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Archive > Volume 20 Issue 3 > 2013,20(3):336-341. DOI:10.3872/j.issn.1007-385X.2013.03.014 Prev Next
Technigue and Method
Comparison of four kinds of NK cell in vitro expansion methods
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Abstract:
Objective:By comparing the cell immunophenotype, the expansion fold and the cytotoxic activity of the expansion products in various cell culture methods to identify a more effective solution for the in vitro expansion of NK cells. Methods: Four methods for expansion of NK cells from peripheral blood were established, including method one, a classical culture protocol for NK cells (IL-2+IL-15), method two (IL-2+IL-15+IL- 18), method three (IL-2+IL-15+IL-7), and method four, a novel NK-specific culture medium (IL- 2+OKT3). 10 patients with advanced solid tumors in Department of Biotherapy, Cancer Hospital Affiliated to Tianjin Medical University from February 2012 to April 2012 were obtained, and PBMCs from those patients were isolated by Ficoll-Hypaque density gradient centrifugation. The proportion of different lymphocyte subsets (especially for NK cells) were detected by flow cytometry on 0, 5, 10 and 15 days. The changes of NK cell expansion fold and the proportion of different lymphocyte subsets were detected among 4 groups after expansion in vitro for 15 days. The anti-tumor ytotoxicity against human K562 cell line among 4 groups were measured using LDH assay. Results: After expansion for 15 days, the expansion folds of total cells in 4 groups were (40.1±2000), (44.08±22.09), (44.82±23.67) and (46.82±25.02), respectively. The proportion of NK cells in 4 groups increased from (20.44±2.23)% on day 0 to (48.30±13.90)%, (54.72±12.25)%, (55.94±12.70)% and (54.5±14.93)% on day 15, respectively. The expansion folds of NK cells in 4 groups were (75.86±28.57), (93.32±32.16), (88.66±24.94) and (58.88±4153), respectively. No significant difference was found on the total cell expansion, NK cell expansion folds among the 4 groups (P<0.05). The cytotoxic activity of the expansion products in methods one, two and three were higher than that of method four in vitro (\[63.40±5.00\]%, \[77.30±940\]%, [62.17±5.60\]% vs \[37.39±10.42\]%，P<0.05). There was no significant difference among the first 3 groups (P >0.05). Conclusion:NK-specific cytokines have great influence on the expansion of NK cells in vitro. However, no significant difference is found among various cytokine combinations. The cytotoxic activity of the expansion products in methods one, two and three against K562 cells are significantly higher than that of method four.
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Project Supported:
Project supported by the General Foundation for Applicational Basic Research from Science and Technology Comminssion of Tianjin City（No. 11JCYBJC13200）, and the Special Foundation for Clinical Trial of Affiliated Tumor Hospital, Tianjin Medical University（No. 11L01）
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