lines and NK cells were added onto the plates and incubated for 4 hours (A) and 6 hours (B) at 37℃. The data from three independent experiments performed in triplicate are expressed as mean±SD.

• 

  Fig. 5 Immunophenotypic features of NK cells after expansion with feeder cells. Expressions of NKG2D and DNAM-1 on NK cells were examined in CD3dep PBMCs after 12 days of coculture with IL-2 and with K562 and Jurkat cells. The data shown are representative of three experiments.

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