The validation of quality attributes in Primary Human Hepatocytes Standard.

Primary human hepatocytes (PHH) are used as the FDA-recognized "gold standard" for liver-related studies in vitro. The world's first PHH group standard (T/CSCB 0008-2021, CSCB standard) was released by Chinese Society for Cell Biology in 2021. In order to justify this standard, six key quality attributes of ten different batches from commercial PHHs, including cell viability, cell morphology, cell markers, albumin secretion, drug metabolism function and bile secretion, were characterized using the designated test methods in the standard. The PHHs from various batches all exhibited typical hepatocytic morphology, high cell viability, and sufficient albumin secretion; whereas, tremendous variations in cell markers, drug metabolism functions, and bile secretion were unexpectedly detected across the board. Flow cytometric assessment of hepatocyte markers revealed the percentages of ALB+ or HNF4A+cells in six batches of PHHs, ranging from 49.4% to 98.9% and from 37.7% to 91.4%, respectively. Single cell transcriptomic analysis also revealed significant cell heterogeneity across the different batches, with the proportions of hepatocytes ranging from 69.2% to 98.9%. Considerable heterogeneity in drug metabolism functions across the batches were also found in substrate clearance rate (SCR) and metabolite formation rate (MFR) for six representative CYP450 enzymes, while the results didn't influence current SCR attribute of CYP3A4. Metabolic capacity and purity are two independent attributes for PHH. The varied biliary excretion indexes around criteria (30%) indicated heterogeneity of PHH biliary excretion capacity. These results confirmed the robustness of most quality attributes in current CSCB standard, while highlighting the need to refine remaining parameters to enhance its practical applicability.