Cell density during differentiation can alter the phenotype of bone marrow-derived macrophages

Abstract

BackgroundBone marrow-derived macrophages (BMDMs) are widely used primary cells for studying macrophage function. However, despite numerous protocols that are currently available, lack of a notable consensus on generating BMDMs may obscure the reliability in comparing findings from different studies or laboratories.FindingsIn this study, we addressed the effect of cell density on the resulting macrophage population. With reference to previously published methods, bone marrow cells from wild type C57BL/6 mice were plated at either 4 × 105 cells or 5 × 106 cells per 10 cm and cultured in 20% L-cell conditioned media for 7 days, after which they were analyzed for cell surface markers, production of proinflammatory cytokines, and responsiveness to polarizing signals. Reproducibly, cells plated at lower density gave a pure population of CD11b+F4/80+ macrophages (97.28 ± 0.52%) with majority being Ly-6C-Ly-6G- and c-Fms+, while those plated at higher density produced less CD11b+F4/80+ cells and a considerably higher proportion of CD11b+F4/80+CD11c+ (68.72 ± 2.52%) and Ly-6C-Ly-6G+ (71.10 ± 0.90%) cells. BMDMs derived from higher plating density also secreted less proinflammatory cytokines such as IL-6, IL-12 and TNF-α and were less phagocytic, and had a different pattern of expression for M1- and M2-related genes upon LPS or IL-4 stimulation.ConclusionsOverall, our findings indicate that altering cell density during BMDM differentiation can give rise to distinct macrophage populations that could vary the outcome of a functional study.

The Cytokine Response to Lipopolysaccharide Does Not Predict the Host Response to Infection

“…Bone marrow cells were incubated in RPMI 1640 with glutamine and 25mM HEPES (Gibco) containing 10% certified FBS (Gibco), 1% antibiotic-antimycotic (Gibco), and 10ng/mL mouse recombinant macrophage colony stimulating factor (M-CSF, R&D Systems, Minneapolis, MN) for 7 days. At the initiation of the growth period, bone marrow cell concentration was 4×10 4 /mL, as this has been found to yield a mature, pure population of macrophages (20). After 7 days, macrophages were washed with PBS and incubated with media containing various TLR ligands for 24 hours.…”

Criticality of plasma membrane lipids reflects activation state of macrophage cells

“…Our hypothesis is that cell density indirectly induces a decrease in T m , perhaps by triggering the production of cytokines with the same effect of IL4. This picture is supported by a study in which M1/M2-like differentiation was induced by the population density Lee and Hu (2013) . Indeed, among other things, denser cell cultures where found less efficient in the production of cytokines than sparser ones after LPS stimulation Lee and Hu (2013) .…”

“…In this picture, the crowded populations, with no need to further recruit cells and promote additional inflammation against possible infections, diminish their cytokine production, thus acting more like M2 cells. This hypothesis is supported by the observation that BMDMs from high density cultures secrete less pro-inflammatory cytokines and have lower phagocytic ability Lee and Hu (2013) . Moreover the number of cells showing typical M2 membrane markers like CD11c and Ly-6CLy-6G increases in dense cultures Lee and Hu (2013) .…”

“…This hypothesis is supported by the observation that BMDMs from high density cultures secrete less pro-inflammatory cytokines and have lower phagocytic ability Lee and Hu (2013) . Moreover the number of cells showing typical M2 membrane markers like CD11c and Ly-6CLy-6G increases in dense cultures Lee and Hu (2013) .…”

Towards the design of 3D multiscale instructive tissue engineering constructs: Current approaches and trends

“…The presence of other cells will affect the nano/micro-environment, e.g., by creating a new profile of cell-secreted cytokines and GFs. (Cooke, et al, 2011;Wang, et al, 2011a) Additionally, variables such as cellular density can affect cell growth (Rodriguez, et al, 2001;Dar, et al, 2002;Wen-tao, et al, 2006) and differentiation (Poumay and Pittelkow, 1995;Masur, et al, 1996;Bitar, et al, 2008;Lee and Hu, 2013).…”