Establishment of A Novel Method to Activate and Expand Human NK Cells Ex Vivo

Abstract

To develop a new method to activate and expand human NK cells ex vivo by using sodium hyaluronate as a major activating agent and to explore its related mechanism. Mononuclear cells were isolated from 3 samples of peripheral blood from three healthy donors. New NK cell culture method and the control method were used to culture NK cells from each samples separately for 14 days. Flow cytometry was used to analyze the ratio of NK cells and CD69 expression. To measure the in vitro cytotoxicity of NK cells cultured by the two methods, the K562 cells were used as the targeting cells and flow cytometry combined with CFSE marker was used as the testing method. After culturing for 14 days, the number of NK cells obtained by new culture method for NK cells expanded by 188.63±3.83 times while the number of NK cells cultured by control method expanded by 152.77±5.77 times. The ratio of NK cells in new cell culture method was above 90%, while the ratio of NK cells in control method was about 70%. The ratio of CD69+ NK cells in new cell culture method was 32.37%±3.22%, while the ratio of CD69+ NK cells in control method was 17.29%±3.79%. The results of cytotoxicity experiment in vitro showed that NK cells cultured by the new method had a higher killing ability to the target cells as compared with NK cells cultured by the control method. New NK cell culture method using sodium hyaluronate as a major activating agent can expand NK cells more efficiently as compared with the cells cultured by control method, which may be related to the direct and/or indirect activation of sodium hyaluronate to NK cells, further causing the dominant expansion of the NK cells.