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

Animal serum is often used to generate human macrophages in vitro. Since fe tal calf serum (FCS) may complicate antigen uptake, processing and presenta tion on HLA molecules, we tested the ability of M-CSF to generate macrophag es at low fetal calf serum conditions. Peripheral blood monocytes from 12 i ndividuals were cultured 1-4 days with 0-100 ng/ml macrophage colony stimul ating factor (M-CSF) at either 1 (low) or 5% (v/v) FCS. Regardless of numbe r 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 ester ase 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 numbe r did not increase, confirming that M-CSF promote macrophage differentiatio n also at low FCS. M-CSF increased the average cell size after 24 or 96 h b y 5.9+/-1.0 (P<0.05) and 8.6+/-0.5 (P<0.001) <mu>m, respectively, without a n increase in 5% FCS, further demonstrating the efficiency of M-CSF to prom ote macrophage generation at low FCS. The culture supernatants were negativ e for IL-LP and TNF-ol, 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 wit h the assay. (C) 2001 Elsevier Science B.V. All rights reserved.