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Cited by 160Cancer, Hypoxia, and MetabolismAmino Acid Enzymes and MetabolismMetabolomics and Mass Spectrometry Studies

Rational Cell Culture Optimization Enhances Experimental Reproducibility in Cancer Cells

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Wright Muelas, Marina, Ortega, Fernando, Breitling, Rainer, Bendtsen, Claus, Westerhoff, Hans V.·2018·Scientific Reports
DOI 10.1038/s41598-018-21050-4PMC PMC5813001

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Abstract

Optimization of experimental conditions is critical in ensuring robust experimental reproducibility. Through detailed metabolomic analysis we found that cell culture conditions significantly impacted on glutaminase (GLS1) sensitivity resulting in variable sensitivity and irreproducibility in data. Baseline metabolite profiling highlighted that untreated cells underwent significant changes in metabolic status. Both the extracellular levels of glutamine and lactate and the intracellular levels of multiple metabolites changed drastically during the assay. We show that these changes compromise the robustness of the assay and make it difficult to reproduce. We discuss the implications of the cells' metabolic environment when studying the effects of perturbations to cell function by any type of inhibitor. We then devised 'metabolically rationalized standard' assay conditions, in which glutaminase-1 inhibition reduced glutamine metabolism differently in both cell lines assayed, and decreased the proliferation of one of them. The adoption of optimized conditions such as the ones described here should lead to an improvement in reproducibility and help eliminate false negatives as well as false positives in these assays.

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  • Existing recommendations that aim to increase the relevance of in vitro cultures to in vivo physiology are available for many of these factors.

    “For many of these factors, existing recommendations that aim to increase the relevance of in vitro cultures to in vivo physiology are available (Baker, 2016; Muelas et al., 2018; Hirsch and Schildknecht, 2019).”

    From Toward Best Practices for Controlling Mammalian Cell Culture Environments (2022) · cites this paper as Muelas et al., 2018

    The Discussion (page unavailable) describes existing reporting standards and recommends documenting culture factors including cell density, medium, nutrients, and pH. It presents these measures as ways to improve reproducibility and assay evaluation, not specifically as recommendations to make in vitro cultures more representative of in vivo physiology. (LLM, conf=0.82)

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  • Medium changes have been shown to influence oxygen supply and cellular metabolism, as well as synthesis of proteins, DNA, and RNA.

    “Medium changes have been shown to influence oxygen supply and cellular metabolism, as well as synthesis of proteins, DNA, and RNA (Al-Ani et al., 2018; Wright Muelas et al., 2018).”

    From In Vitro research Reproducibility: Keeping up High Standards (2019) · cites this paper as Wright Muelas et al., 2018

    The Results section (‘Explanation: The cellular environment is uncontrolled’; page not provided) describes changing culture-medium conditions, including glutamine depletion, that could alter cellular metabolism. The cited paper does not establish that medium changes affect oxygen supply or the synthesis of proteins, DNA, and RNA; searches for those effects found no supporting passage. (LLM, conf=0.86)

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Cited by

Sorted by citations

Authors
Wright Muelas, M., Ortega, F., Breitling, R., Bendtsen, C., & Westerhoff, H. V.
Affiliations
  • University of Manchester (GB)
  • AstraZeneca (United Kingdom) (GB)
ORCID
Venue
Scientific Reports
Publisher
Springer Science and Business Media LLC
Published
2018-02-14
Volume
8
Issue
1
Pages
3029-3029
ISSN
2045-2322 (ISSN-L)
DOI
10.1038/s41598-018-21050-4
PMC
PMC5813001
OpenAlex
W2949384615
Cited by
160
References
14
Citations by year
2026: 4 · 2025: 2 · 2024: 1 · 2023: 2 · 2022: 6 · 2021: 82 · 2020: 42 · 2019: 15 · 2018: 6
FWCI
10.96
Type
journal article
Language
en
Open access
Licence
Locations
  • Scientific Reports (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Research Explorer (The University of Manchester) (publishedVersion, other-oa)
  • VU Research Portal (publishedVersion, other-oa)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • Data Archiving and Networked Services (DANS) (submittedVersion, other-oa)
Funders
  • AstraZeneca
  • Directorate for Biological Sciences
  • Biotechnology and Biological Sciences Research Council (BB/M017702/1, 1088313)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Cancer, Hypoxia, and Metabolism
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biochemistry › Amino Acid Enzymes and Metabolism
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Metabolomics and Mass Spectrometry Studies
Keywords
cell culture optimization, experimental reproducibility, cancer cells, GLS1, glutamine metabolism, lactate, intracellular metabolites, metabolite profiling, assay standardization, cell proliferation, false positives and false negatives
MeSH terms
Animals, Glutaminase, Glutamine, Humans, Neoplasms, Research Design, Thiadiazoles, Reproducibility of Results, Cell Culture Techniques, Cell Line, Tumor, Cell Proliferation, Metabolomics

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