Eicosanoid Content in Fetal Calf Serum Accounts for Reproducibility Challenges in Cell Culture.

Laura Niederstaetter,Benjamin Neuditschko,Lukas Janker,Andrea Bileck,Julia Brunmair,Christopher Gerner,Giorgia Del Favero

doi:10.3390/biom11010113

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.

Highlights

Introduction iationsProblems with the inter-laboratory reproducibility of results obtained with in vitro cell culture models are increasingly being recognized [1,2]
All cells were differentiated using phorbol 12-myristate 13-acetate (PMA) to induce macrophage formation as verified by FACS analysis (Supplementary Figure S1), while three aliquots of each group (FCS batch) were subsequently treated with lipopolysaccharides (LPS) to induce inflammatory stimulation, the other three per group serving as untreated controls
54 eicosanoids and fatty acid precursor molecules (Supplementary Table S4). Whereas most molecules such as the chemokine CCL3 and CXCL5 showed rather little variation between the groups, reproducibility issues of differentiated U937 macrophages were evidenced by fetal calf serum (FCS) batch-dependent significant (FDR < 0.05) differences in the formation of the chemokine CCL5, the cell growth regulator IGFBP2, and the cell migration and fibrinolysis regulator SERPINE1 (PAI1) and MMP1 (Figure 1A)

Summary

Introduction

Problems with the inter-laboratory reproducibility of results obtained with in vitro cell culture models are increasingly being recognized [1,2]. The need to reduce the use of animal models for research purposes relies on the use of accurate in vitro test models [3,4]. Important decisions such as the choice of drug candidates to be evaluated in clinical studies may be based on such experiments [5]. Biological materials and reference materials have been recognized as the main contributors for irreproducibility, resulting in a current focus on the investigation of genetic heterogeneity and genetic instability of cell culture models [6,7]. FCS is commonly used as cell culture supplement sustaining the growth and duplication of mammalian cells in vitro

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Discussion

Conclusion