Low serum conditions for in vitro generation of human macrophages with macrophage colony stimulating factor

Abstract

Animal serum is often used to generate human macrophages in vitro. Since fetal calf serum (FCS) may complicate antigen uptake, processing and presentation on HLA molecules, we tested the ability of M-CSF to generate macrophages at low fetal calf serum conditions. Peripheral blood monocytes from 12 individuals were cultured 1–4 days with 0–100 ng/ml macrophage colony stimulating factor (M-CSF) at either 1 (low) or 5% (v/v) FCS. Regardless of number of days in culture, maximal (50–100 ng/ml) M-CSF stimulation and low FCS induced 65±5% esterase positive cells in all individuals compared to 52±7% without M-CSF ( P<0.001). M-CSF increased the mean proportion of esterase positive cells after 24 or 96 h by 13% ( P<0.005) and 13% ( P<0.005), respectively, in 1% FCS, and 8% ( P<0.05) and 2% (NS), respectively, in 5% FCS, indicating a slight negative interaction between 5% FCS and M-CSF ( P<0.05). All cells were positive for CD14 and HLA class II, but cell number did not increase, confirming that M-CSF promote macrophage differentiation also at low FCS. M-CSF increased the average cell size after 24 or 96 h by 5.9±1.0 ( P<0.05) and 8.6±0.5 ( P<0.001) μm, respectively, without an increase in 5% FCS, further demonstrating the efficiency of M-CSF to promote macrophage generation at low FCS. The culture supernatants were negative for IL-1β and TNF-α, which demonstrates that M-CSF did not activate the macrophages. The generation of human macrophages by M-CSF at low FCS should prove useful in studies where higher FCS concentrations may interfere with the assay.