an-all-human-hepatic-culture-system-for-drug-development-applications

An All-Human Hepatic Culture System for Drug Development Applications

Finding a long-term, human-relevant culture model for primary human hepatocytes (PHHs) for pharmacological and toxicological studies remains a challenge. ...

LifeSciences, LifeNet Health

There remains a significant need for a convenient, phenotypically stable long-term culture platform for primary human hepatocytes (PHHs) for use in pharmacological and toxicological applications. Conventional in vitro models are often inconvenient, burdensome to use, and unable to support a multitude of donor lots or maintain PHH structural and functional properties over extended time. To address these limitations, an all-human cell-based hepatic tri-culture system (HTCS) has been developed comprised of frozen vials of PHHs and feeder cells. Qualified PHHs exhibited healthy morphological characteristics for ≥30 days. Extensive anastomosing networks of bile canaliculi with tight and gap junctions were established early and remained stable and functional throughout the culture period. After 5 culture days, albumin, urea, and basal Phase 1 and Phase 2 metabolic functions were stable for at least 2 weeks and significantly higher in the HTCS PHHs compared to sandwich monoculture PHHs. Induction of CYP functional activity by prototypical receptor agonists was stable after 4 days for at least 2 weeks. Gene expression of Alb and various CYPs in the HTCS PHHs was significantly higher compared to sandwich monoculture PHHs. The HTCS represents a convenient, phenotypically stable, all-human PHH culture platform for pharmacological and toxicological applications.

The morphology, functionality, and longevity of a novel all human hepatic cell-based tri-culture system.

The aryl hydrocarbon receptor (AhR) is an inducible transcription factor whose ligands include the potent environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin. Ligand activated AhR binds to DNA at Dioxin Response Elements (DREs) containing the core motif 5'-GCGTG-3'. However, AhR binding is highly tissue specific. Most DREs in accessible chromatin are not bound by TCDD-activated AhR, and DREs accessible in multiple tissues can be bound in some and unbound in others. As such, AhR functions similarly to many nuclear receptors. Given that AhR possesses a strong core motif, it is suited for a motif-centered analysis of its binding. We developed interpretable machine learning models predicting the AhR binding status of DREs in MCF-7, GM17212, and HepG2 cells, as well as primary human hepatocytes. Cross-tissue models predicting transcription factor-DNA binding generally perform poorly. However, reasons for the low performance remain unexplored. By interpreting the results of individual within-tissue models and by examining the features leading to low cross-tissue performance, we identified sequence and chromatin context patterns correlated with AhR binding. We conclude that AhR binding is driven by a complex interplay of tissue-agnostic DRE flanking DNA sequence and tissue-specific local chromatin context. Additionally, we demonstrate that interpretable machine learning models can provide novel and experimentally testable mechanistic insights into DNA binding by inducible transcription factors.

Edward L. LeCluyse

Research and Development

LifeSciences, LifeNet Health

An All-Human Hepatic Culture System for Drug Development Applications

Edward L. LeCluyse

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LifeSciences, LifeNet Health

An All-Human Hepatic Culture System for Drug Development Applications

Research and Development