Obtaining functionally intact primary human liver sinusoidal endothelial cells (hLSECs)

Protocols published for isolation and cultivation of hLSECs encompass long-term cultivation, and often trypsinization and passaging. Moreover, experiments are generally carried out with hLSECs that have been maintained for... [ view full abstract ]

Protocols published for isolation and cultivation of hLSECs encompass long-term cultivation, and often trypsinization and passaging. Moreover, experiments are generally carried out with hLSECs that have been maintained for long periods in culture. Few if any relevant attempts have been published to verify if cultured hLSECs maintain expression of LSEC specific genes or proteins, or metabolic integrity characteristic of LSECs.

When developing a protocol for isolation of hLSECs we first noticed that the cells lost virtually all gene expression of the characteristic LSEC endocytosis receptors after only a short time (hours) in adherent culture. Although the protein expression of same receptors was retained for a longer time (a few weeks), their uptake efficacy and ability to degrade endocytosed cargo was reduced, but still higher than that of commercially available hLSECs. Based on these observations we used unpassaged early primary cultures to yield functionally intact hLSECs.        

Our protocol is based on collagenase digestion of liver resections from patients with liver metastasis, followed by centrifugation regimes to enrich for hepatocytes and hLSECs. Fc-γ receptor IIb (FcγRIIb)-immunomagnetic selection is used for purification of the hLSECs. Immunofluorescence staining and qRT-PCR on liver tissues and/or isolated hLSECs revealed that the cells are positive for specific LSEC scavenger receptors (stabilin-2, mannose receptor and FcγRIIb). Intact cellular functionality was assessed in situ and in vitro by endocytosis of fluorescently tagged and radiolabelled ligands specific for the signature LSEC scavenger receptors. The morphological LSEC hallmark, fenestration, was preserved up to one month in culture. Trypsin treatment abrogated fenestration.