An inter-laboratory effort to harmonize the cell-delivered in vitro dose of aerosolized materials

November 14, 2022
Anne Bannuscher a, Otmar Schmid b,c, Barbara Drasler a, Alain Rohrbasser a, Hedwig M. Braakhuis d, Kirsty Meldrum e, Edwin P. Zwart d, Eric R. Gremmer d, Barbara Birk f, Manuel Rissel f, Robert Landsiedel f,g, Elisa Moschini h, Stephen J. Evans e, Pramod Kumar b,c, Sezer Orak b,c, Ali Doryab b,c, Johanna Samulin Erdem i, Tommaso Serchi h, Rob J. Vandebriel d, Flemming R. Cassee d,j, Shareen H. Doak e, Alke Petri-Fink a, Shanbeh Zienolddiny i, Martin J. D. Clift e, Barbara Rothen-Rutishauser a,
a Adolphe Merkle Institute, University of Fribourg, Chemin des Verdiers 4, 1700 Fribourg, Switzerland
b Comprehensive Pneumology Center (CPC-M), Helmholtz Zentrum München – Member of the German Center for Lung Research (DZL), Max-Lebsche-Platz 31, 81377 Munich, Germany
c Institute of Lung Health and Immunity, Helmholtz Zentrum München – German Research Center for Environmental Health, 85764 Neuherberg, Germany
d National Institute for Public Health and the Environment (RIVM), PO Box 1, 3720, BA, Bilthoven, the Netherlands
e In Vitro Toxicology Group, Faculty of Medicine, Health and Life Sciences, Medical School, Institute of Life Sciences, Centre for NanoHealth, Swansea University, Singleton Campus, Wales SA2 8PP, UK
f BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen am Rhein, Germany
g Free University of Berlin, Pharmacy, Pharmacology and Toxicology, 14195 Berlin, Germany.
h Department of Environmental Research and Innovation, Luxembourg Institute of Science and Technology (LIST), 41 rue du Brill, L4422 Belvaux, Grand-Duchy of Luxembourg, Luxembourg
i National Institute of Occupational Health (STAMI), N-0033 Oslo, Norway
j Institute for Risk Assessment Sciences, Utrecht University, Utrecht, the Netherlands

Harmonization of SOP for (nano)material aerosolization using the VITROCELL® Cloud12.
Inter-laboratory comparison among seven different laboratories in Europe.

• Method development for (nano-)material deposition data of high reproducibility.
• Harmonization of SOP for (nano)material aerosolization using the VITROCELL® Cloud12.
• Inter-laboratory comparison among seven different laboratories in Europe.
• Efficient and comparable DQ12 and TiO2 NM aerosolization within partner laboratories.


Air-liquid interface (ALI) lung cell models cultured on permeable transwell inserts are increasingly used for respiratory hazard assessment requiring controlled aerosolization and deposition of any material on ALI cells. The approach presented herein aimed to assess the transwell insert-delivered dose of aerosolized materials using the VITROCELL® Cloud12 system, a commercially available aerosol-cell exposure system. An inter-laboratory comparison study was conducted with seven European partners having different levels of experience with the VITROCELL® Cloud12. A standard operating procedure (SOP) was developed and applied by all partners for aerosolized delivery of materials, i.e., a water-soluble molecular substance (fluorescence-spiked salt) and two poorly soluble particles, crystalline silica quartz (DQ12) and titanium dioxide nanoparticles (TiO2 NM-105). The material dose delivered to transwell inserts was quantified with spectrofluorometry (fluorescein) and with the quartz crystal microbalance (QCM) integrated in the VITROCELL® Cloud12 system. The shape and agglomeration state of the deposited particles were confirmed with transmission electron microscopy (TEM).
Inter-laboratory comparison of the device-specific performance was conducted in two steps, first for molecular substances (fluorescein-spiked salt), and then for particles. Device- and/or handling-specific differences in aerosol deposition of VITROCELL® Cloud12 systems were characterized in terms of the so-called deposition factor (DF), which allows for prediction of the transwell insert-deposited particle dose from the particle concentration in the aerosolized suspension. Albeit DF varied between the different labs from 0.39 to 0.87 (mean (coefficient of variation (CV)): 0.64 (28%)), the QCM of each VITROCELL® Cloud 12 system accurately measured the respective transwell insert-deposited dose. Aerosolized delivery of DQ12 and TiO2 NM-105 particles showed good linearity (R2 > 0.95) between particle concentration of the aerosolized suspension and QCM-determined insert-delivered particle dose. The VITROCELL® Cloud 12 performance for DQ12 particles was identical to that for fluorescein-spiked salt, i.e., the ratio of measured and salt-predicted dose was 1.0 (29%). On the other hand, a ca. 2-fold reduced dose was observed for TiO2 NM-105 (0.54 (41%)), which was likely due to partial retention of TiO2 NM-105 agglomerates in the vibrating mesh nebulizer of the VITROCELL® Cloud12.
This inter-laboratory comparison demonstrates that the QCM integrated in the VITROCELL® Cloud 12 is a reliable tool for dosimetry, which accounts for potential variations of the transwell insert-delivered dose due to device-, handling- and/or material-specific effects. With the detailed protocol presented herein, all seven partner laboratories were able to demonstrate dose-controlled aerosolization of material suspensions using the VITROCELL® Cloud12 exposure system at dose levels relevant for observing in vitro hazard responses. This is an important step towards regulatory approved implementation of ALI lung cell cultures for in vitro hazard assessment of aerosolized materials.

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