![]() At higher density on the other hand, more MΦ adopted a high activation state ( 9). Indeed, MΦ were found to resume exponential growth faster ( 12), and to exhibit a more mature phenotype ( 13) and improved viability ( 12) at lower density. Moreover, a quorum sensing mechanism has been observed in mice where the inflammation-resolving effect of nitric oxide (NO) was found to be dependent on the density of NO-producing MΦ ( 7).Īlong with quorum sensing, cell density is vital for MΦ proliferation and viability. In RAW264.7 MΦ, gelsolin was identified as autoinducer of programmed cell death 4 (PDCD4) expression which increased at higher cell density ( 11). Quorum sensing had originally been established for biofilm formation of bacteria ( 8) but has recently also been observed in MΦ ( 7, 9, 11). Quorum sensing, a term adopted from bacteriology, refers to the modulation of gene expression by diffusible molecular cues (autoinducers) which convey information about density between cells ( 8– 10). This is particularly worrisome in view of the reported importance of local contact and context for MΦ viability ( 6) and recent growing evidence of quorum sensing by MΦ ( 7). Even though seeding density is usually mentioned and cells are often seeded at similar densities, differences in growing protocols, experimenters, and experimental conditions may result in density differences during experimentation. However, culture density at point of observation is an often-overlooked factor and either not specified at all or insufficiently substantiated by experimental data. To ensure reproducibility and comparability of results obtained from MΦ cultured in vitro, standardized cultivation protocols optimized for each cell type and assay are essential ( 4, 5). Cell lines like THP-1, J774 or RAW264.7, or primary MΦ derived from bone marrow cells or blood monocytes are commonly used as models of human MΦ in biological assays or screening tools in vitro ( 1). Macrophages (MΦ) have attracted growing interest as a therapeutic target due to their dynamic role in various pathologies, including inflammatory diseases and cancer ( 1– 3). Our findings demonstrate the importance of culture density for MΦ function and demand for awareness of culture density when conducting and interpreting in vitro experiments. In addition, functional differences between monocyte-derived MΦ from different donors could at least partly be attributed to differences in culture density. At the lowest density, the distance to the closest neighboring cells showed greater influence on THP-1 MΦ than monocyte-derived MΦ. These different findings in THP-1 MΦ and monocyte-derived MΦ could be attributed to the colony-forming growth pattern of THP-1 MΦ. With increasing density, monocyte-derived MΦ exhibited progressively increased phagocytosis, increased inflammasome activation, and decreased mitochondrial stress, whereas lipid uptake was unaffected. Culture density was also found to affect monocyte-derived MΦ, with functional implications that were distinct from those observed in THP-1 MΦ, suggesting particular relevance of density effects for cell lines. For THP-1 MΦ, the functional profile displayed a consistent trajectory with increasing density when exceeding a threshold (of 0.2 x 10 3 cells/mm 2), as visualized by principal component analysis. THP-1 MΦ showed increasing phagocytic activity and proliferation with increasing density but decreasing lipid uptake, inflammasome activation, mitochondrial stress, and secretion of cytokines IL-10, IL-6, IL-1β, IL-8, and TNF-α. We assessed 10 core functions of human MΦ derived from the THP-1 cell line as well as primary monocyte-derived MΦ. In this study, we investigated how the functional phenotype of MΦ was influenced by culture density. However, culture density is frequently overlooked in the standardization of culture protocols as well as the interpretation of results obtained in vitro. Recent evidence suggests MΦ to engage in quorum sensing, adapting their functions in response to cues about the proximity of neighboring cells. ![]() Macrophages (MΦ) are commonly cultured in vitro as a model of their biology and functions in tissues. 3Institute for Molecular Cardiovascular Research, RWTH Aachen University, Aachen, Germany.2BHF Centre for Cardiovascular Science, University of Edinburgh, Edinburgh, United Kingdom.1Cardiovascular Research Institute Maastricht (CARIM), Department of Pathology, Maastricht University Medical Center (UMC), Maastricht, Netherlands.Sluimer 1,2, Pieter Goossens 1† and Erik A.L. van Dommelen 1, Jan Nagenborg 1, Chang Lu 1, Judith C. ![]()
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