Broad assessment of primary hepatocyte donors for use in MPS and in vitro assays to model human liver disease

Primary human hepatocytes (PHHs) remain the gold standard for in vitro liver research and drug development, with widespread use across the industry. One key benefit of PHH use in research is the ability to explore the role of patient variability. However, differences in tissue quality and PHH isolation can contribute to data variance and a lack of reproducibility. In this study we aimed to characterise a broad range of PHH donors in both 2D and 3D platforms to identify high quality PHH sources, for use in translational microphysiological systems (MPS). This research would explore whether 2D cell culture can predict PHH performance in MPS, with a particular focus on insulin sensitivity, a key feature of metabolic disease modelling.

PHHs isolated from 23 human donors (from four commercial vendors) were assessed for ability to culture in both 2D, and in a 3D liver MPS platform. Two key parameters were assessed: hepatocyte functionality, through biomarkers albumin and cytochrome P450; and hepatocyte insulin sensitivity, a key marker for metabolic disease modelling. PHHs were assayed after 4 days in 2D culture, and 8 days in Liver MPS for their response to 3 nM insulin challenge, with the production of glucose and phospho-Akt measured.

Of the 23 donors tested, 44% were found to successfully culture in the liver MPS platform with >10 µg/day/million cells of albumin at Day 8; and high levels (>0.5 pmol/min/million cells) of CYP3A4 activity at Day 8. 48% of PHH donors showed insulin sensitivity in the 3D Liver MPS, but importantly the same donors that were insulin sensitive in 2D were not sensitive in the liver MPS, with 2 donors found to be non-responsive to insulin challenge in 2D but significantly responsive in the 3D MPS platform. Additionally, the dynamic range of responses to insulin challenge was significantly greater between the donors in the MPS compared with the cells in 2D.

This work demonstrates that detailed qualification and testing of primary cells is critical in understanding how in vitro studies will perform, allowing establishment of robust disease models such as MASLD/MASH or viral hepatitis. Given the observed differences in results, with regards to PHH donor validation, between 2D culture and 3D Liver MPS, it is important to assay key functions in the context in which they will be used, as PHH that perform well in 2D may not always perform equivalently in 3D.