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

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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 EC50 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 EC50 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 EC50 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 EC50 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.

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The study was supported by the European Union’s Horizon 2020 research and innovation programme (EUToxRisk; no. 681002) with additional contributions of the projects StemCellNet (BMBF, 01EK1604A), Liver Simulator (BMBF, 031A355A), DILI (BMBF, 031L0074F), LiSyM (BMBF, 031Loo45), LivSysTransfer (BMBF, 0101-31Q0517), InnoSysTox (BMBF/EU, 031L0021A), SteatoTox (031L0114B) and DEEP (BMBF, 01KU1216).

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Correspondence to Jan G. Hengstler.

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Suppl. Fig. 1A: Cell-Titer-Blue cytotoxicity data for 30 compounds in primary human hepatocytes after incubation for one, two and seven days. The concentration-dependent curves represent data from three independent experiments corresponding to hepatocytes from three different donors with four technical replicates each. The cell viability for each concentration is presented as the percentage of untreated controls. Gray symbols indicate the viability of each technical replicate normalized to untreated controls; whereas, black symbols represent the mean values of all technical replicates for each concentration. The vertical line indicates the concentration which causes 20% (A–C) or 50% (D–F) loss of viability. The vertical dashed black lines show the 95% confidence interval of the EC50 or EC20s. The exact EC50 or EC20 (or EC80) values are given in the lower left corner of each panel. (A) EC20, one day incubation period; (B) EC20, two days incubation period; (C) EC20, seven days incubation period; (D) EC50, one day incubation period; (E) EC50, two days incubation period; and (F) EC50, seven days incubation period (PDF 130 KB)

Suppl. Fig. 1B (PDF 133 KB)

Suppl. Fig. 1C (PDF 126 KB)

Suppl. Fig. 1D (PDF 130 KB)

Suppl. Fig. 1E (PDF 132 KB)

Suppl. Fig. 1F (PDF 126 KB)


Suppl. Fig. 2: Bar plots of cytotoxicity EC50 and EC20 for 30 compounds in primary human hepatocytes after incubation for one, two and seven days. Note: For day one and day two or both, some of the less toxic compounds did not reduce viability compared to controls. In these cases, the highest tested concentrations were used and bars were labeled by an asterisk (PPTX 2685 KB)

Supplement 1: Detailed donor characteristics given by the supplier (PDF 331 KB)

Supplement 2: Standard operation procedure (SOP) for cultivation of primary human hepatocytes (DOCX 148 KB)

Supplement 3: Standard operation procedure (SOP) for cytotoxicity testing (DOCX 147 KB)


Suppl. Table 1: (A) Stock solutions of the test compounds. For incubation periods of 24 and 48 h, the concentrations of stock solutions given in the light fields were used. For the 7 days incubation period, additional lower concentrations of stock solutions were used as indicated by the dark background. A final concentration of 0.1% DMSO in the culture medium was used with the exception of compounds with low solubility, where 0.5% DMSO was used, indicated by *. Culture medium-soluble compounds are indicated in blue, while compounds dissolved in DMSO are indicated in red. In the present study, concentrations have been selected to study cytotoxicity. To compare in vivo relevant concentrations, peak plasma concentrations of therapeutic doses (or documented human exposure for chemicals not used as drugs) are given. (B) Investigated concentrations of the test compounds. The applied stock solutions are given in Suppl. Table 1. For incubation periods of 24 and 48 h, the concentrations indicated in the white fields were tested. For the seven days incubation period, additional lower concentrations were tested as indicated in the fields with grey background. For water soluble test compounds, the required concentrations could be obtained in the culture medium (indicated by blue colour). For the further compounds (labeled red), stock solutions were prepared in dimethyl sulfoxide (DMSO). The final concentration of DMSO was 0.1%, with the exception of compounds with relatively low DMSO solubility which had a final concentration of 0.5% DMSO (indicated by * in Table 1 and Suppl. Table 1) (DOCX 22 KB)


Suppl. Table 2: Cytotoxicity data (EC50 and EC20 values; mean and median values of three donors) from all test compounds, with ratios of EC50 as well as EC20 values of the different incubation periods (XLSX 15 KB)

Suppl. Table 3: EC50 and EC20 values for each individual donor (XLSX 16 KB)

Suppl. Table 4: Raw data from the CellTiter-Blue test of all individual compounds and donors (XLSX 115 KB)

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Gu, X., Albrecht, W., Edlund, K. et al. Relevance of the incubation period in cytotoxicity testing with primary human hepatocytes. Arch Toxicol 92, 3505–3515 (2018).

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