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LIVER Publication-derived

Biliary Organoids for Drug Screening with Enhanced Maturation

Full SOP with Academic or a plan

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

Authors: Nova Yuli Prasetyo Budi, Wei-Yu Lai, Yen-Hua Huang, Hong-Nerng Ho Duration: 28 days 6 phases Cell source: 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

Phase 1 · Stage 1: Definitive Endoderm (DE) Induction Days 1-4
  1. a Activate definitive endoderm differentiation
  2. b Continue DE maturation with Activin A and bFGF
  3. c Verify DE differentiation by flow cytometry
Phase 2 · Stage 2: Ventral Foregut Endoderm (VFE) Differentiation Days 5-8
  1. a Induce VFE differentiation
  2. b Confirm VFE specification by marker expression
Phase 3 · Stage 3: Hepatoblast (HB) Differentiation Days 9-13
  1. a Activate hepatoblast differentiation pathways
  2. b Characterize hepatoblasts at day 13
Phase 4 · Stage 4: Cholangiocyte Progenitor (CP) Commitment Days 14-17
  1. a Activate Notch and FGF signaling for biliary lineage
  2. b Verify cholangiocyte progenitor specification
Phase 5 · Stage 5: Mature Cholangiocyte Organoid (CO) Generation via 3D Culture Days 18-28
  1. a Dissociate CPs into small clumps for 3D culture
  2. b Culture CP clumps in Matrigel-based 3D system
  3. c Monitor organoid formation and maturation
  4. d Verify mature cholangiocyte markers at day 28
Phase 6 · Functional Assessment of Cholangiocyte Organoids Day 28 (post-harvest)
  1. a Rhodamine-123 MDR1 transporter activity assay
  2. b Wash and image organoids
  3. c Forskolin-induced swelling assay for CFTR function
  4. d Analyze swelling data

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Attribution

This SOP was written by Organthis based on the published method in Budi et al., 2024. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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