Tubular bile duct structure mimicking bile duct morphogenesis for prospective in vitro liver metabolite recovery

Abstract

Background: Liver metabolites are used to diagnose disease and examine drugs in clinical pharmacokinetics. Therefore, development of an in vitro assay system that reproduces liver metabolite recovery would provide important benefits to pharmaceutical research. However, liver models have proven challenging to develop because of the lack of an appropriate bile duct structure for the accumulation and transport of metabolites from the liver parenchyma. Currently available bile duct models, such as the bile duct cyst-embedded extracellular matrix (ECM), lack any morphological resemblance to the tubular morphology of the living bile duct. Moreover, these systems cannot overcome metabolite recovery issues because they are established in isolated culture systems. Here, we successfully established a non-continuous tubular bile duct structure model in an open-culture system, which closely resembled an in vivo structure. This system was utilized to effectively collect liver metabolites separately from liver parenchymal cells.

Results: Triple-cell co-culture of primary rat hepatoblasts, rat biliary epithelial cells, and mouse embryonic fibroblasts was grown to mimic the morphogenesis of the bile duct during liver development. Overlaying the cells with ECM containing a Matrigel and collagen type I gel mixture promoted the development of a tubular bile duct structure. In this culture system, the expression of specific markers and signaling molecules related to biliary epithelial cell differentiation was highly upregulated during the ductal formation process. This bile duct structure also enabled the separate accumulation of metabolite analogs from liver parenchymal cells.

Conclusions: A morphogenesis-based culture system effectively establishes an advanced bile duct structure and improves the plasticity of liver models feasible for autologous in vitro metabolite-bile collection, which may enhance the performance of high-throughput liver models in cell-based assays.

Keywords: Bile duct; Bile recovery; Hierarchical co-culture; Liver metabolite; Morphogenesis.

Fig. 1

Optimum condition for the establishment of tubular bile duct structure. a PDMS–plate treatment and seeding procedure for initial hierarchal co-culture. b Diagram showing cross-section of bile duct hierarchal co-culture after ECM overlay. c Map showing average % of bile duct-like structure surface area (mm²) in all cultures after ≤ 35 days (n = 12; three independent experiments). d Bile duct cysts and tubular bile duct structures. e Gene expression assay comparing bile duct cysts and tubular bile duct structure. Tubular bile duct structures highly expressed Ck19 marker but only weakly expressed hepatoblast (Afp) and hepatocyte (Alb) markers. β-actin was used as a housekeeping gene and primary cells served as a negative control (n = 5; three independent experiments, values are the means ± SD. *P < 0.05; **P < 0.01. Red stars and bars indicate significant differences between bile duct structures)

Fig. 2

Characteristics of tubular bile duct structure established in triple co-culture in ≤ 23 days. a Tubular bile duct structure in triple co-culture expressing significant levels of the bile duct markers Sox9, Cftr, and Sctr. (β-actin was used as the housekeeping gene, n = 5; three independent experiments; values are the means ± SD; *P < 0.05). b Differentiation stages in cyst and tubular bile duct structure. White arrows emphasize the shape. Stages were validated by immunostaining with SOX9 and CFTR at ≤ 17 days. c Immunofluorescence shows the 17 day tubular bile duct slightly expressing VIM and resembling fine ZO1 tight junctions

Fig. 3

In vitro ≤ 23 day triple co-culture, tubular bile duct structure polarity and transporter activity. aAqp1 and Ae2 were significantly upregulated in the tubular structure whereas Mrp3 expression did not significantly change (β-actin was used as the housekeeping gene, n = 5; three independent experiments; values are means ± SD. *P < 0.05; **P < 0.01). b AE2 was abundantly distributed on the apical site. c Rhodamine 123 accumulated in the lumen of the tubular structure. Verapamil blocked MDR1 transporter activity and attenuated green fluorescence in the lumen

Fig. 4

Morphogenesis of tubular bile duct structure established within the 17 day triple co-culture period. a Three-sided imagery of phalloidin and Hoechst staining resembling the tubular structure of an in vitro bile duct (white arrow), displaying continuous lumen structure within the tubular structure. b Tubular structure showed significant upregulation of Jagged1, Notch2, and Cldn15 within 17 days of culture (β-actin was used as the housekeeping gene, n = 5; three independent experiments; values are means ± SD; *P < 0.05)

Fig. 5

Integrated culture system combining in vitro tubular bile duct structure and primary hepatocyte tissue. a Seeding procedure for integrated tubular–hepatocyte culture. b Diagram showing hierarchy of integrated BEC-hepatocyte and tubular–hepatocyte culture established in collagen membrane-culture insert. c FDA assay administrated to hepatocyte-contained compartment. Fluorescein presented by green fluorescence accumulated in tubular bile duct structures located in the upper compartment. d Tubular structures demonstrated the ability to accumulate fluorescein. FDA removal is represented by yellow backlights. (n = 4; two independent experiments; values are the means ± SD; *P < 0.05; **P < 0.01). Red stars and bars indicate significant differences between independent treatments)