6533b822fe1ef96bd127d6b1

RESEARCH PRODUCT

Three-Dimensional Spheroid Primary Human Hepatocytes in Monoculture and Coculture with Nonparenchymal Cells

subject

description

Recent advances in the development of various culture platforms are promising for achieving more physiologically relevant in vitro hepatic models using primary human hepatocytes (PHHs). Previous studies have shown the value of PHHs three-dimensional (3D) spheroid models, cultured in low cell number (1330-2000 cells/3D spheroid), to study long-term liver function as well as pharmacological drug effects and toxicity. In this study, we report that only plateable PHHs aggregate and form compact 3D spheroids with a success rate of 79%, and 96% reproducibility. Out of 3D spheroid forming PHH lots, 65% were considered stable (<50% ATP decrease) over the subsequent 14 days of culture, with reproducibility of a given PHH lot being 82%. We also report successful coculturing of PHHs with human liver nonparenchymal cells (NPCs). Crude P1c-NPC fractions were obtained by low centrifugation of the PHH supernatant fraction followed by a few days of culture before harvesting and cryopreservation. At aggregation of PHHs/P1c-NPCs (2:1 ratio 3D spheroids), liver sinusoidal endothelial cells, Kupffer cells, and hepatic stellate cells were successfully integrated and remained present throughout the subsequent 14-day culture period as revealed by mRNA expression markers and immunostaining. Increased mRNA expression of albumin (ALB), apolipoprotein B (APOB), cytochrome P450 3A4 (CYP3A4), and increased albumin secretion compared to PHH 3D spheroid monocultures highlighted that in a 3D spheroid coculture, configuration with NPCs, PHH functionality is increased. We thus achieved the development of a more integrated coculture model system requiring low cell numbers, of particular interest due to the scarcity of human liver NPCs.