Optimization of the method to cultivate NK cells from abandoned white cells

Abstract

Using abandoned white cells separated from preparation of blood products to cultivate NK cells in vitro, and to optimize the method of cultivation of allogeneic NK cells for clinical application. Abandoned white cells separated from blood production were collected from 15 healthy donors. PBMCs were isolated from the abandoned white cells and cultured for 17 days using culture bottles as previously coated antibodies (group CD3 mAb was coated with CD3 mAb, group CD 16mAb was coated with CD16mAb, and group CD3 mAb+ CD16 mAb was coated with CD3 mAb and CD16 mAb). Flow cytometry was used to determine the ratio of CD3(-)CD56(+) cells, expression of activated cell surface receptors, and secretion of IFN-γ. The anti-tumor cytotoxicity against K562 and Raji cells was determined using LDH cytotoxicity assay and flow cytometry. After expansion for 17 days, the proportions of CD3(-)CD56(+) cells was (15.19±12.22)% in the group CD3 mAb, (83.63±10.63)% in the group CD16 mAb, (49.40±12.64)% in the group CD3 mAb+ CD16 mAb, and it was (16.34±10.51)% before expansion. The total number of NK cells was more than 10(9). The expression ratios of NK cell surface activated receptors NKp30 and NKp46 were significantly increased, while that of the NKG2D was not significantly changed. The NK cells after expansion showed high cytotoxicity activity against K562 cells, reaching up to(76.97±3.16)% when effector-cell-to-target-cell ratio (E∶T ratio) was 40∶1. NK cells can be obtained from abandoned white cells after cultivation for 17 days, with a purity up to 90% and total cell number of more than 10(9). Their activity was reinforced, the anti-tumor cytotoxicity activity was increased, and may meet the standard of clinical therapeutic application.