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LIVER Publication-derived

3D Organoid Cultivation for Cholangiocyte Maturation and Functional Differentiation from Human Pluripotent Stem Cells

Source Budi et al., 2024 · Taipei Medical University · 10.3389/fcell.2024.1361084

👤 Nova Yuli Prasetyo Budi, Wei-Yu Lai, Yen-Hua Huang, Hong-Nerng Ho ⏱ 28 days 📋 6 phases 🧫 Human iPSC, Human ESC

Abstract

This protocol describes a 28-day feeder-free, serum-free differentiation method to generate functional cholangiocyte organoids from human pluripotent stem cells through five sequential differentiation stages. The 3D culture system produces mature cholangiocytes with enhanced expression of biliary markers, region-specific cholangiocyte characteristics, and functional transport activity suitable for modeling cholangiopathies.

Cell source
Human iPSC, Human ESC
Application
Disease modeling, Drug screening, Cell therapy development

Protocol overview

17 steps across 6 phases

Stage 1: Definitive Endoderm (DE) Induction Days 1–4
  1. 1 Activate definitive endoderm differentiation
  2. 2 Continue DE maturation with Activin A and bFGF
  3. 3 Verify DE differentiation by flow cytometry
Stage 2: Ventral Foregut Endoderm (VFE) Differentiation Days 5–8
  1. 1 Induce VFE differentiation
  2. 2 Confirm VFE specification by marker expression
Stage 3: Hepatoblast (HB) Differentiation Days 9–13
  1. 1 Activate hepatoblast differentiation pathways
  2. 2 Characterize hepatoblasts at day 13
Stage 4: Cholangiocyte Progenitor (CP) Commitment Days 14–17
  1. 1 Activate Notch and FGF signaling for biliary lineage
  2. 2 Verify cholangiocyte progenitor specification
Stage 5: Mature Cholangiocyte Organoid (CO) Generation via 3D Culture Days 18–28
  1. 1 Dissociate CPs into small clumps for 3D culture
  2. 2 Culture CP clumps in Matrigel-based 3D system
  3. 3 Monitor organoid formation and maturation
  4. 4 Verify mature cholangiocyte markers at day 28
Functional Assessment of Cholangiocyte Organoids Day 28 (post-harvest)
  1. 1 Rhodamine-123 MDR1 transporter activity assay
  2. 2 Wash and image organoids
  3. 3 Forskolin-induced swelling assay for CFTR function
  4. 4 Analyze swelling data

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Budi et al., 2024. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

This wording is awaiting legal review.

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