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 macrophage 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 spectrometry (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 composition. Efficient uptake of fatty acids from the medium by U937 macrophages and inflammation-induced 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 peroxisomes observed by high-resolution microscopy. In conclusion, the present data demonstrate that FCS-contained eicosanoids, subject to substantial batch-wise variation, may modulate cellular effector functions in cell culture experiments.

High Glucose Shifts the Oxylipin Profiles in the Astrocytes towards Pro-Inflammatory States

“…This was carried out for several reasons. Firstly, oxylipins bind with extracellular proteins [ 11 , 41 ], and these processes may influence oxylipin profiles and cellular responses [ 42 , 43 , 44 ]. Secondly, in our experimental protocol, HG as a stressor has a long-term application, therefore, we added a set of experiments with an acute stressor.…”

Metabolic phenotyping of tear fluid as a prognostic tool for personalised medicine exemplified by T2DM patients

“…Metabolic profiling of minute amounts of sweat as well as of low abundant plasma eicosanoids using the Q Exactive HF hyphenated with an ultra-high-performance LC (UPLC) was already successfully demonstrated by us [10, 11, 44, 45], consequently, this set-up was also applied for tear fluid analysis. Regarding sample preparation, several extraction conditions were tested in order to ensure optimal extraction for both, metabolites and eicosanoids, present in tears (Figure 1B).…”

“…In this basic research project, we have developed a simple and quick sample preparation workflow for the comprehensive investigation of tear fluid, which is based on our previously established liquid chromatography-mass spectrometry (LC-MS) methods for sweat metabolomics [10, 11] and plasma eicosadomics. [44, 45] First, we wanted to systematically investigate and compare tear fluid composition with sweat in order to find differences deriving from the respective secretory glands. Indeed, distinct small molecules were preferably accumulated in tears such as eicosanoids, taurine or epinephrine, while other molecules like amino acids could be found in higher concentrations in sweat, demonstrating that each fluid has its unique set of metabolites.…”

Metabo-tip: a metabolomics platform for lifestyle monitoring supporting the development of novel strategies in predictive, preventive and personalised medicine

“…Up until now, the most comprehensive metabolic studies were conducted using in vitro cell culture model systems. As demonstrated previously, those experiments often suffer from unexpected influencing factors such as the composition of fetal calf serum [ 61 ]. The advantage of metabo-tip analysis is the facilitated use of humans as model system for comprehensive metabolic studies in vivo.…”