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CONCLUSION In conclusion, we developed a technique which causes primary human LSECs and hepatocytes to proliferate up to 40 population doublings whilst still retaining an adult phenotype when cultured at confluence. upcyte® LSECs retained important endothelial cell markers, such as CD31 and vWF, and exhibited functional uptake of LDL, as well as the ability to form tubes in MatrigelTM. LSEC-specific uptake of ligands or the expression of the corresponding uptake receptors (MR, FcɣR2BII and HA/S-R) could be detected. Upcyte® hepatocytes retained typical hepatic markers such as HSA and formation of bile canaliculi. At confluence, phase I and II enzymes were detected and showed donor-specific differences. These data support that upcyte® LSECs & hepatocytes are very uniquely and applicable to cell based assays, such as as co-culture and toxicity studies. Moreover, this technology allows for the generation of large batches of upcyte® cells (up to 12 x 109 cells per donor), enabling a reproducible and standardized experimental setting. INTRODUCTION 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).