Primary human hepatocyte spheroids as an in vitro tool for investigating drug compounds with low clearance

Drug-induced liver injury, including cholestasis, is an important clinical issue and economic burden for pharmaceutical industry and healthcare systems. However, human-relevant in vitro information on the ability of other types of chemicals to induce cholestatic hepatotoxicity is lacking. This work aimed at investigating the cholestatic potential of non-pharmaceutical chemicals using primary human hepatocytes cultured in 3D spheroids. Spheroid cultures were repeatedly (co-) exposed to drugs (cyclosporine-A, bosentan, macitentan) or non-pharmaceutical chemicals (paraquat, tartrazine, triclosan) and a concentrated mixture of bile acids for 4 weeks. Cell viability (adenosine triphosphate content) was checked every week and used to calculate the cholestatic index, an indicator of cholestatic liability. Microarray analysis was performed at specific time-points to verify the deregulation of genes related to cholestasis, steatosis and fibrosis. Despite the evident inter-donor variability, shorter exposures to cyclosporine-A consistently produced cholestatic index values below 0.80 with transcriptomic data partially supporting its cholestatic burden. Bosentan confirmed to be hepatotoxic, while macitentan was not toxic in the tested concentrations. Prolonged exposure to paraquat suggested fibrotic potential, while triclosan markedly deregulated genes involved in different types of hepatotoxicity. These results support the applicability of primary human hepatocyte spheroids to study hepatotoxicity of non-pharmaceutical chemicals in vitro.

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Intermittent Starvation Extends the Functional Lifetime of Primary Human Hepatocyte Cultures

Toxicological Sciences ◽

10.1093/toxsci/kfaa003 ◽

2020 ◽

Vol 174 (2) ◽

pp. 266-277

Author(s):

Matthew D Davidson ◽

Salman R Khetani

Keyword(s):

Drug Exposure ◽

Adenosine Monophosphate ◽

Human Hepatocyte ◽

Toxicity Tests ◽

Drug Induced ◽

Nonparenchymal Cells ◽

Primary Human Hepatocyte ◽

Serum Hormone

Abstract Primary human hepatocyte (PHH) cultures have become indispensable to mitigate the risk of adverse drug reactions in human patients. In contrast to dedifferentiating monocultures, coculture with nonparenchymal cells maintains PHH functions for 2–4 weeks. However, because the functional lifespan of PHHs in vivo is 200–400 days, it is desirable to further prolong PHH functions in vitro toward modeling chronic drug exposure and disease progression. Fasting has benefits on the longevity of organisms and the health of tissues such as the liver. We hypothesized that a culturing protocol that mimics dynamic fasting/starvation could activate starvation pathways and prolong PHH functional lifetime. To mimic starvation, serum and hormones were intermittently removed from the culture medium of micropatterned cocultures (MPCCs) containing PHHs organized onto collagen domains and surrounded by 3T3-J2 murine fibroblasts. A weekly 2-day starvation optimally prolonged PHH functional lifetime for 6+ weeks in MPCCs versus a decline after 3 weeks in nonstarved controls. The 2-day starvation also enhanced the functions of PHH monocultures for 2 weeks, suggesting direct effects on PHHs. In MPCCs, starvation activated 5' adenosine monophosphate-activated protein kinase (AMPK) and restricted fibroblast overgrowth onto PHH islands, thereby maintaining hepatic polarity. The effects of starvation on MPCCs were partially recapitulated by activating AMPK using metformin or growth arresting fibroblasts via mitomycin-C. Lastly, starved MPCCs demonstrated lower false positives for drug toxicity tests and higher drug-induced cytochrome-P450 activities versus nonstarved controls even after 5 weeks. In conclusion, intermittent serum/hormone starvation extends PHH functional lifetime toward enabling clinically relevant drug screening.

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