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.

BioNanomaterials Group Adolphe Merkle Institute University of Fribourg Chemin des Verdiers 4 1700 Fribourg Switzerland

BioNanomaterials Group Adolphe Merkle Institute University of Fribourg Chemin des Verdiers 4 1700 Fribourg Switzerland; Institute of Experimental Medicine of the Czech Academy of Sciences Videnska 1083 14220 Prague Czech Republic

In Vitro Toxicology Group Swansea University Medical School Swansea University SA2 8PP Wales United Kingdom

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