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LIVER Publication-derived

Human branching cholangiocyte organoids recapitulate functional bile duct formation

Source Roos et al., 2022 · Erasmus MC Transplant Institute, University Medical Center Rotterdam, Department of Surgery · 10.1016/j.stem.2022.04.011

👤 Floris J.M. Roos, Gilles S. van Tienderen, Haoyu Wu, Ignacio Bordeu, Dina Vinke, Laura Muñoz Albarinos, Kathryn Monfils, Sabrah Niesten, Ron Smits, Jorke Willemse, Oskar Rosmark, Gunilla Westergren-Thorsson, Daniel J. Kunz, Maurice de Wit, Pim J. French, Ludovic Vallier, Jan N.M. IJzermans, Richard Bartfai, Hendrik Marks, Ben D. Simons, Martin E. van Royen, Monique M.A. Verstegen, Luc J.W. van der Laan ⏱ 14 days 📋 6 phases 🧫 Human adult intrahepatic cholangiocytes (from liver biopsies and transplant specimens)

Abstract

This protocol establishes branching cholangiocyte organoids (BRCOs) from human adult intrahepatic cholangiocytes through a two-stage culture method. BRCOs self-organize into complex tubular structures resembling functional bile ducts in vivo, exhibit cholangiocyte markers and ion transport properties, and recapitulate branching morphogenesis driven by JAG1/NOTCH2 signaling. The protocol enables in vitro modeling of biliary development, disease, and drug responses.

Cell source
Human adult intrahepatic cholangiocytes (from liver biopsies and transplant specimens)
Application
Disease modeling; Developmental study; Drug screening for cholangiocarcinoma

Protocol overview

18 steps across 6 phases

Tissue Dissociation and Initial Organoid Culture Days 0-3
  1. 1 Tissue Digestion
  2. 2 Basement Membrane Embedding
  3. 3 Start-up Expansion Medium Culture
Intrahepatic Cholangiocyte Organoid (ICO) Expansion Days 3-9 (3 passages)
  1. 4 Medium Switch to Canonical-WNT Stimulating Medium
  2. 5 Mechanical Dissociation and Passage
Induction of Branching Morphology (BRCO Formation) Days 9+ (from passage 3 onwards)
  1. 6 Medium Switch to Branching-Initiation Medium
  2. 7 Branching Morphogenesis Culture
  3. 8 Long-term Passaging of BRCOs
Functional Characterization of BRCOs Days 10+ (after branching is established)
  1. 9 Immunofluorescence Staining for Cholangiocyte Markers
  2. 10 Quantitative RT-PCR (qRT-PCR) Analysis
  3. 11 Rhodamine-123 Transport Assay (MDR1 Transporter Function)
  4. 12 Secretin/Somatostatin-Stimulated Swelling Assay
  5. 13 Ussing Chamber Analysis of Ion Channel Activity
  6. 14 Alkaline Phosphatase (ALP) Activity Assay
  7. 15 Gamma-Glutamyltransferase (GGT) Activity Assay
NOTCH Pathway Validation and Branching Morphogenesis Inhibition Days 10+ (after branching is established)
  1. 16 DAPT (Notch Inhibitor) Treatment
Drug Sensitivity Testing (for Cholangiocarcinoma Organoids) Day 14+ (after BRCCAO formation from CCA tissue)
  1. 17 Gemcitabine and Cisplatin Incubation
  2. 18 Dose-Response Analysis and IC50 Determination

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Roos et al., 2022. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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