Quorum sensing: An essential factor in culturing the human promyelocytic leukemia cell line HL-60 and its...

HL-60 cells are frequently employed as a standard in vitro model for neutrophil research and extensively utilized. However, the cultivation of HL-60 cells presents a recurring challenge. Historically, cell culture density has been ignored in the consistency of culture conditions. Here, we optimized the culture protocol and explored the impact of culture density on HL-60 cells. Additionally, we investigated the differentiated rate and antibacterial potential of differentiated HL-60 (dHL-60) neutrophils across varying cell density cultures. The findings revealed a positive correlation between cell proliferation activity and cell density, suggesting that increased density facilitates enhanced cell proliferation. Furthermore, as the density of the cell culture increased, there was a concomitant rise in the differentiation rate of HL-60 cells into neutrophils upon stimulation. Importantly, this elevated density also led to significantly higher levels of mitochondrial reactive oxygen species (ROS) production and bacterial phagocytosis. Further investigation revealed that small extracellular vesicles (sEVs) are crucial communicator in quorum sensing within HL-60 cells. Supplementation of HL60-derived sEVs (hEVs) in low-density cell populations resulted in a restoration of cell proliferation, in dose-dependent tendency. Conversely, the inhibition of EV-secretion in HL-60 cells restrains cell growth and proliferation. Overall, our study not only optimized the HL-60 cell culture protocol but also elucidated the critical role of culture density in enhancing HL-60 cell proliferation and antibacterial activity. This finding offers a noteworthy consideration for in vitro experiments of HL-60 cells and suggests the involvement of a quorum sensing mechanism within the neutrophil microenvironment.