Eicosanoid content in fetal calf serum accounts for reproducibility challenges in cell culture

Abstract

Reproducibility issues regarding in vitro cell culture experiments are related to genetic fluctuations and batch‐wise variations of biological materials such as fetal calf serum (FCS). Genome sequencing may control the former, while the latter may remain unrecognized. Using a U937 mac-rophage model for cell differentiation and inflammation, we investigated whether the formation of effector molecules was dependent on the FCS batch used for cultivation. High resolution mass spec-trometry (HRMS) was used to identify FCS constituents and to explore their effects on cultured cells evaluating secreted cytokines, eicosanoids, and other inflammatory mediators. Remarkably, the FCS eicosanoid composition showed more batch‐dependent variations than the protein composi-tion. Efficient uptake of fatty acids from the medium by U937 macrophages and inflammation‐in-duced release thereof was evidenced using C13‐labelled arachidonic acid, highlighting rapid lipid metabolism. For functional testing, FCS batch‐dependent nanomolar concentration differences of two selected eicosanoids, 5‐HETE and 15‐HETE, were balanced out by spiking. Culturing U937 cells at these defined conditions indeed resulted in significant proteome alterations indicating HETE-induced PPARγ activation, independently corroborated by HETE‐induced formation of peroxi-somes observed by high‐resolution microscopy. In conclusion, the present data demonstrate that FCS‐contained eicosanoids, subject to substantial batch‐wise variation, may modulate cellular ef-fector functions in cell culture experiments.