Human precision-cut cystic duct and gallbladder slices: a novel method for studying cholangiopathies

Abstract

Background and aims: Precision-cut tissue slices (PCTS) are widely used as an ex vivo culture tissue culture technique to study pathogeneses of diseases and drug activities in organs in vitro. A novel application of the PCTS model may be in the field of translational research into cholangiopathies such as biliary atresia. Therefore, the aim of this study was to apply the precision-cut slice technique to human bile duct and gallbladder tissue.

Methods: Cystic duct and gallbladder tissue derived from patients undergoing a cholecystectomy were collected, preserved and used for preparation of precision-cut cystic duct slices (PCCDS) and precision-cut gallbladder slices (PCGS). The PCCDS and PCGS were prepared using a mechanical tissue slicer and subsequently incubated for 24 and 48 h respectively in William's Medium E (WME) culture medium. Viability was assessed based on ATP/protein content and tissue morphology [hematoxylin and eosin (H&E) staining].

Results: It was shown that viability, assessed by the ATP/protein content and morphology, of the PCCDS (n = 8) and PCGS (n = 8) could be maintained over the 24 and 48 h incubation period respectively. ATP/protein content of the PCCDS increased significantly from 0.58 ± 0.13 pmol/µg at 0 h to 2.4 ± 0.29 pmol/µg after 24 h incubation (P = .0003). A similar significant increase from 0.94 ± 0.22 pmol/µg at 0 h to 3.7 ± 0.41 pmol/µg after 24 h (P = .0005) and 4.2 ± 0.47 pmol/µg after 48 h (P = .0002) was observed in the PCGS. Morphological assessment of the PCCDS and PCGS showed viable tissue at 0 h and after 24 and 48 h incubation respectively.

Conclusion: This study is the first to report on the use of the PCTS model for human gallbladder and cystic duct tissue. PCCDS and PCGS remain viable for an incubation period of at least 24 h, which makes them suitable for research purposes in the field of cholangiopathies, including biliary atresia.

Keywords: cholangiopathies; cystic duct; ex vivo culture technique; gallbladder; precision-cut tissue slices (PCTS).

Figure 1

Preparation and slicing of human PCCDS. Remnant of cystic duct was flushed with UW solution, cleaned and as much of the loose connective tissue as possible was removed (A). The segment was placed in the core holder, which was filled with 3% low-gelling-temperature agarose (B). After solidification of the agarose, the core was removed from the core holder and placed in the Krumdieck tissue slicer (C). PCCDS were collected in UW solution during the slicing process (D) and afterwards transferred to culture plates. Tissue was kept at 4 degrees celsius during the whole process.

Figure 2

Preparation and slicing of human PCGS. The gallbladder tissue (A) was first cleaned and fat were removed. Next, the gallbladder was cut into small patches (B). The small patch was then placed in 3% low-gelling-temperature agarose. After solidification of the agarose (C), the core was removed from the core holder (D) and placed in the Krumdieck tissue slicer to prepare the PCGS. After slicing, the PCGS were transferred into 12 well culture plates filled with culture medium.

Figure 3

Viability of PCCDS and PCGS at 0 h and after 24 and 48 h incubation as assessed by the ATP/protein content. The cystic duct and gallbladder specimens from one patient were coded with the same color. P-values denote statistical significance.

Figure 4

Panel A shows H&E stained sections of representative PCCDS before and after incubation from one patient (sample code: DC51) (magnification 10 x, 250 µm and 20 × 100 µm). The arrow points towards the epithelium, which shows erosion. A viable crypt of the epithelium is encircled. The arrow points towards viable epithelium and arrowhead indicates a new epithelial cell layer. In terms of morphological scoring of these PCCDS, the following scores were attributed to the slices at 0 h: slight chronic inflammation (score 1), 100% viability (score 0), 66% of epithelium contained crypts (score 1), moderately erosive surface epithelium (score 2) and viable PBG present (score 0). The other scores of these PCCDS and PCGS can be found in row 7 of the heatmaps in Figure 5 (sample codes DC51). Panel B shows heatmaps of morphological scoring of the PCCDS. Scoring items are shown on the x-axis and the different PCCDS samples on the y-axis. Sample codes of the cystic ducts used to prepare the slices can be found in the y-axis legend. Scores are represented by different colors, whereby 0 = green, 1 = yellow, 2 = orange and 3 = red.

Figure 5

Panel A shows H&E stained sections of representative PCGS before and after incubation from one patient (sample code: GB51) (magnification 10 x, 250 µm and 20 × 100 µm). The arrow points towards the epithelium, which shows erosion. A viable crypt of the epithelium is encircled. The arrow points towards viable epithelium and arrowhead indicates a new epithelial cell layer. Panel B shows heatmaps of morphological scoring of the PCCDS. Scoring items are shown on the x-axis and the different PCGS samples on the y-axis. Sample codes of the gallbladders used to prepare the slices can be found in the y-axis legend. Scores are represented by different colors, whereby 0 = green, 1 = yellow, 2 = orange and 3 = red.

Figure 6

Correlation between morphology score and ATP/protein level of the PCCDS and PCGS. The results are shown for each cystic duct or gallbladder separately (n = 8). Solid circles represent samples after 24 h incubation and transparent samples represent 48 h incubation samples.

Figure 7

Immunohistochemical staining for pan cytokeratin of PCGS and PCCDS. Before incubation, the surface epithelium of the PCGS and PCCDS stains positive (images A,B). After incubation, the new cell layer at the side of the epithelium also stains positive (images C,D). Positive staining indicates the presence of epithelial cells.

Figure 8

Quantification of pan cytokeratin staining of PCCDS and PCGS. The figure shows the percentage of positive cells for each cystic duct and gallbladder separately. In the table, the exact values are shown.