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Research ArticleArticle

Optimization of Canalicular ABC Transporter Function in HuH-7 Cells by Modification of Culture Conditions

Hee Eun Kang, Melina M. Malinen, Chitra Saran, Paavo Honkakoski and Kim L.R. Brouwer
Drug Metabolism and Disposition October 2019, 47 (10) 1222-1230; DOI: https://doi.org/10.1124/dmd.119.087676

Article Figures & Data

Figures

  • Fig. 1.
    Fig. 1.

    Representative cellular morphology and bile canaliculi-like structures in 4-week HuH-7 cultures. (A) Phase-contrast microscopy image of 4-week confluent HuH-7 cells cultured without (standard) and with Matrigel overlay. (B) Localization of the fluorescent canalicular marker CDF, the metabolite of CDFDA, in 4-week standard and Matrigel-overlaid HuH-7 cultures.

  • Fig. 2.
    Fig. 2.

    Effect of Matrigel overlay and DEX on the length and number of bile canaliculi-like structures in confluent 4-week HuH-7 cultures. The (A) length (mean ± S.D.) and (B) number (mean ± S.D.) of phalloidin-stained F-actin-rich tubules (i.e., bile canaliculi) normalized by cell count on the basis of DAPI-labeled nuclei are shown. Each representative field view (212.55 × 212.55 μm) from three different wells per each condition was used for the measurement. *P < 0.05; ***P < 0.001 (one-way ANOVA with Games-Howell test).

  • Fig. 3.
    Fig. 3.

    Expression and distribution of canalicular transporters MDR1, MRP2, and BSEP (green) in 4-week HuH-7 cultures without Matrigel overlay [(A) standard culture Area 1 and (B) Area 2]. Nuclei (blue) and F-actin (red) were labeled with DAPI and Alexa Fluor 594-phalloidin, respectively.

  • Fig. 4.
    Fig. 4.

    Expression and distribution of canalicular transporters (A) MDR1, (B) MRP2, and (C) BSEP (green) in 4-week HuH-7 cultures with Matrigel overlay. HuH-7 cells were overlaid with Matrigel on culture day 22 and supplemented with 0, 0.1, or 1 μM DEX from culture day 8 to 28. Nuclei (blue) and F-actin (red) were labeled with DAPI and Alexa Fluor 594-phalloidin, respectively.

  • Fig. 5.
    Fig. 5.

    Effect of DEX on the expression of MDR1, MRP2, and BSEP in 4-week HuH-7 cultures overlaid with Matrigel on culture day 22. (A) Representative immunoblots of MDR1, MRP2, BSEP, and Na+/K+ ATPase in 4-week Matrigel-overlaid HuH-7 cultures and cryopreserved human hepatocytes (positive control). (B) Relative expression levels (mean ± S.D.) of MDR1, MRP2, and BSEP in 4-week Matrigel-overlaid HuH-7 cultures supplemented with 0, 0.1, and 1 μM DEX from culture day 8 to 28 (n = 2–4 each). ***P < 0.001 (one-way ANOVA with Tukey’s test).

  • Fig. 6.
    Fig. 6.

    Accumulation of CDF in 4-week HuH-7 cultures. HuH-7 cells cultured with or without Matrigel overlay were preincubated with standard or Ca2+-free HBSS for 25 minutes followed by incubation for 20 minutes with CDFDA. Accumulation of CDF, the fluorescent metabolite of CDFDA, within canaliculi-like structures is indicated by arrows.

  • Fig. 7.
    Fig. 7.

    Effect of Matrigel overlay and DEX on digoxin and pravastatin accumulation and canalicular excretion in 4-week HuH-7 cultures. Accumulation of (A) digoxin and (B) pravastatin was measured in cells + bile (preincubation with standard HBSS) and in cells (preincubation with Ca2+-free HBSS). Results represent mean ± S.D. of two independent studies performed in triplicate. BEI (mean, %) was calculated as described in Materials and Methods.

  • Fig. 8.
    Fig. 8.

    Effect of Matrigel overlay and DEX on TCA accumulation and canalicular excretion in 4-week HuH-7 cultures. Accumulation of [3H]-TCA in the cells+bile (preincubation with standard HBSS) and in cells (preincubation with Ca2+-free HBSS). Results represent mean ± S.D. of three independent studies performed in triplicate. BEI (mean ± S.D., %) was calculated as described in Materials and Methods.

Tables

  • TABLE 1

    Summary of biliary excretion index (BEI), apparent in vitro uptake clearance (CLuptake,app) and biliary clearance (CLbiliary,app) values of digoxin, pravastatin, and TCA in various hepatic cell models

    Substrates/ParametersIn Vitro Hepatic Models
    HuH-7Sandwich-Cultured Human HepatocytesaHepaRGa
    Digoxin
     BEI (%)27–297–63b–d
     CLuptake,app (μl/min per milligram protein)1.0–1.30.69–2.8b,d
     CLbiliary,app (μl/min per milligram protein)0.3–0.360.18–2.4b–d
    Pravastatin
     BEI (%)2218–3c,e
     CLuptake,app (μl/min per milligram protein)0.41e
     CLbiliary,app (μl/min per milligram protein)0.0860.25–0.55c,e
    TCA
     BEI (%)26–5030–75b,d–f27–39g–i
     CLuptake,app (μl/min per milligram protein)1.2–2.12.7–20b,d–f2.7–13.8g,h
     CLbiliary,app (μl/min per milligram protein)0.5–0.70.8–25b,d–f1–4g,h

    • ↵a The values were adopted or calculated from the following references:

    • ↵b Bi et al. (2006).

    • ↵c Kimoto et al. (2017).

    • ↵d Swift et al. (2009).

    • ↵e Abe et al. (2009).

    • ↵f Ni et al. (2016).

    • ↵g Le Vee et al. (2013).

    • ↵h Susukida et al. (2016).

    • ↵i Bachour-El Azzi et al. (2015).

Additional Files

  • Data Supplement

    • Supplemental Figures -

      Supplementary Figure 1 -  Bile canaliculi-like structures of confluent 4-week HuH-7 cultures overlaid with Matrigel extracellular matrix.

      Supplementary Figure 2 - Effect of dexamethasone (DEX) on the expression of BSEP in standard HuH-7 cultures.

      Supplementary Figure 3 - Effect of Matrigel lots and phenol red on TCA canalicular excretion in 4-week HuH-7 cultures supplemented with 1 μM dexamethasone (DEX).
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Research ArticleArticle

Canalicular ABC Transporters in HuH-7 Cells

Hee Eun Kang, Melina M. Malinen, Chitra Saran, Paavo Honkakoski and Kim L.R. Brouwer
Drug Metabolism and Disposition October 1, 2019, 47 (10) 1222-1230; DOI: https://doi.org/10.1124/dmd.119.087676
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