Toward achieving harmonization in a nanocytotoxicity assay measurement through an interlaboratory comparison study

Abstract

SummaryDevelopment 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 EC 50 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.

Impact of nanoparticle surface functionalization on the protein corona and cellular adhesion, uptake and transport

“…The deposited fraction of the administered doses of the PSNPs (target cell dose) was calculated using the In vitro Sedimentation, Diffusion and Dosimetry (ISDD) model [ 22 ]. The following parameters were used as input in the ISDD model: the hydrodynamic diameters of the PSNPs in water and DMEM + measured by DLS (Table 1 ), medium column height (10.9 mm), temperature (310°K), media density 1 g/ml and media viscosity 0.0009 N s/m 2 [ 23 , 24 ].…”

Nanomaterials and Oxidative Stress

“…Another common approach is the validation of the assay output with an orthogonal method. Still, a significant variability in the NP test results between laboratories using the equivalent method and similar materials often hamper data comparison [31]. Fortunately, the development of standardized protocols and reference nanomaterials by standards organizations is addressing this critical need [32].…”

Counting Caenorhabditis elegans: Protocol Optimization and Applications for Population Growth and Toxicity Studies in Liquid Medium

“…Sensitivity testing was then used to assess which sources of variability have significant impacts on the overall process. Cause-and-effect diagrams in combination with sensitivity analysis facilitate the adoption of appropriate process controls/measurements to ensure that the processes are performing as expected 13 , 45 .…”

“…In many reports, counts of live C. elegans in liquid cultures are often roughly approximated or not listed. The number of C. elegans in an exposure system can impact biological or toxicological responses; the number of cells in culture, for example, can have a substantial impact on nanotoxicity results and EC 50 values 13 , 14 . It has been reported that the number and stage of live nematodes used during experimentation, including culture expansion and toxicant exposure, is a critical factor in toxicological measurements 1 .…”