Endotoxin-reduced milk oligosaccharide fractions suitable for cell biological studies

Endotoxins (ET) are able to activate leukocytes and other cell types and thus affect cell adhesion studies with regard to biological functions of human milk oligosaccharides (HMO). In our HMO preparations we detected ET in concentrations of 1.1 to 32.7 ng/mg. However, as we found in previous tests, an ET concentration of less than 100 pg/ml was necessary in order to avoid effects on the expression of CD11b and CD62L on human neutrophil granulocytes. Therefore, we used affinity chromatography with a detoxifying gel (polymixin B) which reduces the ET concentration of 414.9 +/- 121.5 (mean +/- SEM) and 47.9 +/- 5.9 pg/mg for the acidic and the neutral HMO, respectively. As only about 50 - 100 microg HMO per ml test solution are usually used in biological assays, the residual ET concentration does not interfere with cellular functions. We conclude that despite structural similarities between ET as lipopolysaccharides and complex HMO, a one step purification of HMO preparations was sufficient to get HMO material that can be used in cell culture systems.