Human Hepatocyte/Kupffer cell 3D Spheroid Co-cultures: Characterization and Application for DILI Studies Patrina Gunness, 1 Albert P. Li 2* 1In Vitro ADMET Laboratories, Malden, MA; 2In Vitro ADMET Laboratories, Columbia, MD Introduction Results • Drug-induced liver injury (DILI), is an unpredicted clinical event that can lead to acute liver failure, and results in the termination Size and viability of human hepatocyte spheroid cultures Prototypical hepatotoxicants induce cytotoxicity in human hepatocyte spheroids of drug development program and the application of regulatory restrictions, including boxed warnings. • The well documented limitations of current 2D in vitro hepatocyte cell culture and in vivo animal models does not allow for the 14 acetaminophen ketoconazole troglitazone accurate prediction of DILI in human. There is an urgent need for improved in vitro human hepatocyte models to address these 12 limitations and allow for more accurate prediction of DILI in humans. 300 250 10 • The 3D cell culture of hepatocytes is a rapidly expanding field in an attempt to recreate, in a controlled, artificial environment; the 200 8 complex 3D microenvironment of the human liver, which is essential for hepatocyte longevity and function. A hepatocyte 150 6 spheroid culture is a 3D cell culture of organized, aggregates of hepatocytes. (µM) 100 4 The goal of the study was to develop a procedure to reproducibly culture human hepatocytes and Kupffer cells as spheroids and • 50 2 Spheroid diameter diameter Spheroid (pmol per per spheroid) (pmol to characterize these long-term cultures for their suitability for DILI studies. contentt ATP Intracellular 0 0 • Results show that these long-term spheroid cultures (1) have liver-like morphology and function, (2) are sensitive to prototypical 7 14 21 7 14 21 hepatotoxicants, (3) respond to an inflammatory stimulus and (4) can distinguish between hepatotoxic and non-hepatotoxic Culture Day Culture Day compounds. • Taken all together, the data suggest that these long-term spheroid cultures may be a useful tool to study DILI. Human hepatocyte spheroid cultures have liver-like morphology H&E CD68 Ki67 Materials & Methods IC 50 : 2219 (1347-3656) µmol/L IC 50 : 11.85 (8.90-15.79) µmol/L IC 50 : 27.60 (9.90-76.92) µmol/L Culture of human hepatocyte spheroids: Human hepatocyte spheroids can distinguish between a hepatotoxic and non-hepatotoxic compound trovafloxacin levofloxacin 4-5 days 200 - LPS + LPS 150 • Use of cryopreserved • Cells seeded in an ultra- • Cell aggregation • Compact single spheroid formation 100 cells low attachment plate • Spheroids ready for use 50 0 0.1 1 10 100 1000 Albumin production IL-6 production [levofloxacin] ( mol/L) Size assessment: The size of the spheroids was assessed by measuring their diameter, using a stage micrometer. The assessment was 0.35 performed in replicates of 3-4. The data is presented as the mean ± SD. 100.00 Compound IC 50 0.30 90.00 Cell viability: Cell viability was assessed by measuring the intracellular ATP content (CellTiter® Glo 2.0 assay, Promega) in spheroids. 80.00 -LPS +LPS The luminescence was measured using a plate reader (1420 Multilabel Counter VICTOR 3 V, Perkin Elmer). The assay was performed 0.25 70.00 ™ 0.20 60.00 trovafloxacin 53.77(17.96-161.0) 23.43 (15.23-36.06) in replicates of 3-4. the data is presented as the mean ± SD. 0.15 50.00 40.00 levofloxacin N/A N/A Histology: Pooled spheroids were fixed, embedded in paraffin and subsequently subjected to immunohistochemistry (IHC) 0.10 30.00 (pg/mL) 0.05 20.00 procedures for H&E, CD68 and Ki67 staining. IL-6production (µg/day/10⁶cells) 10.00 Albumin production: Albumin production was assessed in medium supernatant (Human Albumin ELISA, Bethyl Laboratories, Inc.). Albumin production 0.00 0.00 7 14 21 - LPS (0 µg/mL) + LPS (10 µg/mL) The assay was performed in replicates of 3. The data is presented as the mean ± SD. Culture day Conclusions IL-6 production : Spheroids were exposed to LPS (10 µg/mL) for 72 hours. Following the exposure, IL-6 production was assessed in medium supernatant (Human IL-6 ELISA, Boster Biological Technology Co., Ltd.). The assay was performed by pooling medium Long-term phase I P450 enzyme activity in human hepatocyte spheroids • Human hepatocyte spheroid cultures have long-term liver-like morphology & functionality. supernatant from 4 spheroids. Phase I P450 & phase II enzyme activity: Spheroids were incubated with respective enzyme substrates for 6 hours. Following the • Human hepatocyte/Kupffer cell spheroid co-cultures respond to an inflammatory stimulus; suggesting that it 30.00 3.00 CYP2B6 CYP2D6 incubation period, the medium supernatant was collected and subjected to LC/MS analyses (API 5000 mass spectrometer with an CYP1A2 CYP2C9 CYP3A4 may be useful tool for inflammation-mediated toxicity. electrospray ionization source (AB SCIEX) connected to Acquity UPLC). The assay was performed in replicates of 3. The data is 25.00 2.50 • Prototypical hepatotoxicantsinduce cytotoxicity in human hepatocyte spheroid cultures. presented as the mean ± SD. 20.00 2.00 • Exposure to an inflammatory stimulus increases the sensitivity of hepatocyte/Kupffer cell spheroid co-cultures Short-term cytotoxicity studies: Spheroids were exposed to respective hepatotoxicants for 5 days. Cytotoxicity was assessed by 15.00 1.50 to trovafloxacin exposure. measuring cell viability (as previously described). The assay was performed in replicates of 3. The data is presented as % control. IC 50 curves were generated using GraphPad Prism® 6. The data is presented as the mean ± SD. 10.00 1.00 • Human hepatocyte spheroid cultures can distinguish between a hepatotoxic and a non-hepatotoxiccompound. • trovafloxacin/levofloxacin ± LPS: Spheroids were pre-treated with LPS (0, 10 µg/mL) for 72 hours, followed by co-treatment 5.00 0.50 • Taken all together, the data suggest that these long-term 3D hepatocyte/Kupffer cell spheroid cultures may be a CYP enzyme enzyme CYP activity enzyme CYP activity with trovafloxacin or levofloxacin for 5 days. Following the exposure period, cytotoxicity was assessed by measuring cell (pmol/min/10⁶cells) (pmol/min/10⁶cells) 0.00 0.00 useful tool for preclinical drug metabolism and toxicity studies, including inflammation-mediated hepatotoxicity. viability (as previously described). The assay was performed in replicates of 3. The data is presented as % control. IC 50 curves 7 14 21 7 14 21 were generated using GraphPad Prism® 6 measured. The data is presented as the mean ± SD. Culture day Culture day *Corresponding author: Albert Li ([email protected])).