In April 2018, the International Standardization Organization (ISO) published standard ISO 19007:2018, “Nanotechnologies — In vitro MTS assay for measuring the cytotoxic effect of nanoparticles.” The standard specifies a method for evaluating the effects of nano-objects and their aggregates and agglomerates on cellular viability using the MTS assay. The assay design includes performance requirements and control experiments to identify and manage variability in the assay results. In general, according to ISO, changes in absorption intensity are directly proportional to cell number although assay conditions that alter reductase activity or reagent availability can result in colourmetric changes that are not directly due to changes in cell viability (i.e., cell number). ISO states that the MTS reagents are directly added to cell culture well, allowing for rapid evaluation of potential intrinsic toxicity of nanoparticles. Due to the potential interference effects that can occur with nanoparticles and colourmetric assays, ISO notes that it is important control experiments with the nanoparticles and the MTS reagents are performed before the assay results are accepted. Direct microscopic observation of cells after treatment also provides an orthogonal method to validate an MTS assay result. According to ISO, the normalized protocol presented here is limited to adherent cell types, but it could be modified to be used with suspension cells.
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