Phenotypic Characterization of Liver Sinusoidal Endothelial Cells on the Human Liver-Chip for Potential in vitro Therapeutic Antibody Pharmacology Applications

Abstract

AbstractLiver plays a vital role in the human immune system, in the internalization and catabolic clearance of therapeutic antibodies and antibody-bound immune complexes via Fc-receptor (FcR) binding on the hepatic reticuloendothelial system cells. This Fc portion of the antibody binding to FcR in the liver initiates the clearance of these antibodies or immune complexes, which is vital in the context of half-life, dosing interval, efficacy, and safety of therapeutic antibodies. The liver sinusoidal endothelial cells (LSECs) express scavenging receptors that recognize, bind, and internalize an enormous diversity of extracellular ligands. The Fc gamma receptor FcγRIIB or CD32B on LSECs is responsible for the clearance of a large majority of IgG-bound immune complexes in the liver. Investigating the pharmacological effects of antibody clearance via human liver in vitro has been challenging due to the lack of reliable long-term LSEC culture protocols. Human LSECs downregulate the expression of CD32B rapidly in vitro in traditional 2D LSEC mono- and co-cultures,. We describe a Liver-Chip model with a co-culture of primary human LSECs and hepatocytes to recreate the liver microenvironment and extend the viability and function of LSECs, including CD32B expression levels, for a duration that is relevant for assessing the pharmacokinetics (PK) of therapeutic antibodies. Our results show that the expression of CD32B can differ based on experimental variables such as the source of primary cells (donor), passage number or source of detection antibodies used to visualize CD32B and shear stress. The CD32B expression was maintained for 14 days on the Liver-Chip in a donor-dependent but passage number independent manner. The Scanning Electron Microscopy (SEM) imaging showed the presence of fenestrae structures - one of the hallmarks of LSEC function. Key LSEC markers, including CD32B expression, were validated through flow cytometry.