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Eicosanoid Content in Fetal Calf Serum Accounts for Reproducibility Challenges in Cell Culture
Biomolecules ( IF 5.5 ) Pub Date : 2021-01-15 , DOI: 10.3390/biom11010113
Laura Niederstaetter 1 , Benjamin Neuditschko 1, 2 , Julia Brunmair 1 , Lukas Janker 1 , Andrea Bileck 1, 3 , Giorgia Del Favero 4, 5 , Christopher Gerner 1, 3
Affiliation  

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.

中文翻译:

胎牛血清中的类花生酸含量解释了细胞培养中的再现性挑战

有关体外细胞培养实验的可再现性问题与遗传波动和生物材料(例如胎牛血清)的分批变化有关。基因组测序可以控制前者,而后者则可能仍然无法识别。使用用于细胞分化和炎症的U937巨噬细胞模型,我们研究了效应分子的形成是否依赖于用于培养的FCS批次。高分辨率质谱(HRMS)用于鉴定FCS成分,并探讨它们对培养细胞的影响,从而评估分泌的细胞因子,类花生酸和其他炎症介质。值得注意的是,FCS类花生酸的成分比蛋白质成分显示出更多的批次依赖性变异。使用C13标记的花生四烯酸证明了U937巨噬细胞从培养基中有效吸收了脂肪酸并引起了炎症诱导的释放,从而突出了脂质的快速代谢。对于功能测试,通过加标平衡了两种选定的类花生酸5-HETE和15-HETE的FCS批次依赖性纳摩尔浓度差异。在这些确定的条件下培养U937细胞确实会导致蛋白质组显着改变,表明HETE诱导的PPARγ激活,而高分辨显微镜观察到的HETE诱导的过氧化物酶体形成独立地证实了这一点。总而言之,本数据表明,FCS含有的类花生酸类化合物可能会发生批量变化,可能会在细胞培养实验中调节细胞效应子的功能。
更新日期:2021-01-15
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