Endotoxin contamination in fetal bovine serum and its influence on tumor necrosis factor production by macrophage-like cells J774.1 cultured in the presence of the serum

Trace amounts of endotoxin (lipopolysaccharide:LPS) are assumed to contaminate commercially available fetal bovine serum (FBS) for tissue or cell culture during the manufacturing process. We examined how cultured cells were affected by the endotoxin and how much endotoxin was in the FBS. Macrophage-like J774.1 cells maintained in RPMI1640 medium supplemented with FBS containing low doses of LPS for 15 or 21 days showed less TNF production in response to LPS than the cells maintained under LPS-free conditions, and the affected responses of the cells were not recovered by an additional 21 day culture in medium with LPS-free FBS. Concentrations of LPS in 40 lots of FBS obtained from 13 international manufacturers were measured by a highly sensitive and LPS-specific chromogenic limulus assay. The median of endotoxin levels in these lots was 46 ng/ml and the maximum was 38.8 ng/ml. Relatively higher concentrations of LPS (> 1 ng/ml) or lower levels (< 10 pg/ml) were found in 9 and 6 lots, respectively. The majority of the FBS lots contained various levels of (1 → 3)-β-d-glucan, and all lots contained high-density lipoprotein (HDL). However, no correlation was found between LPS and (1 → 3)-β-d-glucan or HDL level in the lots. Each FBS was added to macrophage-like J774.1 cells which had been maintained in LPS-free medium. Five lots of FBS induced significant TNF production by the cells without addition of any stimulant. These active 5 FBS contained relatively higher levels of LPS and pretreatment of the FBS with polymyxin B eliminated their ability to induce TNF production. No correlation was found between (1 → 3)-β-d-glucan levels in FBS and the TNF-inducing capability of FBS. These results show that considerable lots of FBS contain significant levels of LPS, which must affect cell culture.