Expansion Of NK-92 Cells In Vitro With Genetically Modified K562 Cell Lines

Objective: Using three lentiviral vectors constructed by molecular cloning technology to prepare the genetically modified K562 cell lines,thus they play the role of feeder cells to amplify high cytotoxic activity of NK-92 cells in vitro,so as to provide theoretical basis for subsequent amplification of CAR-NK-92 cells in vitro.Methods: Three lentiviral expression vectors consist of the target gene interleukin-15(IL-15)or interleukin-21(IL-21)or 4-1BB ligand(4-1BBL)were constructed on the basis of the lentiviral vector p WPI which contains the reporter gene green fluorescent protein(GFP).The lentiviral envelope protein VSV-G,the packaging plasmid ps PAX2,the lentiviral expression vector 4-1BBL and the other lentiviral expression vector IL-15 or IL-21 were co-transfected into HEK293 FT cells.Afterwards,the two kinds of cell culture supernatants were concentrated to obtain lentiviral fluid to infecte the K562 cells respectively.The K562 cell lines that stably expressing GFP were selected and expanded by micromanipulator technique,they played the role of feeder cells later to expand NK-92 cells in vitro after treatment with Mitomycin C.Cytotoxicity was detected by CCK-8 kit,the activity of NK-92 cells was detected by lactate dehydrogenase release method.The level of interferon-? in the supernatant was detected by ELISA,consequently,the function and safety of NK-92 cells after amplification were verified.Results: The results of restriction enzyme digestion and sequencing confirmed that the three lentiviral vectors were successfully constructed.The expression of GFP could be observed by fluorescence microscopy after transfection of two groups of lentiviral expression vectors into HEK293 FT cells and K562 cells infected with lentivirus.The flow cytometry results showed that the positive expression rate of 4-1BBL was higher in both groups.K562 cells treated with MMC solution at a final concentration of 10 ?g/ml for 3 h could obtain good feeder cells.The K562 cell lines prepared by IL-15 and4-1BBL amplified more NK-92 cells as feeder cells.The results of CCK-8 suggested that with the increase of the effective target ratio,the NK-92 cells in IL-15 group were more cytotoxic than other groups after expansion(P<0.05).When the ratio of effective target was 10:1,the activity of LDH and the content of IFN-? in the supernatant of IL-15 group were increased(P<0.05).Conclusion: The data demonstrate that it is feasible to generate K562 cells infected by two kinds of lentiviral solution prepared by lentiviral vectors containing GFP,4-1BBL,IL-15 or IL-21 combined with lentiviral packaging vectors.The K562 cell lines expressing green fluorescent protein and the target gene IL-21/4-1BBL are successfully constructed.K562 cells prepared by the combination of IL-15 and 4-1BBL can be served as feeder layers to amplify NK-92 cells with high cytotoxic activity and effective anti-tumor A549 cell activity in vitro.