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Dosing Methods to Enable Cell-Based In Vitro Testing of Complex Substances: A Case Study with a PAH Mixture
Alexandra C Cordova1,2, Lucie C Ford1,2, Alan Valdiviezo1,2
1Interdisciplinary Faculty of Toxicology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843, USA.
Toxics
|January 20, 2023
Summary
Testing complex mixtures in cell-based assays is challenging. The media accommodated fraction (MAF) method effectively delivers polycyclic aromatic hydrocarbons (PAHs) for in vitro toxicity testing, improving accuracy.
Area of Science:
- Toxicology
- In vitro methods
- Chemical analysis
Background:
- Cell-based testing of complex mixtures is hindered by challenges in substance delivery and representation of original composition.
- Existing in vitro methods often use non-biocompatible solvents or fail to accurately reflect the test article's characteristics.
- Traditional aquatic toxicity dosing methods are unsuitable for small-volume mammalian cell-based in vitro studies.
Purpose of the Study:
- To evaluate scaled-down dosing methods for high-throughput in vitro testing of complex mixtures.
- To compare passive dosing via silicone micro-O-rings, cell culture media-accommodated fraction (MAF), and traditional solvent extraction.
- To determine the most suitable method for delivering polycyclic aromatic hydrocarbons (PAHs) in mammalian cell-based assays.
Main Methods:
- Comparison of three dosing methods: passive dosing (silicone micro-O-rings), media accommodated fraction (MAF), and dimethyl sulfoxide (DMSO) solvent extraction.
- Gas chromatography-tandem mass spectrometry (GC-MS/MS) for analyzing PAH kinetics, recovery, and protein binding.
- In vitro cytotoxicity assays using EA.hy926 and HepG2 human cell lines to assess bioavailability.
Main Results:
- The media accommodated fraction (MAF) method allowed the highest delivery of starting polycyclic aromatic hydrocarbons (PAHs) into cell culture media.
- Analysis confirmed varying kinetics of PAH absorption to micro-O-rings and differential protein binding across methods.
- Cytotoxicity assays demonstrated differences in bioavailability depending on the dosing method employed.
Conclusions:
- The media accommodated fraction (MAF) is the most appropriate method for cell-based in vitro testing of complex substances and mixtures, particularly those containing polycyclic aromatic hydrocarbons (PAHs).
- MAF enables effective concentration-response testing by maximizing the delivery of test substances into cell culture media.
- This method addresses limitations of traditional approaches, facilitating more accurate in vitro toxicity assessments.
