Influence of seeding density and extracellular matrix on bile Acid transport and mrp4 expression in sandwich-cultured mouse hepatocytes

Abstract

This study was undertaken to examine the influence of seeding density, extracellular matrix and days in culture on bile acid transport proteins and hepatobiliary disposition of the model bile acid taurocholate. Mouse hepatocytes were cultured in a sandwich configuration on six-well Biocoat plates with an overlay of Matrigel (BC/MG) or gelled-collagen (BC/GC) for 3 or 4 days at seeding densities of 1.0, 1.25, or 1.5 x 10(6) cells/well. The lower seeding densities of 1.0 and 1.25 x 10(6) cells/well resulted in good hepatocyte morphology and bile canalicular network formation, as visualized by 5-(and 6)-carboxy-2',7'-dichlorofluorescein accumulation. In general, taurocholate cellular accumulation tended to increase as a function of seeding density in BC/GC; cellular accumulation was significantly increased in hepatocytes cultured in BC/MG compared to BC/GC at the same seeding density on both days 3 and 4 of culture. In general, in vitro intrinsic biliary clearance of taurocholate was increased at higher seeding densities. Levels of bile acid transport proteins on days 3 and 4 were not markedly influenced by seeding density or extracellular matrix except for multidrug resistance protein 4 (Mrp4), which was inversely related to seeding density. Mrp4 levels decreased approximately 2- to 3-fold between seeding densities of 1.0 x 10(6) and 1.25 x 10(6) cells/well regardless of extracellular matrix; an additional approximately 3- to 5-fold decrease in Mrp4 protein was noted in BC/GC between seeding densities of 1.25 x 10(6) and 1.5 x 10(6) cells/well. Results suggest that seeding density, extracellular matrix and days in culture profoundly influence Mrp4 expression in sandwich-cultured mouse hepatocytes. Primary mouse hepatocytes seeded in a BC/MG configuration at densities of 1.25 x 10(6) cells/well and 1.0 x 10(6), and cultured for 3 days, yielded optimal transport based on the probes studied. This work demonstrates the applicability of the sandwich-cultured model to mouse hepatocytes.

Figure 1

Effect of seeding density and extracellular matrix on cell morphology and bile canalicular network formation in day 3 sandwich-cultured mouse hepatocytes. Representative (n =3) CDF fluorescence (A – F) and light microscopy (G – L) images of mouse hepatocytes cultured in a Biocoat^™/Matrigel^™ (BC/MG; A–C & G–I) or Biocoat^™/gelled-collagen (BC/GC; D–F & J–L) sandwich configuration in six-well plates (maintained with DMEM for 3 days); seeding densities (1.0, 1.25 or 1.5 × 10⁶ cells/well) are noted at the top of each column. Sandwich-cultured mouse hepatocytes were incubated with 2 μM CDFDA for 10 min.

Figure 1

Effect of seeding density and extracellular matrix on cell morphology and bile canalicular network formation in day 3 sandwich-cultured mouse hepatocytes. Representative (n =3) CDF fluorescence (A – F) and light microscopy (G – L) images of mouse hepatocytes cultured in a Biocoat^™/Matrigel^™ (BC/MG; A–C & G–I) or Biocoat^™/gelled-collagen (BC/GC; D–F & J–L) sandwich configuration in six-well plates (maintained with DMEM for 3 days); seeding densities (1.0, 1.25 or 1.5 × 10⁶ cells/well) are noted at the top of each column. Sandwich-cultured mouse hepatocytes were incubated with 2 μM CDFDA for 10 min.

Figure 2

Effect of seeding density and extracellular matrix on cell morphology and bile canalicular network formation in day 4 sandwich-cultured mouse hepatocytes. Representative (n = 3) CDF fluorescence (A – F) and light microscopy (G – L) images of mouse hepatocytes cultured in a Biocoat^™/Matrigel^™ (BC/MG; A–C & G–I) or Biocoat^™/gelled-collagen (BC/GC; D–F & J–L) sandwich configuration in six-well plates (maintained with DMEM for 4 days); seeding densities (1.0, 1.25 or 1.5 × 10⁶ cells/well) are noted at the top of each column. Sandwich-cultured mouse hepatocytes were incubated with 2 μM CDFDA for 10 min.

Figure 2

Effect of seeding density and extracellular matrix on cell morphology and bile canalicular network formation in day 4 sandwich-cultured mouse hepatocytes. Representative (n = 3) CDF fluorescence (A – F) and light microscopy (G – L) images of mouse hepatocytes cultured in a Biocoat^™/Matrigel^™ (BC/MG; A–C & G–I) or Biocoat^™/gelled-collagen (BC/GC; D–F & J–L) sandwich configuration in six-well plates (maintained with DMEM for 4 days); seeding densities (1.0, 1.25 or 1.5 × 10⁶ cells/well) are noted at the top of each column. Sandwich-cultured mouse hepatocytes were incubated with 2 μM CDFDA for 10 min.

Figure 3

Influence of seeding density, extracellular matrix and day in culture on transport protein levels in sandwich-cultured mouse hepatocytes. Representative immunoblots of Bcrp, Ntcp, Mrp4, Mrp3, Mrp2, Bsep, and Mdr1a/1b in mouse hepatocytes cultured in Biocoat^™/Matrigel^™ (BC/MG) or Biocoat^™/gelled-collagen (BC/GC) sandwich configuration in six-well plates and maintained with DMEM for 3 or 4 days (n = 2). B-actin was used as a loading control.

Figure 4

Relative expression of Mrp4 protein compared with β-actin in sandwich-cultured mouse hepatocytes cultured in Biocoat^™/Matrigel^™ (BC/MG) or Biocoat^™/gelled-collagen (BC/GC) sandwich configuration in six-well plates and maintained with DMEM for 3–4 days. Densitometry was performed with Quantity One software (version 4.1; Bio-Rad, Hercules, CA).

Figure 5

Accumulation in cells+bile (black bars) and cells (white bars) after a 10-min incubation with 1 μM [³H]taurocholate in mouse hepatocytes cultured in Biocoat^™/Matrigel^™ (BC/MG) or Biocoat^™/gelled-collagen (BC/GC) sandwich configuration in six-well plates and maintained with DMEM for 3 or 4 days (Mean ± S.E.M., n=3). Cellular accumulation (white bars) of [³H]taurocholate was significantly increased on day 3 compared to day 4, and significantly greater when cultured in BC/MG configuration compared to the BC/GC configuration on both days 3 and 4 of culture. * p < 0.05