Drug-induced liver injury in biopsy-scaled Precision-Cut Liver Slices

Background and Aims:
Repeated consumption of drugs can lead to hepatocyte damage, and drug-induced liver injury (DILI) is one of the major safety concerns for pharma companies and clinicians. An important challenge is the lack of methods to predict adverse effects of drugs on the liver. While rodent models are used for pre-clinical tests, these cannot predict DILI risk in humans. Human precision-cut liver slices (PCLS) have emerged as a promising ex-vivo model with great potential to replicate liver functionality. PCLS retains the liver's native microarchitecture and cellular diversity while offering a simpler preparation process than other 3D culture models. However, the usual diameter of PCLS cultures is rather big (5-8 mm) so large liver resections are needed to culture human PCLS which become scarce with the current evolution of parenchyma-sparing liver surgery.
We aim to develop biopsy-scaled (1mm) PCLS cultures for the evaluation of DILI.

Method:
Healthy male BALB/c mice (Charles River) were used for baseline studies and in-house bred BALB/c-Tg(Pdgfrb-GFP) strain for live imaging. Livers of mice are excised, cut in 250um thick slices using a Leica 1200S vibratome, punched using 1/3mm biopsy punchers (Kai®), and cultured in 24/96-well plates. Samples are snap-frozen at different times/conditions. RNA extraction is performed using TRIzol®. QuantStudio™ 3 Real-Time PCR System (ThermoFisher) is used for qPCR. QuantSeq 3’ mRNA-Seq method (Lexogen) is used for RNA library and sequenced on a NovaSeq (Illumina). Whole-slice immunostaining was carried out on formalin-fixed samples. Imaging was done using a Zeiss LSM® 800 confocal and an EVOS M7000® microscope.

Results:
Comparison of hepatocyte marker expression between 1mm and 3mm PCLS over 5 days suggests that a smaller diameter better preserves hepatocyte functionality in mouse cultures. GSEA analysis of RNAseq data on day 0 and 5 slices revealed high enrichment of ECM remodeling pathways and wound healing responses on day 5. Using BALB/c-Tg(Pdgfrb-GFP) mice we confirmed hepatic stellate cell activation over time demonstrated by an increase in the GFP signal. Exposure to pro-inflammatory cytokines further increased the signal showing the responsiveness of the cultures. Finally, 1mm slices allowed paracetamol-driven liver damage observed on RNA and immunostainings. We optimized the culture of PCLS in 96-well plates which facilitated the determination of IC50 concentrations of APAP and Omeprazole. Part of our findings in mice could be replicated in human 1mm PCLS cultures.

Conclusion:
PCLS of 1mm diameter from both human and mouse liver samples demonstrated good preservation of hepatocyte functionality in culture compared to larger slices. Use of BALB/c-Tg(Pdgfrb-GFP) transgenic mice facilitates the evaluation of DILI drugs and fibrosis-inducing agents. 1 mm slices will allow for testing human liver biopsy-derived slice cultures for DILI and pro- and anti-fibrotic drugs and thus hold promise for personalized medicine approaches.