Standard Protocols for Characterising Primary and In Vitro-Generated Human Hepatocytes

Hepatocyte-like cells (HLCs) derived from pluripotent stem cells (PSCs) or direct reprogramming are an unlimited source of human hepatocytes for biomedical applications. HLCs are used to model human diseases, develop precise drugs and establish groundbreaking regenerative cell-based therapies. Primary human hepatocytes are the gold standard for studying human liver biology and pathology. However, their widespread use is limited by their rapid dedifferentiation in vitro, reliance on transplant-rejected donor organs, poor scalability and significant batch-to-batch variations. Therefore, high-quality 'off-the-shelf' HLCs are needed to overcome those limitations. Basic stepwise differentiation protocols have been developed to generate HLCs from PSCs. To evaluate the quality of the in vitro generated products, HLCs have been phenotyped using various methods. This review discusses various biological assays and methods available for the robust evaluation of HLC quality, emphasising the importance of using 24-h cultured primary human hepatocytes (PHHs) as a reference standard for comparison.