Co-Culture of Liver-Derived Mesenchymal Stromal Cells and Intrahepatic Cholangiocyte Organoids in an Extracellular Matrix Environment: a Step Towards Complex Liver Models

Abstract

To this day, liver transplantation is the only effective treatment for end-stage liver failure. Unfortunately, the scarcity of liver donors leads to waitlist mortality, pushing the need for alternative treatment options. In vitro models are essential tools in research on alternatives for transplantation. One of the promising models is the hepatobiliary organoid model because these three-dimensional cultures model (elements of) the native tissue structure and function. However, a limitation of these models is that they currently resemble only one liver cell type. The aim of this research was therefore to combine different cell types, i.e., biliary organoids and mesenchymal stromal cells (MSCs), in one culture model.

The organoids were obtained from liver-biopsy-derived intrahepatic biliary epithelium and are therefore named intrahepatic cholangiocyte organoids (ICOs). The liver-specific MSCs (L-MSCs) were isolated from perfusion fluid of donor livers collected at liver transplantation procedures. Decellularized liver tissue was used to include extracellular matrix (ECM) in the co-culture models. To assess the most optimal culture conditions, three experimental setups were tested. In the first setup, ICOs were cultured in different concentrations of L-MSC derived conditioned medium (CM) in commercially available basement membrane extract (BME) (1A). This setup was also used to differentiated the ICOs towards hepatocytes (1B). In the second setup, various concentrations of ICOs and L-MSCs were cultured in an indirect (2A) and direct (2B) BME model. The third setup included liver-derived ECM to replace the BME. Cells were cultured similar to model 2B (3A) and ICOs were added after a pre-culture of L-MSCs (3B). The morphology of both cell types, the visible interaction between cell types, and the expression of hepatocyte, cholangiocyte, MSC and proliferation markers were analyzed.

The effect of L-MSCs and L-MSC-derived CM on ICO formation, morphology, and growth patterns, was small. Direct cell-cell contact was rare in the BME co-cultures, but instances of close contact between two cell types were observed in both the indirect (2A) and direct (2B) setups. In the ECM models (3A-B), ICO cells formed polarized monolayers that pushed the L-MSCs aside, indicating that the cell types did not mix well. ICOs expressed the cholangiocyte markers cytokeratin (CK) 7 and 19 in both mono- and co-cultures, whereas L-MSCs were most likely CK7 and CK19 negative. Variation in gene expression levels of hepatocyte, cholangiocyte, MSC, and proliferation markers was observed, but no statistically significant differences were found between conditions. Nevertheless, the down-regulatory effect of CM on the expression of hepatocyte markers in differentiated ICOs showed a consistent trend. Additionally, the presence of L-MSCs resulted in a higher mean expression of MSC markers CD90, CD105 and Vimentin, and proliferation marker KI-67.

Although no clear effects of L-MSCs on ICO growth were observed in this study, the created co-culture models form a promising base for future research. The models include adult human liver-derived components and are therefore a step towards the reconstruction of the hepatic microenvironment. In addition, if other (liver-derived) cell types were to be introduced to the co-culture models, L-MSCs have the potential to enhance the cell function of these new cells. Last, the models can be used as a base for future ICO co-culture studies, since the L-MSCs can be replaced by other cell types.