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Towards characterization of cell culture conditions for reliable proteomic analysis: in vitro studies on A549, differentiated THP-1, and NR8383 cell lines

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Title: Towards characterization of cell culture conditions for reliable proteomic analysis: in vitro studies on A549, differentiated THP-1, and NR8383 cell lines
Authors: Rico Ledwith, Tobias Stobernack, Antje Bergert, Aileen Bahl, Mario Pink, Andrea Haase, Verónica I. Dumit
Source: Arch Toxicol
Archives of Toxicology
Publisher Information: Springer Science and Business Media LLC, 2024.
Publication Year: 2024
Subject Terms: Proteomics, Proteome, Proteomics/methods [MeSH], Macrophages/drug effects [MeSH], Humans [MeSH], Cell Differentiation/drug effects [MeSH], Cell Line [MeSH], Rats [MeSH], Macrophages/metabolism [MeSH], THP-1 Cells [MeSH], Animals [MeSH], New approach methodologies (NAMs), Toxicity mechanisms, Cell culture conditions, Passage numbers, Toxicogenomics and Omics Technologies, Reproducibility of Results [MeSH], Cell Culture Techniques [MeSH], Oxidative Stress/drug effects [MeSH], THP-1 differentiation process, Proteome [MeSH], THP-1 Cells, Macrophages, Cell Culture Techniques, Reproducibility of Results, Cell Differentiation, Cell Line, Rats, Oxidative Stress, Biowissenschaften, Biologie, Humans, Animals
Description: Proteomic investigations result in high dimensional datasets, but integration or comparison of different studies is hampered by high variances due to different experimental setups. In addition, cell culture conditions can have a huge impact on the outcome. This study systematically investigates the impact of experimental parameters on the proteomic profiles of commonly used cell lines—A549, differentiated THP-1 macrophage-like cells, and NR8383—for toxicity studies. The work focuses on analyzing the influence at the proteome level of cell culture setup involving different vessels, cell passage numbers, and post-differentiation harvesting time, aiming to improve the reliability of proteomic analyses for hazard assessment. Mass-spectrometry-based proteomics was utilized for accurate protein quantification by means of a label-free approach. Our results showed that significant proteome variations occur when cells are cultivated under different setups. Further analysis of these variations revealed their association to specific cellular pathways related to protein misfolding, oxidative stress, and proteasome activity. Conversely, the influence of cell passage numbers on the proteome is minor, suggesting a reliable range for conducting reproducible biological replicates. Notable, substantial proteome alterations occur over-time post-differentiation of dTHP-1 cells, particularly impacting pathways crucial for macrophage function. This finding is key for the interpretation of experimental results. These results highlight the need for standardized culture conditions in proteomic-based evaluations of treatment effects to ensure reliable results, a prerequisite for achieving regulatory acceptance of proteomics data.
Document Type: Article
Other literature type
Language: English
ISSN: 1432-0738
0340-5761
DOI: 10.1007/s00204-024-03858-4
DOI: 10.17169/refubium-44950
Access URL: https://pubmed.ncbi.nlm.nih.gov/39264451
https://repository.publisso.de/resource/frl:6508286
Rights: CC BY
Accession Number: edsair.doi.dedup.....be96bc8693eccefcd5a25f9b9f6a8e4a
Database: OpenAIRE
Description
Abstract:Proteomic investigations result in high dimensional datasets, but integration or comparison of different studies is hampered by high variances due to different experimental setups. In addition, cell culture conditions can have a huge impact on the outcome. This study systematically investigates the impact of experimental parameters on the proteomic profiles of commonly used cell lines—A549, differentiated THP-1 macrophage-like cells, and NR8383—for toxicity studies. The work focuses on analyzing the influence at the proteome level of cell culture setup involving different vessels, cell passage numbers, and post-differentiation harvesting time, aiming to improve the reliability of proteomic analyses for hazard assessment. Mass-spectrometry-based proteomics was utilized for accurate protein quantification by means of a label-free approach. Our results showed that significant proteome variations occur when cells are cultivated under different setups. Further analysis of these variations revealed their association to specific cellular pathways related to protein misfolding, oxidative stress, and proteasome activity. Conversely, the influence of cell passage numbers on the proteome is minor, suggesting a reliable range for conducting reproducible biological replicates. Notable, substantial proteome alterations occur over-time post-differentiation of dTHP-1 cells, particularly impacting pathways crucial for macrophage function. This finding is key for the interpretation of experimental results. These results highlight the need for standardized culture conditions in proteomic-based evaluations of treatment effects to ensure reliable results, a prerequisite for achieving regulatory acceptance of proteomics data.
ISSN:14320738
03405761
DOI:10.1007/s00204-024-03858-4