VALIDATION OF A CYTOTOXICITY ASSAY USING ADULT HUMAN MESENCHYMAL STEM CELLS AS AN ALTERNATIVE TO THE USE OF ANIMALS

Abstract

<h3>Background/Objective</h3> Russell and Burch, in 1959, made the first mention of the 3R‘s strategy involving animals in research: Reduce, Replace or Refine. In vitro methods with stem cells are being developed to predict toxic drug potential as an alternative to animal use. Based on the assumption that a substance can disrupt the differentiation capacity, we investigated the ability of mesenchymal stem cells derived from adipose tissue (ADSCs) to predict drug toxicity, allowing the initial dose estimation the acute toxic class (ATC) method. <h3>Methods</h3> We evaluated ten chemicals representing the categories of toxicity 1-5 and unclassified toxicity of the Global Harmonized Chemical Classification and Labeling System (GHS) and determined the dose-response curves using the Hill function with a R2≥0.85. The resulting IC50 (half-maximal inhibitory concentration of a chemical) values were used to predict LD50 (amount of a chemical which causes the death of 50% of a group of test animals) and estimate the ATC method‘s initial dose. Hybrid microplate reader (HMR) and High-content imaging microscope (HCI) were tested. HMR reading was optimized using the drug Sodium Dodecyl Sulfate (SDS) as control. <h3>Results</h3> Our results indicated that using the adipogenesis inhibition assay to estimate the initial dose for the ATC method would decrease the use of animals for 7 out of 10 tested substances, including all the most toxic substances (GHS 1-3 categories). We observed lower IC50 intensity and differentiation area parameters compared to nuclei quantification. Additionally, concentrations considered non-toxic inhibited adipogenesis, indicating that adipogenesis evaluation may be a sensitive method to evaluate in vitro toxicity. Regarding the standardization of reading in HMR, the ADSCs responded in a dose-dependent way to the SDS to evaluate adipogenesis. Both IC50 and LD50 were reproducible within the expected toxicity range, enabling reading in 3 × 3 fields (reading faster and less bulky). <h3>Conclusion</h3> These results show that the proposed method may be a complementary alternative assay to assess in vitro toxicity.