The experimental study on the expansion and high purity of human NK cells stimulated by different cytokines combination in vitro

Objective This study explored the method of effective amplification of human natural killer (NK) cells in vitro, in order to obtain an optimized quality of NK cells for clinical applications. Methods CD3-CD56+ CD16+ NK cells were negatively selected using immunomagnetic bead sorting (MACS) from normal peripheral blood mononuclear cells (PBMCs). Both purified NK cells and PBMCs were cultured in X-VIVO medium containing interleukin-2(IL-2) and anti-CD3 monoclonal antibody .Various cytokines including IL-7, IL-12, IL-15 and IL-21 were added to the culture in different combination. After 18 days of culture, the folds of NK cell expansion and the purity of NK cells were analyzed by cell counting and flow cytometry, respectively. Results After 18 days in culture and in the present of IL-2 and anti-CD3 MoAb, IL-7/IL-15/IL-21 in combination was able to stimulate the purified NK cells to expand in 124 folds and PBMCs in 108 folds. The purity of CD3-CD56+ CD16+ NK and CD3+ CD16+ CD56+ NK cells can reach to 81.5% and 10.3% , respectively , in the purified NK cells, while the purity of CD3-CD56+ CD16+ NK and CD3+ CD16+ CD56+ NK cells can reach to 22.1% and 33.5% , respectively, in PBMC cells. The number and purity of NK cells obtained by the addition of cytokine IL-7, IL-15 or IL-21 individually were various. Conclusion Using X-VIVO medium containing IL-2, anti-CD3 MoAb and IL-7/IL-15/IL-21 in combination, we obtained the best effect of NK cell expansion and purity. The cell number, purity and quality of the NK cells produced by this method can meet the needs for cellular immunotherapy against tumors in clinical applications. Key words: CD3-CD56+ CD16+ NK; MACS; Cytokines; Cell expansion