DUCT: Double Resin Casting followed by Micro-Computed Tomography for 3D Liver Analysis

Abstract

The liver is the biggest internal organ in humans and mice, and high auto-fluorescence presents a significant challenge for assessing the three-dimensional (3D) architecture of the organ at the whole-organ level. Liver architecture is characterized by multiple branching lumenized structures, which can be filled with resin, including vascular and biliary trees, establishing a highly stereotyped pattern in the otherwise hepatocyte-rich parenchyma. This protocol describes the pipeline for performing double resin casting micro-computed tomography, or "DUCT". DUCT entails injecting the portal vein and common bile duct with two different radiopaque synthetic resins, followed by tissue fixation. Quality control by clearing one lobe, or the entire liver, with an optical clearing agent, allows for pre-screening of suitably injected samples. In the second part of the DUCT pipeline, a lobe or the whole liver can be used for micro-computed tomography (microCT) scanning, (semi-)automated segmentation, and 3D rendering of the portal venous and biliary networks. MicroCT results in 3D coordinate data for the two resins allowing for qualitative as well as quantitative analysis of the two systems and their spatial relationship. DUCT can be applied to postnatal and adult mouse liver and can be further extended to other tubular networks, for example, vascular networks and airways in the lungs.

Spatially segregated defects and IGF1‐responsiveness of hilar and peripheral biliary organoids from a model of Alagille syndrome

“…Hilar bile ducts are present around P15 in Jag1 Ndr/Ndr mice, 21 while most peripheral bile ducts are de novo ‐generated later, and we therefore expected Jag1 Ndr/Ndr pICOs to exhibit the most divergent signatures. However, Jag1 Ndr/Ndr pICOs, derived from peripheral tortuous de novo ‐generated bile ducts, 21,51 were more similar to Jag1 +/+ ICOs than Jag1 Ndr/Ndr hICOs (Figure 1D). Contrary to our expectations, Jag1 Ndr/Ndr hICOs were highly divergent (Figures 1D,F, 2–6), less proliferative (Figure 2B–E), and hepatocyte‐like (Figures 5 and 6).…”