Culture Media and Serum Conditions Alter Primary Human Macrophage Morphology and Inflammatory Function
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1183319 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:Macrophages are central to innate immunity and defining the mechanisms by which these cells mediate inflammation is critical to understanding in situ homeostatic and pathophysiologic roles. Human monocyte derived macrophages (hMDM) are widely used to examine these processes. Stimuli that influence macrophage function could influence these studies, including differences in culture environment that have been suggested significantly impact macrophage function. We examined baseline hMDM activity and the response to the TLR4 agonist, lipopolysaccharide (LPS) in different culture media in the presence and absence of serum. We focused on the most common macrophage medias, DMEM and RPMI 1640 Medium +/- fetal bovine serum (FBS), as well as Macrophage Serum Free Media (M-SFM), a media specifically formulated for serum free culture. Cells cultured in these five distinct media were treated with vehicle (H20) or LPS and evaluated for changes in morphology, transcriptomic profile, and alterations in inflammatory response. Culture in M-SFM resulted in the greatest morphological differences (area, length-to-width, perimeter-to-area ratio) and while there were few transcriptomic differences between DMEM and RPMI with FBS, both the absence of FBS supplementation and culture in M-SFM significantly altered the baseline transcriptome. Culture in M-SFM also significantly reduced macrophage sensitivity to LPS as measured by NF-kB activation. All hMDM conditions showed a dose dependent phagocytic response and elicited cytokine and chemokine secretion in response to LPS stimulation, but the magnitude of these effects varied between conditions, with different trends depending on the cytokine. These findings suggest that culture media and the presence of serum alters baseline activation and the response to stimulus in macrophages, potentially obscuring or driving artifactual results. These studies confirm and expand on previous findings showing that in vitro microenvironment is not a benign component of experimental design and demonstrate the need to optimize experimental conditions for such plastic cells.
2025-07-16 | GSE281287 | GEO
Project description:HEK293T cells grown to confluence in media +10% fetal bovine serume. Media was removed and replaced with serum free media, and cultured for 3 days. RNA was harvested from day0 (serum supplemented), control, and day3 (serum starved) cultures, experiment.
2014-12-13 | GSE64117 | GEO
Project description:Neurons are almost exclusively cultured in media containing glucose at much higher concentration than found in the brain. To test whether “standard” hyperglycaemic culture conditions affect neuronal respiration compared to near- euglycemic conditions, we grew neuronal cultures with minimal glial contamination from hippocampus and cortex of neonatal C57bl6 mice in standard commercially available media (25 mM Glucose) and in identical media with 5 mM glucose. Neurons grew well in both glucose concentrations until at least 14 days in vitro, with similar morphology and synaptogenesis. Neurons grown in high glucose were more dependent on glycolysis as their primary source of ATP, measured using ATP luminescence and cellular respirometry assays. In contrast, neurons grown in 5 mM glucose showed a more balanced dependence on glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) and greater reserve mitochondrial respiration capacity, relative to standard media. Changes in gene and protein expression levels corroborate these changes in function. Our results suggest that neurons cultured in high glucose media preferentially use glycolysis, opposite to what is known for neurons in vivo as the primary pathway for ATP maintenance. We suggest using neuronal culture systems in 5 mM glucose to better represent physiologically relevant neuronal respiration.
2025-06-30 | PXD059008 | Pride
Project description:Most cell culture experiments utilize media containing fetal calf serum. Results are often interpreted regarding importance to human pathways. We studied gene expression in mouse macrophages grown in the absence of serum, and in fetal calf serum, mouse serum, and human serum using genome wide expression systems in resting conditions and after stimulation with lipopolysaccharide.
2011-09-23 | GSE32328 | GEO
Project description:In the research field of extracellular vesicles (EVs), the use of EV-depleted fetal bovine serum (FBS) for in vitro studies is highly recommended to eliminate the confounding effects of media derived EVs. EV-depleted FBS may either be prepared by ultracentrifugation or bought commercially, nevertheless these depletion methods do not guarantee an RNA-free preparation. In this study we have addressed the RNA contamination issue in FBS, ultracentrifuged EV-depleted FBS, commercially available EV-depleted FBS, and also from our recently developed filtration based EV depleted FBS. Commercially available serum-free, xeno-free defined media were also screened for RNA contamination.
2019-04-30 | GSE120594 | GEO
Project description:Questioning the purity of the media - Extracellular small non-coding RNA contaminants in fetal bovine serum and serum-free media
| PRJNA493652 | ENA
Project description:Cancer cell culture models frequently rely on fetal bovine serum as a source of protein and lipid factors that support cell survival and proliferation; however, serum-containing media imperfectly mimics the in vivo cancer environment. Recent studies suggest that typical serum-containing cell culture conditions can mask cancer dependencies, for example on cholesterol biosynthesis enzymes, that exist in vivo and emerge when cells are cultured in media that provides more realistic levels of lipids. Here we describe a high-throughput screen that identified fenretinide and ivermectin as small molecules whose cytotoxicity is greatly enhanced in lipid-restricted media formulations. Mechanism of action studies indicate that the ivermectin-induced cell death involves oxidative stress, while fenretinide likely targets DEGS1, a lipid desaturase necessary for ceramide synthesis, to induce cell death. Notably, both fenretinide and ivermectin have previously demonstrated in vivo anticancer efficacy despite their low cytotoxicity under typical cell culture conditions. These studies reveal ceramide synthesis as a targetable vulnerability of cancer cells cultured under lipid-restricted conditions and suggest a general screening strategy for identifying additional cancer dependencies masked by culture conditions unrepresentative of the in vivo environment.
2024-11-14 | GSE247900 | GEO
Project description:Most porcine embryonic stem cells (pESC) derived from in vitro blastocysts Still require complex media compositions and feeder layers which make difficulty to adapt their broad and routine use. We derived pESC using simplified culture media (FIW) consisting of small molecules FGF2, IWR-1, and WH-4-023 in serum-free medium. We established several pESC-FIW lines capable of single cell passaging with short cell doubling time (about 12 hours). These cells expressed pluripotency markers, OCT4, SOX2, and NANOG, as well as cell surface markers, SSEA1, SSEA4, and TRA-1-60. pESC-FIW showed stable proliferation rate and normal karyotype even after long-term culture over 50 passages. Established pESC showed formative characteristics based on the negative expression for the naive marker KLF4 and primed marker T, whereas strong expression for the formative marker OTX2 with high levels of related genes. Transcriptome analysis showed that pESC-FIW had similar cellular identities to reported pESC maintained in complex media formulations and had characteristics of gastrulating epiblast cells. In addition, pESC-FIW could be maintained for multiple passages in a feeder-free condition on fibronectin coated plate using mTeSR™, a commercial medium for feeder-free culture. These results indicate that the canonical WNT (IWR-1) inhibition and the SRC inhibition (WH-4-023) are sufficient to establish pESC capable of single cell passaging and feeder-free expansion under the serum-free condition. Easy to maintain-pESC can be a tool for gene editing application useful for agriculture and biomedicine, and lineage commitment studies.
2024-08-07 | GSE240454 | GEO
Project description:Production of cultured meat requires the robust differentiation of satellite cells into mature muscle fibers without the use of animal-derived components. Current protocols induce myogenic differentiation in vitro through serum starvation, an abrupt reduction in serum concentration. Here, we used RNA sequencing to investigate the transcriptomic remodelling of bovine satellite cells during myogenic differentiation induced by serum starvation. We characterized canonical myogenic gene expression, and identified surface receptors upregulated during the early phase of differentiation. Supplementation of ligands to these receptors enabled the formulation of a chemically defined media that induced differentiation in the absence of serum starvation and/or transgene expression. Serum-free myogenic differentiation was of similar extent to that induced by serum starvation, as evaluated by transcriptome analysis, protein expression and the presence of a functional contractile apparatus. Moreover, the serum-free differentiation media supported the fabrication of mature three-dimensional bioartificial muscle constructs, demonstrating its suitability for cultured beef production.
2021-10-22 | GSE173198 | GEO
Project description:Production of cultured meat requires the robust differentiation of satellite cells into mature muscle fibers without the use of animal-derived components. Current protocols induce myogenic differentiation in vitro through serum starvation, an abrupt reduction in serum concentration. Here, we used RNA sequencing to investigate the transcriptomic remodelling of bovine satellite cells during myogenic differentiation induced by serum starvation. We characterized canonical myogenic gene expression, and identified surface receptors upregulated during the early phase of differentiation. Supplementation of ligands to these receptors enabled the formulation of a chemically defined media that induced differentiation in the absence of serum starvation and/or transgene expression. Serum-free myogenic differentiation was of similar extent to that induced by serum starvation, as evaluated by transcriptome analysis, protein expression and the presence of a functional contractile apparatus. Moreover, the serum-free differentiation media supported the fabrication of mature three-dimensional bioartificial muscle constructs, demonstrating its suitability for cultured beef production.
2021-10-22 | GSE173196 | GEO