Elliott John T, Rösslein Matthias, Song Nam Woong, Toman Blaza, Kinsner-Ovaskainen Agnieszka, Maniratanachote Rawiwan, Salit Marc L, Petersen Elijah J, Sequeira Fatima, Romsos Erica L, Kim Soo Jin, Lee Jieun, von Moos Nadia R, Rossi François, Hirsch Cordula, Krug Harald F, Suchaoin Wongsakorn, Wick Peter
Biosystems and Biomaterials Division, Material Measurement Laboratory, National Institute of Standards and Technology (NIST), Gaithersburg, MD, USA.
EMPA, Swiss Federal Laboratories for Material Testing and Research, Particles-Biology Interactions Laboratory, St. Gallen, Switzerland.
ALTEX. 2017;34(2):201-218. doi: 10.14573/altex.1605021. Epub 2016 Sep 29.
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.
开发可靠的基于细胞的纳米毒理学检测方法对于评估潜在危险的工程纳米材料非常重要。制定可靠的检测方案面临的挑战包括处理纳米颗粒分散液和活细胞系，以及存在纳米相关干扰效应的可能性。在此，我们展示了一种96孔板设计的应用，该设计带有多个测量对照，以及一项涉及五个实验室的实验室间比较研究，以表征基于A549细胞系的纳米细胞毒性MTS细胞活力检测方法的稳健性。对于带正电荷的聚苯乙烯纳米颗粒，在无血清和有血清条件下的共识半数有效浓度（EC50）值分别为22.1 mg/L（95%置信区间为16.9 mg/L至27.2 mg/L）和52.6 mg/L（44.1 mg/L至62.6 mg/L）；对于阳性化学对照硫酸镉，在无血清和有血清条件下的EC50值分别为49.7 μmol/L（47.5 μmol/L至51.5 μmol/L）和77.0 μmol/L（5 ,4.3 μmol/L至99.4 μmol/L）。测量对照的结果可用于评估实验室内部和实验室之间变异性的来源及其相对大小。这些信息揭示了可能需要修改的检测方案步骤，以提高整体稳健性和精度。结果表明，诸如细胞系标识、培养基更换、细胞处理和纳米颗粒分散等检测方案细节对于确保纳米细胞毒性检测方案的稳健性和检测结果的可比性至关重要。系统对照测量和实验室间比较数据的结合产生了单独采用任何一种方法都无法获得的见解。
