Cytotoxicity evaluation using cryopreserved primary human hepatocytes in various culture formats

Richert, Lysiane; Baze, Audrey; Parmentier, Céline; Gerets, Helga; Sison-Young, Rowena; M, Dorau; Lovatt, Cerys A.; Czich, Andreas; Goldring, Christopher E.; Park, B. Kevin; Juhila, Satu; Foster, Alison J.; Williams, Dominic P.

doi:10.1016/j.toxlet.2016.06.1127

Cited by 23 publications

(15 citation statements)

References 22 publications

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“…Previous studies have reported only minor inter-individual differences in PHH 2D monolayer cultures when using EC 50 values as readout ( Sison-Young et al ., 2017 ) and, in agreement with these findings, we did not detect significant differences between donors for any of the compounds tested. When applying a daily treatment protocol and using the highest non-cytotoxic concentration as readout, inter-donor variability was previously seen with APAP and diclofenac after short-term (72 h) treatment of PHH in 2Dsw ( Richert et al , 2016 ). This was not seen in the present study after long-term treatment, most probably due the gradual loss of CYP activities that play a role in the cytotoxicity of both compounds ( Figure 4 ).…”

Section: Discussionmentioning

confidence: 90%

Comparison of Hepatic 2D Sandwich Cultures and 3D Spheroids for Long-term Toxicity Applications: A Multicenter Study

Bell

Dankers

Lauschke

et al. 2018

Toxicological Sciences

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Primary human hepatocytes (PHHs) are commonly used for in vitro studies of drug-induced liver injury. However, when cultured as 2D monolayers, PHH lose crucial hepatic functions within hours. This dedifferentiation can be ameliorated when PHHs are cultured in sandwich configuration (2Dsw), particularly when cultures are regularly re-overlaid with extracellular matrix, or as 3D spheroids. In this study, the 6 participating laboratories evaluated the robustness of these 2 model systems made from cryopreserved PHH from the same donors considering both inter-donor and inter-laboratory variability and compared their suitability for use in repeated-dose toxicity studies using 5 different hepatotoxins with different toxicity mechanisms. We found that expression levels of proteins involved in drug absorption, distribution, metabolism, and excretion, as well as catalytic activities of 5 different CYPs, were significantly higher in 3D spheroid cultures, potentially affecting the exposure of the cells to drugs and their metabolites. Furthermore, global proteomic analyses revealed that PHH in 3D spheroid configuration were temporally stable whereas proteomes from the same donors in 2Dsw cultures showed substantial alterations in protein expression patterns over the 14 days in culture. Overall, spheroid cultures were more sensitive to the hepatotoxic compounds investigated, particularly upon long-term exposures, across testing sites with little inter-laboratory or inter-donor variability. The data presented here suggest that repeated-dosing regimens improve the predictivity of in vitro toxicity assays, and that PHH spheroids provide a sensitive and robust system for long-term mechanistic studies of drug-induced hepatotoxicity, whereas the 2Dsw system has a more dedifferentiated phenotype and lower sensitivity to detect hepatotoxicity.

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Section: Discussionmentioning

confidence: 90%

Comparison of Hepatic 2D Sandwich Cultures and 3D Spheroids for Long-term Toxicity Applications: A Multicenter Study

Bell

Dankers

Lauschke

et al. 2018

Toxicological Sciences

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241

203

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“…However, it should be noted that the predictive power of long term hepatocytes cultures is still in question. Richert et al postulated that exposure periods longer than 72 hours in human hepatocytes do not increase the DILI‐prediction rate compared with shorter exposure times. Contrary to this Proctor et al recently reported higher predictivity of liver toxicants by 3D hLiMT (14‐day treatment) vs plated D2 primary human hepatocytes (48h‐treatment).…”

Section: Resultsmentioning

confidence: 99%

Mechanistic investigations of the liver toxicity of the free fatty acid receptor 1 agonist fasiglifam (TAK875) and its primary metabolites

Ackerson

Amberg

Atzrodt

et al. 2019

J Biochem & Molecular Tox

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For fasiglifam (TAK875) and its metabolites the substance‐specific mechanisms of liver toxicity were studied. Metabolism studies were run to identify a putatively reactive acyl glucuronide metabolite. In vitro cytotoxicity and caspase 3/7 activation were assessed in primary human and dog hepatocytes in 2D and 3D cell culture. Involvement of glutathione (GSH) detoxication system in mediating cytotoxicity was determined by assessing potentiation of cytotoxicity in a GSH depleted in vitro system. In addition, potential mitochondrial liabilities of the compounds were assessed in a whole‐cell mitochondrial functional assay. Fasiglifam showed moderate cytotoxicity in human primary hepatocytes in the classical 2D cytotoxicity assays and also in the complex 3D human liver microtissue (hLiMT) after short‐term treatment (24 hours or 48 hours) with TC50 values of 56 to 68 µM (adenosine triphosphate endpoint). The long‐term treatment for 14 days in the hLiMT resulted in a slight TC50 shift over time of 2.7/3.6 fold lower vs 24‐hour treatment indicating possibly a higher risk for cytotoxicity during long‐term treatment. Cellular GSH depletion and impairment of mitochondrial function by TAK875 and its metabolites evaluated by Seahorse assay could not be found being involved in DILI reported for TAK875. The acyl glucuronide metabolites of TAK875 have been finally identified to be the dominant reason for liver toxicity.

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“…PHH suspension cultures are mainly useful for the prediction of metabolic clearance and CYP inhibition-mediated DDIs (207)(208)(209)(210), while assessments of drug hepatotoxicity and CYP induction are not feasible due to their limited life span (i.e. a couple hours) (211). When seeded on a layer of rat-tail collagen as two-dimensional (2D) monolayers, PHH can be kept viable for several days in culture.…”

Section: Conventional In Vitro Modelsmentioning

confidence: 99%

Inter-individual differences in the susceptibility of primary human hepatocytes towards drug-induced cholestasis are compound and time dependent

Parmentier¹,

Hendriks

Heyd

et al. 2018

Toxicology Letters

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Cytotoxicity evaluation using cryopreserved primary human hepatocytes in various culture formats

Cited by 23 publications

References 22 publications

Comparison of Hepatic 2D Sandwich Cultures and 3D Spheroids for Long-term Toxicity Applications: A Multicenter Study

Comparison of Hepatic 2D Sandwich Cultures and 3D Spheroids for Long-term Toxicity Applications: A Multicenter Study

Mechanistic investigations of the liver toxicity of the free fatty acid receptor 1 agonist fasiglifam (TAK875) and its primary metabolites

Inter-individual differences in the susceptibility of primary human hepatocytes towards drug-induced cholestasis are compound and time dependent

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