TOWARDS IN-VITRO DIAGNOSTIC IMAGING OF HEPA-RG SPHEROIDS BY DESI TANDEM QUADRUPOLE MS

Significance of the topic

In vitro models that closely mimic human organ function are essential for early drug development and toxicity screening. HepaRG spheroids represent a three-dimensional liver model that better reflects the microenvironment and metabolic activity of human liver tissue. Imaging these spheroids with high sensitivity supports assessment of drug distribution, uptake, and potential hepatotoxic effects without relying on animal studies.

Objectives and study overview

This work aims to demonstrate the capability of desorption electrospray ionization tandem quadrupole mass spectrometry (DESI-MS/MS) for targeted imaging of drug compounds and their metabolites in intact HepaRG spheroids. Three model hepatotoxic drugs—perhexiline, carbamazepine and warfarin—were dosed at therapeutically relevant concentrations to evaluate detection limits, spatial distribution and spheroid integrity.

Methodology and instrumentation

HepaRG spheroids were cultured for seven days and dosed with either perhexiline (10–0.004 μM), carbamazepine (1000–0.4 μM) or warfarin (1000–0.4 μM), using 0.5% DMSO vehicle. After a 72-hour incubation, spheroids were washed in PBS, spotted onto polylysine-coated glass slides and dried under vacuum.

DESI imaging was performed on a Waters Xevo TQ Absolute triple quadrupole MS equipped with a Waters DESI XS source and high-performance sprayer. Optimized parameters included:

• Solvent flow rate 1.5 μL/min (95% methanol)
• Gas pressure 10 psi, capillary voltage 0.66 kV (positive), 0.60 kV (negative)
• Pixel size 100×100 μm, scan rate 5 Hz for dilution series, 10 Hz for spheroid imaging

Multiple reaction monitoring (MRM) transitions for parent drugs and key metabolites were selected based on literature and preliminary MS/MS experiments.

Key results and discussion

Dilution series indicated detection limits of 0.04 μM for perhexiline, 0.4 μM for warfarin and carbamazepine, corresponding to low-picogram quantities loaded per spot. DESI images revealed successful localization of all three compounds within spheroids, with clear contrast against blank PBS and DMSO controls when signal from vehicle was subtracted. At high dosing, spheroids displayed signs of structural deformation, consistent with known hepatotoxic effects. Perhexiline showed elevated signal in the spheroid core, suggesting differential penetration or accumulation. No significant epoxide metabolite signal was observed for carbamazepine under these conditions.

Benefits and practical applications

Targeted DESI-MS imaging enables:

• Mapping of drug distribution and relative concentration within intact 3D liver models
• Detection of parent compounds and selected metabolites at therapeutically relevant levels
• Non-destructive analysis of spheroid integrity and drug-induced morphological changes

This approach supports safer candidate selection and reduces reliance on animal dosing by providing spatially resolved chemical information in human cell-based models.

Future trends and potential applications

Further developments may include:

• Comparison of sectioned versus whole spheroid imaging to enhance depth profiling
• Expansion to full metabolomic profiling for discovery of novel biomarkers of toxicity
• Integration with high-resolution optical microscopy for correlated structural and chemical mapping
• Application to other 3D tissue models such as organoids from diverse organ systems

Conclusion

DESI-MS/MS imaging with a DESI XS source and Xevo TQ Absolute demonstrates high sensitivity and spatial resolution for mapping drug compounds in HepaRG spheroids. The method detects low-micromolar dosing levels and provides insight into compound distribution and spheroid response, offering a valuable tool for in vitro safety assessment and drug development workflows.

Reference

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