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Cited by 84Nanoparticles: synthesis and applicationsAnimal testing and alternativesGraphene and Nanomaterials Applications

Toward achieving harmonization in a nanocytotoxicity assay measurement through an interlaboratory comparison study

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Elliott, John·2017·ALTEX
DOI 10.14573/altex.1605021
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Abstract

Development of reliable cell-based nanotoxicology assays is important for evaluation of potentially hazardous engineered nanomaterials. Challenges to producing a reliable assay protocol include working with nanoparticle dispersions and living cell lines, and the potential for nano-related interference effects. Here we demonstrate the use of a 96-well plate design with several measurement controls and an interlaboratory comparison study involving five laboratories to characterize the robustness of a nanocytotoxicity MTS cell viability assay based on the A549 cell line. The consensus EC50 values were 22.1 mg/L (95% confidence intervals 16.9 mg/L to 27.2 mg/L) and 52.6 mg/L (44.1 mg/L to 62.6 mg/L) for positively charged polystyrene nanoparticles for the serum-free and serum conditions, respectively, and 49.7 μmol/L (47.5 μmol/L to 51.5 μmol/L) and 77.0 μmol/L (54.3 μmol/L to 99.4 μmol/L) for positive chemical control cadmium sulfate for the serum-free and serum conditions, respectively. Results from the measurement controls can be used to evaluate the sources of variability and their relative magnitudes within and between laboratories. This information revealed steps of the protocol that may need to be modified to improve the overall robustness and precision. The results suggest that protocol details such as cell line ID, media exchange, cell handling, and nanoparticle dispersion are critical to ensure protocol robustness and comparability of nanocytotoxicity assay results. The combination of system control measurements and interlaboratory comparison data yielded insights that would not have been available by either approach by itself.

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  • Short tandem repeat sequencing revealed a partial chromosome deletion in one of the cell cultures.

    “Short tandem repeat sequencing revealed a partial chromosome deletion in one of the cell cultures.”

    From In Vitro research Reproducibility: Keeping up High Standards (2019) · cites this paper as Elliott et al., 2017

    Section 3.1 reports that STR genotyping found a missing allele at a marker on chromosome 5 in the A549-A cell line. It does not establish a partial chromosome deletion or describe the method as STR sequencing. (LLM, conf=0.86)

    Experimental — accuracy not yet measured

  • Even under strict conditions, EC50 values of two A549 cultures upon CdSO4 treatment differed by a factor of two in all laboratories.

    “Even under such strict conditions, EC50 values of the two A549 cultures upon CdSO4 treatment differed by a factor of two in all laboratories.”

    From In Vitro research Reproducibility: Keeping up High Standards (2019) · cites this paper as Elliott et al., 2017

    The Discussion reports that the CdSO4 EC50 for A549-B was twice that for A549-A, but specifies that this finding was observed under serum-free conditions. Figure 10 identifies the serum-free A549-B result from Laboratory A as an outlier, so the paper does not establish a twofold difference in every laboratory. (LLM, conf=0.85)

    Experimental — accuracy not yet measured

  • Simple cell handling steps, such as PBS washing, were identified to significantly change assay outcomes.

    “For example, simple cell handling steps, such as PBS washing, were identified to significantly change assay outcomes.”

    From In Vitro research Reproducibility: Keeping up High Standards (2019) · cites this paper as Elliott et al., 2017

  • Cell line authentication was discovered to be one of the main factors influencing assay results.

    “In the course of these investigations, cell line authentication was discovered to be one of the main factors influencing assay results.”

    From In Vitro research Reproducibility: Keeping up High Standards (2019) · cites this paper as Elliott et al., 2017

  • Elliott et al. 2017 assessed reproducibility of MTS-tetrazolium reduction assay results as indicators of cell viability in an international inter-laboratory comparison study with five independent laboratories.

    “An exemplary illustration of this transparency is a publication by Elliott and colleagues (Elliott et al., 2017) assessing the reproducibility of MTS-tetrazolium reduction assay results as indicators of cell viability in an international inter-laboratory comparison study with five independent laboratories.”

    From In Vitro research Reproducibility: Keeping up High Standards (2019) · cites this paper as Elliott et al., 2017

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Authors
Elliott, J.
Affiliations
National Institute of Standards and Technology (US)
Corresponding author
Elliott, John
ORCID
Elliott, John
Venue
ALTEX
Publisher
ALTEX Edition
Published
2017
Volume
34
Issue
2
Pages
201-218
ISSN
  • 0946-7785 (ISSN-L)
  • 1868-596X
DOI
10.14573/altex.1605021
OpenAlex
W2426712039
Cited by
84
References
4
Citations by year
2026: 9 · 2025: 3 · 2024: 5 · 2023: 5 · 2022: 19 · 2021: 11 · 2020: 12 · 2019: 7 · 2018: 8 · 2017: 4
FWCI
2.53
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • ALTEX (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DORA Empa (Swiss Federal Laboratories for Materials Science and Technology (Empa)) (submittedVersion, cc-by)
  • Infoscience (Ecole Polytechnique Fédérale de Lausanne) (submittedVersion)
  • Infoscience (Ecole Polytechnique Fédérale de Lausanne) (submittedVersion, cc-by-nc-nd)
Funders
  • Ministry of Science, ICT and Future Planning (2014M3A7B6020163, NRF-2014M3A7B6020163)
  • Competence Centre for Materials Science and Technology
  • National Institute of Standards and Technology
  • Center for Nanoscale Science and Technology
Topics
  • Physical Sciences › Materials Science › Materials Chemistry › Nanoparticles: synthesis and applications
  • Health Sciences › Veterinary › Small Animals › Animal testing and alternatives
  • Physical Sciences › Engineering › Biomedical Engineering › Graphene and Nanomaterials Applications
Keywords
interlaboratory comparison, assay harmonization, A549 cells, polystyrene nanoparticles, cadmium sulfate, EC50, measurement control, assay robustness, engineered nanomaterials, 96-well plate assay
MeSH terms
A549 Cells, Humans, Laboratories, Polystyrenes, Reproducibility of Results, Hazardous Substances, Toxicity Tests, Nanoparticles

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