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. 2017 Jun;91(6):2315-2330.
doi: 10.1007/s00204-016-1897-2. Epub 2016 Dec 10.

Pan-European inter-laboratory studies on a panel of in vitro cytotoxicity and pro-inflammation assays for nanoparticles

Jean-Pascal Piret  1 , Olesja M Bondarenko  2 , Matthew S P Boyles  3 , Martin Himly  3 , Ana R Ribeiro  4 , Federico Benetti  5   6 , Caroline Smal  7 , Braulio Lima  8 , Annegret Potthoff  9 , Monica Simion  10 , Elise Dumortier  11 , Paulo Emilio C Leite  4 , Luciene Bottentuit Balottin  4 , José Mauro Granjeiro  4 , Angela Ivask  2   12 , Anne Kahru  2 , Isabella Radauer-Preiml  3 , Ulrike Tischler  3 , Albert Duschl  3 , Christelle Saout  11 , Sergio Anguissola  13 , Andrea Haase  14 , An Jacobs  15 , Inge Nelissen  15 , Superb K Misra  16 , Olivier Toussaint  17
Affiliations

Pan-European inter-laboratory studies on a panel of in vitro cytotoxicity and pro-inflammation assays for nanoparticles

Jean-Pascal Piret et al. Arch Toxicol. 2017 Jun.

Abstract

The rapid development of nanotechnologies and increased production and use of nanomaterials raise concerns about their potential toxic effects for human health and environment. To evaluate the biological effects of nanomaterials, a set of reliable and reproducible methods and development of standard operating procedures (SOPs) is required. In the framework of the European FP7 NanoValid project, three different cell viability assays (MTS, ATP content, and caspase-3/7 activity) with different readouts (absorbance, luminescence and fluorescence) and two immune assays (ELISA of pro-inflammatory cytokines IL1-β and TNF-α) were evaluated by inter-laboratory comparison. The aim was to determine the suitability and reliability of these assays for nanosafety assessment. Studies on silver and copper oxide nanoparticles (NPs) were performed, and SOPs for particle handling, cell culture, and in vitro assays were established or adapted. These SOPs give precise descriptions of assay procedures, cell culture/seeding conditions, NPs/positive control preparation and dilutions, experimental well plate preparation, and evaluation of NPs interference. The following conclusions can be highlighted from the pan-European inter-laboratory studies: Testing of NPs interference with the toxicity assays should always be conducted. Interference tests should be designed as close as possible to the cell exposure conditions. ATP and MTS assays gave consistent toxicity results with low inter-laboratory variability using Ag and CuO NPs and different cell lines and therefore, could be recommended for further validation and standardization. High inter-laboratory variability was observed for Caspase 3/7 assay and ELISA for IL1-β and TNF-α measurements.

Keywords: In vitro assays; Inter-laboratory studies; Interference; Nanoparticles; Standard operating procedures.

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