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 con taminate commercially available fetal bovine serum (FBS) for tissue or cell culture during the manufacturing process. We examined how cultur ed cells were affected by the endotoxin and how much endotoxin was in the FBS. Macrophage-like J774.1 cells maintained in RPMI1640 medium su pplemented with FBS containing low doses of LPS for 15 or 21 days show ed less TNF production in response to LPS than the cells maintained un der 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 internat ional manufacturers were measured by a highly sensitive and LPS-specif ic 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 co ncentrations of LPS (> 1 ng/ml) or lower levels (< 10 pg/ml) were foun d in 9 and 6 lots, respectively. The majority of the FBS lots containe d various levels of (1 --> 3)-beta-D-glucan, and all lots contained hi gh-density lipoprotein (HDL). However, no correlation was found betwee n LPS and (1 --> 3)-beta-D-glucan or HDL level in the lots. Each FBS w as added to macrophage-like J774.1 cells which had been maintained in LPS-free medium. Five lots of FBS induced significant INF production b y the cells without addition of any stimulant. These active 5 FBS cont ained relatively higher levels of LPS and pretreatment of the FBS with polymyxin B eliminated their ability to induce TNF production. No cor relation was found between (1 --> 3)-beta-D-glucan levels in FBS and t he TNF-inducing capability of FBS. These results show that considerabl e lots of FBS contain significant levels of LPS, which must affect cel l culture. (C) 1997 International Society for Immunopharmacology.