Simultaneous isolation of primary human hepatocytes and non-parenchymal liver cells for the establishment of functional co-culture and tissue engineering liver models

Primary human hepatocytes (PHH) are parenchymal liver cells and represent ˜60 to 70% of total liver cells. Additionally non-parenchymal cells (NPC) like Kupffer cells (KC), Stellate cells (SC), cholangiocytes (CC) and sinusoidal endothelial cells (SEC) are present in the liver. NPC play a central role in many pathophysiologies of the liver, such as drug induced liver diseases, inflammation, liver fibrosis and cirrhosis as well as in regenerative processes. Many in vitro liver models are based on a monoculture of PHH and thus, the influence of NPC is missing. In recent years, NPC have become more relevant for the development of liver co-culture models and in tissue engineering. However, only few protocols are dealing with the isolation of NPC are available in literature. Therefore the aim of the present study was the establishment of protocols for the simultaneous isolation of NPC from human tissue in a high quality and purity.

Human liver cells were isolated from healthy liver tissue after surgical liver interventions by a two-step EDTA/collagenase perfusion technique. For the separation of parenchymal and the NPC containing fraction, the crude cell suspension was initially centrifuged at 50xg. The cell fractions were purified by a Percoll density gradient centrifugation. NPC were separated using specific adherence properties as well as magnetic activated cell sorting (MACS). Isolated cells were cultured at 37 °C for 12h in cell type specific culture medium. Subsequently the cells were identified and characterized by immunofluorescence stainings.

Our protocol allows the isolation of a high amount of PHH in a good quality. Beside PHH isolation we were able to separate the hepatic NPC. KC were obtained by their ability to adhere short term on cell culture plastics. They were identified by immunofluorescence staining for CD68 and their affinity for the phagocytosis of latex beads. This cell fraction was counted with 1,25 × 10⁶ cells/g liver and a purity of more than 80%. Endothelial cells were separated from the remaining cell suspension using MACS beads for endothelial surface protein CD31. We received a CD31 positive cell fraction with an amount of 2,5 × 10⁵ SEC/g liver and a purity of about 83%. CD31 negative cells were identified as SC using immunofluorescent staining for GFAP and Vimentin as well as their specific autofluorescence of retinol. SC were isolated with a cell count of 3,4 × 10⁵ cells/g liver and a purity of more than 70%. CC were not detectable in the isolated fractions.

In the present work we established a protocol for the simultaneous isolation of parenchymal and most non-parenchymal human liver cells. The specific identification revealed that the different cell populations were separated with a high purity. Further investigations for the characterisation, the establishment of culture conditions and the creation of functional co-culture models are in progress.

This study was supported by the virtual liver network,BMBF 0315741