Studying the adverse effects of polypharmacy on liver sinusoidal endothelial cells (LSEC) in mouse liver-derived in vitro models: advancing from 2D to 3D systems

Abstract

Polypharmacy, the concurrent use of multiple medications, is a rising phenomenon due to the aging population. In vivo studies in humans and mice show that polypharmacy reduces cognitive and physical function, which effects cannot be explained by single drugs alone. These studies suggest that the total drug load is key, but the precise source of dysfunction remains unclear. One proposed mechanism is impaired hepatic clearance, leading to drug accumulation. Liver sinusoidal endothelial cells (LSEC), which filter blood and deliver substances to hepatocytes for metabolism, are emerging as potential contributors. LSECs possess fenestrations, which are small pores facilitating transfer, and scavenger receptors that clear specific compounds by endocytosis. Disruption in either of these features impairs filtration and reduces delivery to hepatocytes, promoting systemic accumulation.

This study seeks to evaluate whether polypharmacy treatment leads to drug induced disruption of LSEC that impairs the solute exchange between the blood and hepatocytes. Primary mouse LSEC were cultured in 2D monocultures, and in 3D spheroids to better mimic in vivo conditions. Cells were treated with citalopram, oxybutynin, metoprolol, and oxycodone at serum and first-pass concentrations, both as monotherapies and as a high DBI polypharmacy cocktail. Cell viability, scavenger receptor-mediated endocytosis, and morphology, including fenestrations, were assessed.

The treatments did not affect LSEC viability, indicating that polypharmacy-related effects are not due to cell loss. Instead, both monotherapies and polypharmacy induced similar functional and structural changes, including altered endocytosis activity, cytoskeletal remodelling, and reduced fenestration frequency and porosity. These findings suggest that drug-induced defenestration and cytoskeletal changes may impair hepatic filtration and drug clearance.

The 3D spheroid model shows potential as an in vitro liver research tool. However, the technically demanding spheroid model requires extensive optimisation and hands-on training before it can be implemented as a standard method in this field. Using spheroids consisting of only hepatocytes and of hepatocytes combined with LSEC allowed for indication of the drug effect on the two different cell types. Ultimately, the aim is to extend all 2D experiments to 3D models to better reflect physiological conditions.