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

Abstract

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 P1-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/P1-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.

Human Liver Spheroids as a Model to Study Aetiology and Treatment of Hepatic Fibrosis

“…Cryopreserved PHH and crude NPCs were obtained from Bioreclamation IVT (BioIVT, NY, USA), KaLy-Cell (KLC, Plobsheim, France), and Lonza (Basel, Switzerland). PHH were seeded, as previously described [18,33], into Corning 96-well ultra-low attachment plates with or without the addition of NPCs seeded at a 4:1 ratio of PHH:NPC (1500:375). Initial titration experiments, at ratios ranging from 2:1 to 6:1 using fixed numbers of PHH, were conducted.…”

“…Historically, in vitro liver cultures lacked hetero-cellular interactions, however, co-cultures improve hepatocyte functionality and allow the study of PHH-NPC interactions [27][28][29]. Furthermore, bio-printed or spheroid co-cultures which modulate extracellular matrix [30,31] and inflammatory responses [32,33] have provided promising proof-of-principle for modelling NAFLD but currently lack critical high-throughput compatibility. In this study, a high-throughput screening (HTS)-compatible human liver spheroid system was established, using commercially available donor PHH and NPCs, and applied to model the fibrotic aspects of NASH.…”

“…In this study, a high-throughput screening (HTS)-compatible human liver spheroid system was established, using commercially available donor PHH and NPCs, and applied to model the fibrotic aspects of NASH. This PHH spheroid system has previously been shown to mimic the human liver in vivo with respect to proteomics [18], transcriptomics, and metabolomics [19], with the feasibility of co-cultures previously demonstrated [32,33]. We characterized hetero-cellular spheroids from eight different donors with matched and non-matched, ratio controlled NPCs, and interrogated their use in modelling fibrotic phenotypes and applications for anti-NASH compound screening.…”

3D Primary Hepatocyte Culture Systems for Analyses of Liver Diseases, Drug Metabolism, and Toxicity: Emerging Culture Paradigms and Applications

“…Importantly, the spheroid culture paradigm supports the coculture of hepatocytes with other nonparenchymal liver cells, such as Kupffer cells (KCs), liver sinusoidal endothelial cells (LSECs), and hepatic stellate cells (HSCs). Coculture of hepatocytes with nonparenchymal cells (NPCs) in the spheroids has been shown to be stable for many weeks and has been suggested to further support hepatic functionality of PHH in spheroid cultures . Hepatic cocultures open up possibilities to study aspects of liver biology that require the interplay between the different hepatic cell types.…”

“…Coculture of hepatocytes with nonparenchymal cells (NPCs) in the spheroids has been shown to be stable for many weeks [9] and has been suggested to further support hepatic functionality of PHH in spheroid cultures. [12] Hepatic cocultures open up possibilities to study aspects of liver biology that require the interplay between the different hepatic cell types. Combined, the presented studies demonstrate that the spheroid method constitutes a versatile experimental paradigm that supports the long-term culture of PHH and other hepatic cell types with physiologically relevant molecular phenotypes.…”