In vitro evaluation of magnetite nanoparticles in human mesenchymal stem cells: comparison of different cytotoxicity assays

Abstract

This work was aimed at defining the suitable test for evaluating Fe₃O₄ NPs cytotoxicity after short-term exposure in human mesenchymal stem cells (hMSCs) using different viability tests, namely NRU, MTT and TB assays, paralleled by cell morphology analyses for cross checking. MTT and NRU data (culture medium with/without hMSCs plus Fe₃O₄NPs) indicated artificial/false increments in cell viability after Fe₃O₄NPs. These observations did not fit with the morphological analyses showing reduced cell density, loss of monolayer features, and morphological alterations at Fe₃O₄NPs ≥50 μg/ml. Fe₃O₄NPs alone induced a substantial increased absorbance at the wavelength required for MTT and NRU. A significant death (25%) of hMSC at Fe₃O₄NPs ≥10 μg/ml, with a maximum effect (45%) at 300 μg/ml after 24 h, exacerbated after 48 h, was observed when applying TB test. These results paralleled the effects on cell morphology. The optical properties and stability of Fe₃O₄NP suspension (tendency to agglomerate in a specific culture medium) represent factors that limit in vitro result interpretation. These findings suggest the non applicability of the spectrophotometric assays for hMSC culture conditions, while TB is an accurate method for determining cell viability after Fe₃O₄NP exposure in this model. In relation to NPs safety assessment: cell-based assays must be considered on case-by-case basis; selection of relevant cell models is also important for predictive toxicological studies; application of a testing strategy is fundamental for understanding the toxicity pathways driving cellular responses.

Keywords:

• Fe₃O₄ nanoparticles
• environmental and occupational exposure
• safety assessment
• cell-based assay
• toxicity-testing strategies
• human in vitro cell model

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by grant from the Italian Ministries of Health, Research and Education.