Toward achieving harmonization in a nano-cytotoxicity assay measurement through an interlaboratory comparison study.
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.
Biosystems and Biomaterials Division, Material Measurement Laboratory, National Institute of Standards and Technology (NIST), Gaithersburg, MD, USA.
John T
JT
Elliott
EMPA, Swiss Federal Laboratories for Material Testing and Research, Particles-Biology Interactions Laboratory, St. Gallen, Switzerland.
Matthias
M
Rösslein
Center for Nanosafety Metrology, Korea Research Institute of Standards and Science (KRISS), Daejeon, Republic of Korea.
Nam Woong
NW
Song
Statistical Engineering Division, Information Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, MD, USA.
Blaza
B
Toman
European Commission, Joint Research Centre (JRC), Institute for Health and Consumer Protection, Nanobiosciences Unit, Ispra, Italy.
Agnieszka
A
Kinsner-Ovaskainen
National Nanotechnology Center (NANOTEC), National Science and Technology Development Agency (NSTDA), Pathum Thani, Thailand.
Rawiwan
R
Maniratanachote
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.
Marc L
ML
Salit
Biosystems and Biomaterials Division, Material Measurement Laboratory, National Institute of Standards and Technology (NIST), Gaithersburg, MD, USA.
Elijah J
EJ
Petersen
Biosystems and Biomaterials Division, Material Measurement Laboratory, National Institute of Standards and Technology (NIST), Gaithersburg, MD, USA.
Fatima
F
Sequeira
Biomolecular Measurement Division, Material Measurement Laboratory, National Institute of Standards and Technology, Gaithersburg, MD, USA.
Erica L
EL
Romsos
Center for Nanosafety Metrology, Korea Research Institute of Standards and Science (KRISS), Daejeon, Republic of Korea.
Soo Jin
SJ
Kim
Center for Nanosafety Metrology, Korea Research Institute of Standards and Science (KRISS), Daejeon, Republic of Korea.
Jieun
J
Lee
Powder Technology Laboratory, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.
Nadia R
NR
von Moos
European Commission, Joint Research Centre (JRC), Institute for Health and Consumer Protection, Nanobiosciences Unit, Ispra, Italy.
François
F
Rossi
EMPA, Swiss Federal Laboratories for Material Testing and Research, Particles-Biology Interactions Laboratory, St. Gallen, Switzerland.
Cordula
C
Hirsch
EMPA, Swiss Federal Laboratories for Material Testing and Research, International Research Cooperations Manager, St. Gallen, Switzerland.
Harald F
HF
Krug
National Nanotechnology Center (NANOTEC), National Science and Technology Development Agency (NSTDA), Pathum Thani, Thailand.
Wongsakorn
W
Suchaoin
EMPA, Swiss Federal Laboratories for Material Testing and Research, Particles-Biology Interactions Laboratory, St. Gallen, Switzerland.
Peter
P
Wick
D000072283:A549 Cells; D015386:Hazardous Substances / Q000633:toxicity*; D006801:Humans; D007753:Laboratories / Q000592:standards / Q000706:statistics & numerical data*; D053758:Nanoparticles / Q000633:toxicity*; D011137:Polystyrenes / Q000633:toxicity*; D015203:Reproducibility of Results; D018675:Toxicity Tests / Q000592:standards / Q000706:statistics & numerical data*
D015386:Hazardous Substances; D011137:Polystyrenes
MTS assay; alternatives to animal testing; interlaboratory comparison; nanotoxicology; polystyrene nanoparticles