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LIVER Publication-derived

Alcohol-Related Liver Disease Organoids for Drug Screening

Full SOP with Academic or a plan

Source Feng et al., 2024 · Shanxi Medical University · 10.3724/abbs.2024074

Authors: Zhiwei Feng, Bingrui Zhou, Qizhi Shuai, Yunliang Wei, Ning Jin, Xiaoling Wang, Hong Zhao, Zhizhen Liu, Jun Xu, Jianbing Mu, Jun Xie Duration: 50 days 8 phases Cell source: Human iPSC

Abstract

This protocol describes the differentiation of human induced pluripotent stem cells (hiPSCs) into three-dimensional liver organoids containing hepatocytes, cholangiocytes, hepatic stellate cells, and Kupffer cells. The resulting organoids are treated with ethanol to establish an in vitro model of alcoholic liver disease (ALD) that recapitulates key pathological features including hepatocyte steatosis, mitochondrial damage, elevated reactive oxygen species, and cell necrosis. The model demonstrates applicability for drug screening and therapeutic evaluation.

Cell source
Human iPSC
Application
Disease modeling (Alcoholic Liver Disease) and drug screening

Protocol overview

25 steps across 8 phases

Phase 1 · hiPSC Culture and Maintenance Ongoing (passages 30-50)
  1. a Preparation of culture plates with Matrigel coating
  2. b Seed hiPSC-B1 cells on Matrigel-coated plates
  3. c Culture hiPSCs in mTeSR medium
  4. d Passage hiPSCs
Phase 2 · Definitive Endoderm (DE) Differentiation Days 1-3
  1. a Initiate DE differentiation on Day 1
  2. b Change medium on Day 2
  3. c Change medium on Day 3
Phase 3 · Foregut Endoderm (FG) Differentiation Days 4-6
  1. a Culture in FG differentiation medium
Phase 4 · 3D Hepatic Progenitor Cell Expansion Days 7-8
  1. a Dissociate foregut spheroids on Day 7
  2. b Embed dissociated cells in Matrigel
  3. c Culture for 2 days (Days 7-9)
Phase 5 · Retinoic Acid (RA) Treatment for Hepatic Maturation Days 9-12
  1. a Replace medium with RA-containing medium on Day 9
  2. b Incubate for 4 days. Renew the medium on Day 11.
Phase 6 · Hepatocyte Maturation and Organoid Consolidation Days 13-25
  1. a Switch to hepatocyte culture medium on Day 13
  2. b Isolate liver organoids from Matrigel on Day 16
  3. c Passage organoids on Day 16
  4. d Change medium every 3 days
  5. e Organoids ready for use (Days 22-25)
Phase 7 · Ethanol Treatment for Alcoholic Liver Disease (ALD) Model Induction Days 27-30
  1. a Initiate ethanol treatment on Day 27
  2. b Maintain ethanol treatment for 72 hours
  3. c Harvest organoids after 72 hours ethanol treatment
Phase 8 · Drug Treatment and Efficacy Assessment (Optional) Days 27-30 (concurrent with ethanol treatment)
  1. a Prepare ALD model with concurrent drug treatment
  2. b Define drug dosing (example doses)
  3. c Maintain cultures for 3 days with drug treatment
  4. d Assess drug efficacy and organoid viability

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Attribution

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

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