Article Figures & Data
Figures
- 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.
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.
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.
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.
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.
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.
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.
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/Parameters In Vitro Hepatic Models HuH-7 Sandwich-Cultured Human Hepatocytesa HepaRGa Digoxin BEI (%) 27–29 7–63b–d CLuptake,app (μl/min per milligram protein) 1.0–1.3 0.69–2.8b,d CLbiliary,app (μl/min per milligram protein) 0.3–0.36 0.18–2.4b–d Pravastatin BEI (%) 22 18–3c,e CLuptake,app (μl/min per milligram protein) 0.4 1e CLbiliary,app (μl/min per milligram protein) 0.086 0.25–0.55c,e TCA BEI (%) 26–50 30–75b,d–f 27–39g–i CLuptake,app (μl/min per milligram protein) 1.2–2.1 2.7–20b,d–f 2.7–13.8g,h CLbiliary,app (μl/min per milligram protein) 0.5–0.7 0.8–25b,d–f 1–4g,h
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).
- Supplemental Figures -
