Culture of NK Cells in Umbilical Cord Blood after Cryopreservation and Preliminary Study on the Function of CAR-NK
ZHANG Yueqin1,2, MENG Lu3, ZHAO Ri4, MAO Juan5, LIU Qin1, ZHOU Dan3, LIU Yang1, ZOU Qiang3*
This study was to optimize the culture conditions of cord blood NK cells after cryopreservation, construct CD19-targeting CAR-NK cells, and explore the killing effect of CD19-CAR-NK on CD19+ B-cell lymphoma. First, the cryopreserved umbilical cord blood mononuclear cells were recovered and allowed to stand for 18 h, NK cells were purified by CD56 magnetic beads, and the proportion of NK cells in CBMC increased from 13.8% to 96.6% after purification by magnetic beads. And then the effects of different cytokines and their combinations on NK proliferation, differentiation, apoptosis and function were compared, both IL-2 and IL-15 could activate NK cells, but NK cells proliferated better when cultured with IL-2 compared with IL-15 (P<0.05). NK cells under IL2+15 accounted for the largest proportion of CD56dim (74.33±5.03)%, the least apoptosis (4.6±0.14)%, and the cumulative proliferation was (62.67±3.21) times, which was significantly better than other cytokine combinations (P<0.05). In this study, the PDK1 inhibitor BX795 was used to improve the transduction efficiency of CAR-19 lentiviral vector on NK cells, 6 μmol/L BX795 could increase the viral transduction efficiency of NK cells by 3.6 times, and the transduction efficiency (36.4±1.91)% was the highest at this concentration. Finally this study verified the killing ability of CD19-CAR-NK on CD19+ tumor cells in vitro. CD19-CAR-NK could enhance the killing effect of NK cells on Nalm6, but did not affect the killing of Nalm6-CD19KO. In conclusion, this study concluded that IL-2+15 was the best cytokine combination for umbilical cord blood NK cells after cryopreservation. NK cells modified by CAR can specifically recognize CD19 antigen and kill CD19+ tumor cells.
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