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

Duk Seong Bae; Jae Kwon Lee

doi:10.5045/br.2014.49.3.154

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Original Article

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Blood Res 2014; 49(3):

Published online September 25, 2014

https://doi.org/10.5045/br.2014.49.3.154

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

Duk Seong Bae, and Jae Kwon Lee^*

Author Info. +

Department of Biology Education, College of Education, Chungbuk National University, Cheongju, Korea.

Correspondence to : Correspondence to Jae Kwon Lee, Ph.D. Department of Biology Education, College of Education, Chungbuk National University, 776, 1sunhwan-ro, Seowon-gu, Cheongju 361-763, Korea. Tel: +82-43-261-2734, Fax: +82-43-260-3361, chemokine@cbnu.ac.kr

Received: July 22, 2014; Revised: August 19, 2014; Accepted: August 27, 2014

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

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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 naïve 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.

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

Article

Original Article

Blood Res 2014; 49(3): 154-161

Published online September 25, 2014 https://doi.org/10.5045/br.2014.49.3.154

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

Duk Seong Bae, and Jae Kwon Lee^*

Department of Biology Education, College of Education, Chungbuk National University, Cheongju, Korea.

Correspondence to: Correspondence to Jae Kwon Lee, Ph.D. Department of Biology Education, College of Education, Chungbuk National University, 776, 1sunhwan-ro, Seowon-gu, Cheongju 361-763, Korea. Tel: +82-43-261-2734, Fax: +82-43-260-3361, chemokine@cbnu.ac.kr

Received: July 22, 2014; Revised: August 19, 2014; Accepted: August 27, 2014

Abstract

Background

Methods

Results

Conclusion

Keywords: Natural killer cells, Feeder cell, K562, Cytotoxic activity, DNAM-1, NKG2D

Abstract

Fig 1.

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Figure 1.

Comparison of total PBMC expansion with and without feeder cells. CD3^dep 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.

Blood Research 2014; 49: 154-161https://doi.org/10.5045/br.2014.49.3.154

Fig 2.

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Figure 2.

Optimization of NK cell expansion. CD3^dep 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.

Blood Research 2014; 49: 154-161https://doi.org/10.5045/br.2014.49.3.154

Fig 3.

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Figure 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.

Blood Research 2014; 49: 154-161https://doi.org/10.5045/br.2014.49.3.154

Fig 4.

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Figure 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.

Blood Research 2014; 49: 154-161https://doi.org/10.5045/br.2014.49.3.154

Fig 5.

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Figure 5.

Immunophenotypic features of NK cells after expansion with feeder cells. Expressions of NKG2D and DNAM-1 on NK cells were examined in CD3^dep PBMCs after 12 days of coculture with IL-2 and with K562 and Jurkat cells. The data shown are representative of three experiments.

Blood Research 2014; 49: 154-161https://doi.org/10.5045/br.2014.49.3.154

Table 1 . Comparison of perforin and granzyme B content inside of NK cells..

Abbreviation: MFI, mean fluorescence intensity..