In vitro cell models offer a unique opportunity for conducting toxicology research, and the human lung adenocarcinoma cell line A549 is commonly used for toxicology testing strategies. It is essential to determine whether the response of these cells grown in different laboratories is consistent. In this study, A549 cells were grown under both submerged and air-liquid interface (ALI) conditions following an identical cell seeding protocol in two independent laboratories. The cells were switched to the ALI after four days of submerged growth, and their behaviour was compared to submerged conditions. The membrane integrity, cell viability, morphology, and (pro-)inflammatory response upon positive control stimuli were assessed at days 3, 5, and 7 under submerged conditions and at days 5, 7, and 10 at the ALI. Due to the high variability of the results between the two laboratories, the experiment was subsequently repeated using identical reagents at one specific time point and condition (day 5 at the ALI). Despite some variability, the results were more comparable, proving that the original protocol necessitated improvements. In conclusion, the use of detailed protocols and consumables from the same providers, special training of personnel for cell handling, and endpoint analysis are critical to obtain reproducible results across independent laboratories.
- Klíčová slova
- Alveolar epithelial cells, In vitro model, Inter-laboratory assessment, Lung model, Standard operating procedure,
- MeSH
- buněčná diferenciace účinky léků MeSH
- buněčné kultury * MeSH
- buňky A549 MeSH
- epitelové buňky * účinky léků MeSH
- interleukin-6 metabolismus MeSH
- interleukin-8 metabolismus MeSH
- laboratoře MeSH
- lidé MeSH
- lipopolysacharidy farmakologie MeSH
- proliferace buněk účinky léků MeSH
- reprodukovatelnost výsledků MeSH
- TNF-alfa farmakologie MeSH
- viabilita buněk účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- CXCL8 protein, human MeSH Prohlížeč
- IL6 protein, human MeSH Prohlížeč
- interleukin-6 MeSH
- interleukin-8 MeSH
- lipopolysacharidy MeSH
- TNF-alfa MeSH
Following advancements in the field of genotoxicology, it has become widely accepted that 3D models are not only more physiologically relevant but also have the capacity to elucidate more complex biological processes that standard 2D monocultures are unable to. Whilst 3D liver models have been developed to evaluate the short-term genotoxicity of chemicals, the aim of this study was to develop a 3D model that could be used with the regulatory accepted in vitro micronucleus (MN) following low-dose, longer-term (5 days) exposure to engineered nanomaterials (ENMs). A comparison study was carried out between advanced models generated from two commonly used liver cell lines, namely HepaRG and HepG2, in spheroid format. While both spheroid systems displayed good liver functionality and viability over 14 days, the HepaRG spheroids lacked the capacity to actively proliferate and, therefore, were considered unsuitable for use with the MN assay. This study further demonstrated the efficacy of the in vitro 3D HepG2 model to be used for short-term (24 h) exposures to genotoxic chemicals, aflatoxin B1 (AFB1) and methyl-methanesulfonate (MMS). The 3D HepG2 liver spheroids were shown to be more sensitive to DNA damage induced by AFB1 and MMS when compared to the HepG2 2D monoculture. This 3D model was further developed to allow for longer-term (5 day) ENM exposure. Four days after seeding, HepG2 spheroids were exposed to Zinc Oxide ENM (0-2 µg/ml) for 5 days and assessed using both the cytokinesis-block MN (CBMN) version of the MN assay and the mononuclear MN assay. Following a 5-day exposure, differences in MN frequency were observed between the CBMN and mononuclear MN assay, demonstrating that DNA damage induced within the first few cell cycles is distributed across the mononucleated cell population. Together, this study demonstrates the necessity to adapt the MN assay accordingly, to allow for the accurate assessment of genotoxicity following longer-term, low-dose ENM exposure.
- MeSH
- aflatoxin B1 toxicita MeSH
- biologické modely MeSH
- buněčné kultury metody MeSH
- buněčné linie MeSH
- buněčné sféroidy * MeSH
- buňky Hep G2 MeSH
- hepatocyty účinky léků MeSH
- játra účinky léků MeSH
- lidé MeSH
- methylmethansulfonát toxicita MeSH
- mikrojaderné testy metody MeSH
- mutageny toxicita MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- aflatoxin B1 MeSH
- methylmethansulfonát MeSH
- mutageny MeSH
