John T. Elliott
Biosystems and Biomaterials Division, Material Measurement Laboratory, National Institute of Standards and Technology (NIST), Gaithersburg, MD, USA
Matthias Rösslein
EMPA, Swiss Federal Laboratories for Material Testing and Research, Particles-Biology Interactions Laboratory, St. Gallen, Switzerland
Nam Woong Song
Center for Nanosafety Metrology, Korea Research Institute of Standards and Science (KRISS), Daejeon, Republic of Korea
Blaza Toman
Statistical Engineering Division, Information Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, MD, USA
Agnieszka Kinsner- Ovaskainen
European Commission, Joint Research Centre (JRC), Institute for Health and Consumer Protection, Nanobiosciences Unit, Ispra, Italy
Rawiwan Maniratanachote
National Nanotechnology Center (NANOTEC), National Science and Technology Development Agency (NSTDA), Pathum Thani, Thailand
Marc L. Salit
Biosystems and Biomaterials Division, Material Measurement Laboratory, National Institute of Standards and Technology (NIST), Gaithersburg, MD, USA; Department of Bioengineering, Stanford University, Stanford, CA, USA
Elijah J. Petersen
Biosystems and Biomaterials Division, Material Measurement Laboratory, National Institute of Standards and Technology (NIST), Gaithersburg, MD, USA
Fatima Sequeira
Biosystems and Biomaterials Division, Material Measurement Laboratory, National Institute of Standards and Technology (NIST), Gaithersburg, MD, USA
Erica Romsos
Biomolecular Measurement Division, Material Measurement Laboratory, National Institute of Standards and Technology, Gaithersburg, MD, USA
Soo Jin Kim
Center for Nanosafety Metrology, Korea Research Institute of Standards and Science (KRISS), Daejeon, Republic of Korea
Jieun Lee
Center for Nanosafety Metrology, Korea Research Institute of Standards and Science (KRISS), Daejeon, Republic of Korea
Nadia R. von Moos
Powder Technology Laboratory, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland
François Rossi
European Commission, Joint Research Centre (JRC), Institute for Health and Consumer Protection, Nanobiosciences Unit, Ispra, Italy
Cordula Hirsch
EMPA, Swiss Federal Laboratories for Material Testing and Research, Particles-Biology Interactions Laboratory, St. Gallen, Switzerland
Harald F. Krug
EMPA, Swiss Federal Laboratories for Material Testing and Research, International Research Cooperations Manager, St. Gallen, Switzerland
Wongsakorn Suchaoin
National Nanotechnology Center (NANOTEC), National Science and Technology Development Agency (NSTDA), Pathum Thani, Thailand
Peter Wick
EMPA, Swiss Federal Laboratories for Material Testing and Research, Particles-Biology Interactions Laboratory, St. Gallen, Switzerland
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Abstract
Development of reliable cell-based nanotoxicology assays is important for evaluation of potentially hazardous engineered nanomaterials. Challenges to producing a reliable assay protocol include working with nanoparticle dispersions and living cell lines, and the potential for nano-related interference effects. Here we demonstrate the use of a 96-well plate design with several measurement controls and an interlaboratory comparison study involving five laboratories to characterize the robustness of a nanocytotoxicity MTS cell viability assay based on the A549 cell line. The consensus EC50 values were 22.1 mg/L (95% confidence intervals 16.9 mg/L to 27.2 mg/L) and 52.6 mg/L (44.1 mg/L to 62.6 mg/L) for positively charged polystyrene nanoparticles for the serum-free and serum conditions, respectively, and 49.7 μmol/L (47.5 μmol/L to 51.5 μmol/L) and 77.0 μmol/L (54.3 μmol/L to 99.4 μmol/L) for positive chemical control cadmium sulfate for the serum-free and serum conditions, respectively. Results from the measurement controls can be used to evaluate the sources of variability and their relative magnitudes within and between laboratories. This information revealed steps of the protocol that may need to be modified to improve the overall robustness and precision. The results suggest that protocol details such as cell line ID, media exchange, cell handling, and nanoparticle dispersion are critical to ensure protocol robustness and comparability of nanocytotoxicity assay results. The combination of system control measurements and interlaboratory comparison data yielded insights that would not have been available by either approach by itself.