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LIVER Publication-derived

Alcohol-Related Liver Disease Organoids for Developmental Studies

Full SOP with Academic or a plan

Source Wang et al., 2019 · Beijing Institute of Health Service and Transfusion Medicine · 10.1038/s41422-019-0242-8

Authors: Shuyong Wang, Xuan Wang, Zuolong Tan, Yuxin Su, Juan Liu, Mingyang Chang, Fang Yan, Jie Chen, Tao Chen, Chuanjiang Li, Jie Hu, Yunfang Wang Duration: 36 days 7 phases Cell source: Human ESC

Abstract

This protocol describes the generation of human embryonic stem cell-derived expandable hepatic organoids (hEHOs) using chemically defined, serum-free media. The hEHOs maintain bipotential hepatic stem/progenitor cell phenotypes, expand for over 20 passages, and can differentiate into functional hepatocytes or cholangiocytes. When co-cultured with human fetal liver mesenchymal cells (hFLMCs), the resulting hFLMC/hEHO model system recapitulates alcoholic liver disease-associated pathophysiology including oxidative stress, steatosis, inflammation, and fibrosis.

Cell source
Human ESC
Application
Disease modeling and therapeutic liver repopulation

Protocol overview

30 steps across 7 phases

Phase 1 · Definitive Endoderm (DE) Formation 0-3 days
  1. a Maintain H9 human ESCs in feeder-free conditions
  2. b Induce definitive endoderm differentiation—Day 1
  3. c Continue DE induction—Days 2-3
Phase 2 · Hepatic Specification (HS) Lineage Formation 3-8 days
  1. a Initiate hepatic specification with BMP4 and FGF2
  2. b Dissociate HS cells into single cells
Phase 3 · 3D Hepatic Organoid (hEHO) Formation and Expansion 8-20+ days (20 passages)
  1. a Embed HS cells in Matrigel droplets
  2. b Add organoid formation medium (NBNiGNaEWRAF)
  3. c Monitor organoid formation
  4. d Passage hEHOs every 7-9 days
  5. e Cryopreserve hEHOs
Phase 4 · In Vitro Differentiation of hEHOs into Mature Hepatocytes (hEHO-Heps) 20-36 days (relative to start of organoid formation)
  1. a Pretreat hEHOs with growth factors
  2. b Dissociate pretreated hEHOs and form 3D aggregates
  3. c Culture aggregates in hepatocyte maturation medium—Phase 1 (3 days)
  4. d Culture aggregates in hepatocyte maturation medium—Phase 2 (8 days)
Phase 5 · In Vitro Differentiation of hEHOs into Mature Cholangiocytes (hEHO-Clcs) 10-24 days (from start of organoid formation)
  1. a Dissociate hEHOs into single cells
  2. b Resuspend cells in type I collagen and prepare culture wells
  3. c Culture in cholangiocyte differentiation medium
Phase 6 · Co-culture of hEHOs with Human Fetal Liver Mesenchymal Cells (hFLMCs) for ALD Modeling Days 0-14 (organoid formation and maturation)
  1. a Isolate and prepare hFLMCs
  2. b Coat wells with sterile agarose
  3. c Dissociate hEHOs and hFLMCs; resuspend at 2:1 ratio
  4. d Seed cells in agarose-coated wells and culture—Phase 1 (7 days)
  5. e Switch to maturation medium—Phase 2 (7 days)
  6. f Treat hFLMC/hEHO organoids with ethanol (EtOH) for ALD modeling
  7. g Assess ALD-related pathophysiology at day 7 of EtOH treatment
Phase 7 · In Vivo Transplantation of hEHOs into FRG Mice Variable (3 months post-transplantation for analysis)
  1. a Prepare FRG mice and NTBC treatment
  2. b Dissociate hEHOs and prepare cell suspension
  3. c Transplant hEHOs into liver parenchyma
  4. d Initiate NTBC withdrawal schedule
  5. e Monitor survival, weight, and serum markers
  6. f Harvest liver samples at 3 months post-transplantation

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Attribution

This SOP was written by Organthis based on the published method in Wang et al., 2019. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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