Generation of proliferating human liver sinusoidal endothelial cells

Isolated liver cells, such as hepatocytes, liver sinusoidal endothelial cells (LSECs), Kupffer Cells and hepatic stellate cells, are frequently used to study hepatic metabolism, toxicity and diseases. The current in vitro culture models, however, have several disadvantages e.g. short culture longevity and artificial culture conditions that focus mainly on a single cell type in 2D culture. The use of primary cells in vitro is compromised by the limited quantity of cells that can be isolated from one donor, a lack of or very restricted proliferation capacity (hepatocytes and LSECs) and/or the change from a quiescent to an activated state (hepatic stellate cells).

LSECs are highly specialized endothelial cells lining the walls of hepatic sinusoids. Their key roles include the transfer of substrates between blood and liver parenchyma, rapid internalization of blood-borne macromolecules, liver regeneration and immune tolerance. Despite their substantial contribution to liver homeostasis, LSECs are often overlooked during hepatotoxicity assays, due to insufficient cell numbers after isolation and a limited proliferation capacity in vitro. How can we bypass the lack of proliferation?

To address this issue, we employed our previously described upcyte® technology for expansion of primary LSECs by lentiviral transduction of proliferation inducing genes. We were able to significantly enhance the proliferation of primary LSECs up to 35 additional population doublings compared to naïve LSECs. Generated upcyte® LSECs expressed typical endothelial cell markers (CD31, van-Willebrandt-factor), expressed several LSEC-specific receptors (mannose receptor, LYVE-1 and L-SIGN) and showed uptake of several macromolecule ligands (e.g. serum albumin, aggregated IgG).

Since LSECs are involved in drug-induced liver injury, we evaluated and characterized mono and co-culture with hepatocytes in regard to finding the appropriate medium, morphology, functionality and stability. Further we did a comparison of 2D and 3D cultivation to see if we can further enhance characteristics such as CYP function and the formation of bile canaliculi.

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 screenings under reproducible and standardized conditions.