Relevance of the incubation period in cytotoxicity testing with primary human hepatocytes

Abstract

Primary human hepatocytes (PHHs) remain the gold standard for in vitro testing in the field of pharmacology and toxicology. One crucial parameter influencing the results of in vitro tests is the incubation period with test compounds. It has been suggested that longer incubation periods may be critical for the prediction of repeated dose toxicity. However, a study that systematically analyzes the relationship between incubation period and cytotoxicity in PHHs is not available. To close this gap, 30 compounds were tested in a concentration-dependent manner for cytotoxicity in cultivated cryopreserved PHHs (three donors per compound) for 1, 2 and 7 days. The median of the EC 50 values of all compounds decreased 1.78-fold on day 2 compared to day 1, and 1.89-fold on day 7 compared to day 1. Median values of EC 50 ratios of all compounds at day 2 and day 7 were close to one but for individual compounds the ratio increased up to almost six. Strong correlations were obtained for EC 50 on day 1 and day 7 (R = 0.985; 95% CI 0.960-0.994), day 1 and day 2 (R = 0.964; 95% CI 0.910-0.986), as well as day 2 and day 7 (R = 0.981; 95% CI 0.955-0.992). However, compound specific differences also occurred. Whereas, for example, busulfan showed a relatively strong increase on day 7 compared to day 1, cytotoxicity of acetaminophen did not increase during longer incubation periods. To validate the observed correlations, a publicly available data set, containing data on the cytotoxicity of human hepatocytes cultivated as spheroids for incubation periods of 5 and 14 days, was analyzed. A high correlation coefficient of EC 50 values at day 5 and day 14 was obtained (R = 0.894; 95% CI 0.798-0.945). In conclusion, the median cytotoxicity of the test compounds increased between 1 and 2 days of incubation, with no or only a minimal further increase until day 7. It remains to be studied whether the different results obtained for some individual compounds after longer exposure periods would correspond better to human-repeated dose toxicity. Keywords Hepatotoxicity • Primary human hepatocyte • Incubation period • Cell-titer-blue Abbreviations APAP Acetaminophen ASP Aspirin BOS Bosentan BPR Buspirone Xiaolong Gu and Wiebke Albrecht shared first authorship.

Prediction of human drug-induced liver injury (DILI) in relation to oral doses and blood concentrations

“…Another question addressed in this study was how long cultivated hepatocytes should be exposed to test compounds, and whether repeated exposures are superior to a single addition of test compound (Gu et al 2018;Godoy et al 2013). Our results demonstrated that a two-day exposure period of the test compound (with only one addition of test compound at the morning after thawing and plating) lead to a higher TSI and TEI compared to 24 h or 7-day exposure with repeated culture medium changes containing fresh test compound.…”

“…In the present study, an incubation period of 48 h was used. Additionally, incubations of 24 h and 7 days (with repeated culture medium changes with fresh test compound) were performed as previously reported (Gu et al 2018). A higher TSI was obtained for the 48 h compared to the 24 h and 7 day incubation periods (Fig.…”

“…Our results demonstrated that a two-day exposure period of the test compound (with only one addition of test compound at the morning after thawing and plating) lead to a higher TSI and TEI compared to 24 h or 7-day exposure with repeated culture medium changes containing fresh test compound. The better results obtained after 2 days compared to a single day of exposure is plausible since some test compounds require more than 24 h to develop their full toxic potential (Gu et al 2018). However, it was surprising that the longer incubation protocol (7 days) and repeated exposures did not further improve the test performance.…”

“…The cytotoxicity assay with PHH was performed using the CellTitre-Blue (CTB) assay as described in Gu et al (2018) according to the SOP in Supplement 3A. The same cytotoxicity assay was also used for HepG2 cells (Supplement 3A).…”

Highlight report: New applications of chimeric mice with humanized livers

“…It remains open to which degree the transplanted human hepatocytes differ when transplanted into mouse livers compared to their state in the human microenvironment. A well-established alternative system to humanized mouse livers is cultivated human hepatocytes (Gu et al 2018;Deharde et al 2016;Reif et al 2015;Ghallab et al 2016;Kim et al 2015;Frey et al 2014), a cell system well established for the purpose of metabolite generation and identification (Hewitt et al 2007;Godoy et al 2013).…”