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

Significance of the Topic

Three-dimensional liver models such as HepaRG spheroids provide a more physiologically relevant environment for assessing drug distribution and toxicity compared to traditional two-dimensional cultures. High-sensitivity mass spectrometry imaging enables detailed mapping of drug uptake and metabolite localization within these spheroids, supporting improved in vitro-to-in vivo translation and reducing reliance on animal studies.

Study Objectives and Overview

• Demonstrate targeted DESI tandem quadrupole MS imaging of HepaRG spheroids dosed with known hepatotoxic drugs.
• Establish detection limits and spatial distribution of parent compounds and metabolites.
• Evaluate structural changes in spheroids at therapeutic dosing levels.

Methodology

HepaRG spheroids were cultured for seven days and dosed with perhexiline (0.004–10 µM), carbamazepine and warfarin (0.4–1000 µM). After 72 hours, spheroids were washed in PBS, spotted onto polylysine-coated slides, and dried. A dilution series for each analyte was prepared on Teflon-spotted slides to assess limits of detection.

Used Instrumentation

Analysis was performed on a Waters Xevo TQ Absolute triple quadrupole mass spectrometer equipped with a Waters DESI XS source. Key conditions:

• Flow rate: 1.5 µL/min, solvent 95% methanol
• Gas pressure: 10 PSI, capillary voltage 0.66 kV (positive), 0.6 kV (negative)
• Pixel size: 100 × 100 µm, scan rates 5 Hz (dilution) and 10 Hz (spheroids)
• Optimized MRM transitions for each compound and main metabolites

Results and Discussion

Dilution series revealed detection down to 0.4 µM for warfarin and carbamazepine, and 0.04 µM for perhexiline (approx. 9 pg on target). DESI-MS imaging of spheroids confirmed localization of parent drugs and cis-hydroxyperhexiline within the 3D structure. At higher dosing levels, spheroid integrity was compromised, indicating compound-induced cellular damage. Lipid m/z 782.6 served as an anatomical marker to overlay drug images, revealing higher perhexiline accumulation at spheroid centers. Signal subtraction of DMSO controls improved unambiguous detection at low dosing levels.

Benefits and Practical Applications of the Method

• High sensitivity imaging of drug and metabolite distribution at typical therapeutic concentrations.
• Non-destructive analysis of intact spheroids, preserving 3D spatial context.
• Visual differentiation of concentration gradients and structural changes induced by toxic exposure.
• Potential to refine early drug screening and toxicity assessment workflows.

Future Trends and Potential Applications

Future developments may include comparative imaging of sectioned versus intact spheroids, integration with untargeted metabolomic profiling to correlate drug localization with cellular responses, expansion to other 3D tissue models, and application of advanced data analytics or machine learning to automate feature extraction and high-throughput screening.

Conclusion

This study demonstrates the capability of targeted DESI-TQ imaging to map drug distribution and metabolites within HepaRG spheroids at pharmacologically relevant levels. The approach offers a powerful tool for in vitro toxicity screening, supporting mechanistic insights while adhering to the 3R principles.

References

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3. Mei Zhang et al. Journal of Chromatography B. 2009;877:3025–3030.