Transferability and reproducibility of exposed air-liquid interface co-culture lung models

May 18, 2023

Hedwig M. Braakhuis a, Eric R. Gremmer a, Anne Bannuscher b, Barbara Drasler b, Sandeep Keshavan b, Barbara Rothen-Rutishauser b, Barbara Birk c, Andreas Verlohner c, Robert Landsiedel c,i, Kirsty Meldrum d, Shareen H. Doak d, Martin J.D. Clift d, Johanna Samulin Erdem e, Oda A.H. Foss e, Shanbeh Zienolddiny-Narui e, Tommaso Serchi f, Elisa Moschini f, Pamina Weber f, Sabina Burla f, Pramod Kumar g,h, Otmar Schmid g,h, Edwin Zwart a, Jolanda P. Vermeulen a, Rob J. Vandebriel a
a National Institute for Public Health & the Environment (RIVM), the Netherlands
b Adolphe Merkle Institute (AMI), University of Fribourg, Switzerland
c BASF SE, Ludwigshafen, Germany
d Swansea University Medical School (SU), Wales, UK
e National Institute of Occupational Health (STAMI), Norway
f Luxembourg Institute of Science and Technology (LIST), Grand Duchy of Luxembourg, Luxembourg
g Comprehensive Pneumology Center (CPC-M) with the CPC-M bioArchive, Helmholtz Center Munich – Member of the German Center for Lung Research (DZL), Munich, Germany
h Institute of Lung Health and Immunity, Helmholtz Center Munich – German Research Center for Environmental Health, Neuherberg, Germany
i Free University of Berlin, Pharmacy – Pharmacology and Toxicology, Berlin, Germany

The establishment of reliable and robust in vitro models for hazard assessment, a prerequisite for moving away from animal testing, requires the evaluation of model transferability and reproducibility. Lung models that can be exposed via the air, by means of an air-liquid interface (ALI) are promising in vitro models for evaluating the safety of nanomaterials (NMs) after inhalation exposure. We performed an inter-laboratory comparison study to evaluate the transferability and reproducibility of a lung model consisting of the human bronchial cell line Calu-3 as a monoculture and, to increase the physiologic relevance of the model, also as a coculture with macrophages (either derived from the THP-1 monocyte cell line or from human blood monocytes). The lung model was exposed to NMs using the VITROCELL® Cloud12 system at physiologically relevant dose levels.
Overall, the results of the 7 participating laboratories are quite similar. After exposing Calu-3 alone and Calu-3 co-cultures with macrophages, no effects of lipopolysaccharide (LPS), quartz (DQ12) or titanium dioxide (TiO2) NM-105 particles on the cell viability and barrier integrity were detected. LPS exposure induced moderate cytokine release in the Calu-3 monoculture, albeit not statistically significant in most labs. In the co-culture models, most laboratories showed that LPS can significantly induce cytokine release (IL-6, IL-8 and TNF-α). The exposure to quartz and TiO2 particles did not induce a statistically significant increase in cytokine release in both cell models probably due to our relatively low deposited doses, which were inspired by in vivo dose levels. The intra- and inter-laboratory comparison study indicated acceptable interlaboratory variation for cell viability/ toxicity (WST-1, LDH) and transepithelial electrical resistance, and relatively high inter-laboratory variation for cytokine production.
The transferability and reproducibility of a lung co-culture model and its exposure to aerosolized particles at the ALI were evaluated and recommendations were provided for performing inter-laboratory comparison studies. Although the results are promising, optimizations of the lung model (including more sensitive read-outs) and/or selection of higher deposited doses are needed to enhance its predictive value before it may be taken further towards a possible OECD guideline.

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