Abstract
Human hepatoma cell lines are useful for evaluation of drug-induced hepatotoxicity, hepatic drug disposition, and drug-drug interactions. However, their applicability is compromised by aberrant expression of hepatobiliary transporters. This study was designed to evaluate whether extracellular matrix (Matrigel) overlay and dexamethasone (DEX) treatment would support cellular maturation of long-term HuH-7 hepatoma cell cultures and improve the expression, localization, and activity of canalicular ATP-binding cassette (ABC) transporters, multidrug resistance protein 1 (MDR1/P-glycoprotein/ABCB1), multidrug resistance-associated protein 2 (MRP2/ABCC2), and bile salt export pump (BSEP/ABCB11). Matrigel overlay promoted the maturation of HuH-7 cells toward cuboidal, hepatocyte-like cells displaying bile canaliculi-like structures visualized by staining for filamentous actin (F-actin), colocalization of MRP2 with F-actin, and by accumulation of the MRP2 substrate 5(6)-carboxy-29,79-dichlorofluorescein (CDF) within the tubular canaliculi. The cellular phenotype was rather homogenous in the Matrigel-overlaid cultures, whereas the standard HuH-7 cultures contained both hepatocyte-like cells and flat epithelium-like cells. Only Matrigel-overlaid HuH-7 cells expressed MDR1 at the canaliculi and excreted the MDR1 probe substrate digoxin into biliary compartments. DEX treatment resulted in more elongated and branched canaliculi and restored canalicular expression and function of BSEP. These findings suggest that hepatocyte polarity, elongated canalicular structures, and proper localization and function of canalicular ABC transporters can be recovered, at least in part, in human hepatoma HuH-7 cells by applying the modified culture conditions. SIGNIFICANCE STATEMENT We report the first demonstration that proper localization and function of canalicular ABC transporters can be recovered in human hepatoma HuH-7 cells by modification of cell culture conditions. Matrigel overlay and dexamethasone supplementation increased the proportion of hepatocyte-like cells, strongly augmented the canalicular structures between the cells, and restored the localization and function of key canalicular ABC transporters. These results will facilitate the development of reproducible, economical, and easily achievable liver cell models for drug development.
| Original language | English |
|---|---|
| Pages (from-to) | 1222-1230 |
| Number of pages | 9 |
| Journal | Drug Metabolism and Disposition |
| Volume | 47 |
| Issue number | 10 |
| DOIs | |
| State | Published - 2019 |
Bibliographical note
Funding Information:This work was supported by the National Institutes of Health National Institute of General Medical Sciences under Award Numbers R01 GM041935 and R35 GM122576 to Prof. K.L.R.B. Prof. H.E.K. was supported, in part, by a National Research Foundation of Korea grant funded by the Korean government (MSIT) (No. 2019R1F1A1052243) and by the Catholic University of Korea, Research Fund, 2018. Dr. M.M.M. was supported, in part, by the Finnish Cultural Foundation and Orion Research Foundation. Prof. P.H. was supported in part by the ERASMUS+Global exchange program and the University of Eastern Finland. Disclosure: Dr. K.L.R.B. is a coinventor of the sandwich-cultured hepatocyte technology for quantification of biliary excretion (B-CLEAR) and related technologies, which have been licensed exclusively to Qualyst Transporter Solutions, recently acquired by BioIVT. https://doi.org/10.1124/dmd.119.087676. s This article has supplemental material available at dmd.aspetjournals.org.
Publisher Copyright:
© 2019 by The American Society for Pharmacology and Experimental Therapeutics.