Expanded primary human liver sinusoidal endothelial cells as a tool for complex hepatotoxicity studies

Liver sinusoidal endothelial cells (LSECs) are highly specialized endothelial cells lining the walls of hepatic sinusoids. Their key roles include liver regeneration, the transfer of substrates between blood and liver parenchyma, rapid internalization of blood-borne macromolecules as well as immune tolerance. Despite their substantial contribution to liver homeostasis, LSECs are often overlooked during hepatotoxicity assays due to insufficient cell yields after isolation and a restricted proliferation capacity in vitro.

To address these issues, we expanded primary LSECs derived from 3 donors by lentiviral transduction with proliferation inducing genes (upcyte® technology). Transduced LSECs performed 28 – 45 population doublings in a donor-dependent manner until senescence occurred. Generated upcyte LSECs expressed typical endothelial cell markers (CD31, von Willebrand factor) and showed marked binding of UEA-1 (Ulex Europaeus Agglutinin I). In addition, we found expression of several LSEC-associated receptors including MMR (mannose receptor), LYVE-1 (lymphatic vessel endothelial hyaluronan receptor 1) and FCGR2B (inhibitory receptor for the Fc region of immunoglobulin gamma). Expanded LSECs further revealed marked uptake of macromolecule ligands (ovalbumin, acetylated low density lipoprotein) and were capable of tube formation when cultured in Matrigel.

Since LSECs are involved in drug-induced liver injury, we challenged the cells with several hepatotoxic model compounds. Interestingly, upcyte LSECs were more susceptible to e.g. acetaminophen and imipramine-induced toxicity when compared to upcyte hepatocytes, indicating that these cells constitute a useful tool to complement hepatotoxicity evaluation.

Taken together, our data suggest that upcyte LSECs combine many characteristics of primary LSECs with the advantage of an extended lifespan, facilitating their use in hepatotoxicity assays under reproducible and standardized conditions. Future applications include e.g. in vitro uptake assays of ADCs (antibody drug conjugates) or triggering the hepatic immune response via the inclusion of T-cells and their antigen presenting capabilities to the LSECs.