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LIVER Publication-derived

Fatty Liver Disease Organoids

Full SOP with Academic or a plan

Source Qin et al., 2025 · Department of Gastroenterology and Hepatology, Guangzhou Digestive Disease Center, the Second Affiliated Hospital, School of Medicine, South China University of Technology · 10.1186/s13287-025-04865-7

Authors: Changlu Qin, Haibin Wu, Peilin Liao, Wenjiao Yan, Xinyi Shi, Jinghe Xie, Shoupei Liu, Sen Chen, Xiangting Cao, Yongjian Zhou, Yuyou Duan Duration: 15 days 7 phases Cell source: Human ESC (H9 line)

Abstract

This protocol describes the differentiation of human embryonic stem cells (hESC) into mature polarized hepatocyte organoids (P-hep-orgs) and their use as an in vitro model of metabolic dysfunction-associated steatotic liver disease (MASLD). P-hep-orgs are exposed to free fatty acids (FFAs) to recapitulate key pathological features of MASLD progression including lipid accumulation, oxidative stress, apoptosis, and hepatic dysfunction. The model is validated for drug screening applications using known antioxidant and lipid-lowering agents.

Cell source
Human ESC (H9 line)
Application
Disease modeling; Drug screening

Protocol overview

18 steps across 7 phases

Phase 1 · hESC Culture and Maintenance Ongoing
  1. a hESC Culture Setup
  2. b hESC Passaging
Phase 2 · Definitive Endoderm (DE) Differentiation Days 0-3
  1. a hESC Dissociation and Aggregate Formation
  2. b Y-27632 Removal
  3. c DE Differentiation Day 0-1
  4. d DE Differentiation Day 1-2
  5. e DE Differentiation Day 2-3
Phase 3 · Hepatoblast (HB) Sphere Formation Days 3-9
  1. a HB Sphere Medium Preparation and Culture
Phase 4 · Hepatoblast Organoid (HB-org) Expansion Days 9+
  1. a HB Sphere Dissociation and HB-org Expansion Medium Preparation
  2. b Matrigel Addition and ECM Support
  3. c Daily Medium Replacement and Long-term Culture
  4. d HB-org Passaging
Phase 5 · Polarized Hepatocyte Organoid (P-hep-org) Maturation Days 9-15 (6 days maturation)
  1. a Initiation of P-hep-org Maturation
  2. b Medium Replacement During Maturation
Phase 6 · MASLD Induction with Free Fatty Acids (FFAs) Variable (2-10 days based on experimental endpoint)
  1. a FFA Preparation and Application
  2. b MASLD Phenotype Development
Phase 7 · Drug Treatment and Efficacy Assessment Variable (concurrent with FFA treatment)
  1. a Drug Preparation and Cytotoxicity Testing
  2. b Co-treatment with FFAs and Drugs

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Attribution

This SOP was written by Organthis based on the published method in Qin et al., 2025. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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