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LIVER Publication-derived

Alcohol-Induced Liver Injury Organ-on-Chip Models

Full SOP with Academic or a plan

Source Yang et al., 2025 · Shanghai University of Traditional Chinese Medicine · 10.1002/advs.202511169

Authors: Kangdi Yang, Xiayan Chu, Xuerui Wang, Wenkun Zhang, Jinnuo Lu, Chuting Xu, Shoucheng Hu, Guoyu Pan, Chih-Tsung Yang, Xiaohui Zhang, Shaojin Li, Zhaobin Guo, Hanyang Liu, Guangbo Ge Duration: 26 days 14 phases Cell source: Human reprogrammed hepatocyte-like cells (hrHL) co-cultured with HUVECs and HUMSCs

Abstract

This protocol describes the construction of tri-culture vascularized liver organoids (3HLOs) by co-culturing human reprogrammed hepatocyte-like cells (hrHLs) with endothelial cells (HUVECs) and mesenchymal stem cells (HUMSCs). The resulting organoids develop functional vascular networks (CD31+ endothelial cells), biliary structures (CK19+), and enhanced hepatic functions including glycogen storage, albumin secretion, and drug metabolism. The 3HLOs-on-chip platform enables modeling of alcohol-induced liver injury (ALI) pathogenesis and testing of hepatoprotective agents with high physiological relevance.

Cell source
Human reprogrammed hepatocyte-like cells (hrHL) co-cultured with HUVECs and HUMSCs
Application
Disease modeling (alcohol-induced liver injury), drug screening, regenerative therapy

Protocol overview

65 steps across 14 phases

Phase 1 · Cell Preparation and Seeding Day 0
  1. a Count hrHL cells
  2. b Pre-label cells with fluorescent dyes (optional)
  3. c Prepare cell mixture for 3HLOs
  4. d Seed cells into ultra-low-adhesion plates
Phase 2 · Organoid Formation and Maturation Day 0-5
  1. a Incubate organoids at 37°C with 5% CO₂
  2. b Monitor organoid formation and integration
  3. c Change culture medium if required
Phase 3 · Characterization of Organoid Structure and Function Day 5
  1. a Perform immunofluorescence staining for vascular markers
  2. b Assess hepatic maturation markers
  3. c Measure glycogen storage via PAS staining
  4. d Assess ICG uptake and excretion
  5. e Assess Rho-123 uptake and efflux
  6. f Perform H&E staining to visualize lumen structures
Phase 4 · Transcriptomic and Metabolomic Analysis Day 5
  1. a Extract RNA and perform transcriptomic analysis
  2. b Perform metabolomic analysis
Phase 5 · Microfluidic Organoid-on-Chip (OOC) Construction Day 0-7
  1. a Fabricate microfluidic chip device
  2. b Prepare collagen gel and embed organoids
  3. c Remove needle and establish microvessel channel
  4. d Seed HUVECs into the microvessel channel
Phase 6 · Assessment of Functional Anastomosis In Vitro Day 1-11 post-OOC setup
  1. a Monitor organoid-microvessel integration over time
  2. b Perform H&E staining of organoid-microvessel interface
  3. c Assess HUVEC monolayer integrity
  4. d Perform vascular permeability assay using FITC-dextran
Phase 7 · Subcutaneous Implantation and In Vivo Vascularization Day 0-10 post-implantation
  1. a Prepare organoids for implantation
  2. b Subcutaneously implant organoids in nude mice
  3. c Monitor organoid growth and integration
  4. d Harvest organoids and assess vascularization
  5. e Perform immunohistochemistry for vascular markers
Phase 8 · Biosafety Profiling of Implanted Organoids Day 0-10 post-implantation
  1. a Monitor body weight and organoid volume
  2. b Harvest organs and assess histopathology
  3. c Measure serum biomarkers for organ injury
  4. d Assess blood glucose levels
Phase 9 · Alcohol-Induced Liver Injury (ALI) Modeling In Vitro Day 5-8 (organoid-based assay)
  1. a Divide organoids into control and ethanol-exposure groups
  2. b Expose organoids to ethanol for 72 hours
  3. c Assess ROS accumulation using DCFH-DA probe
  4. d Assess mitochondrial depolarization using JC-10
  5. e Perform live/dead assay using Calcein-AM and PI
  6. f Quantify liver injury markers in culture supernatant
Phase 10 · Alcohol-Induced Liver Injury (ALI) Modeling Using 3HLOs-on-Chip Day 1-4 post-EtOH exposure
  1. a Establish ALI in 3HLOs-on-chip with perfused ethanol
  2. b Perform ROS, JC-10, and viability assays at 72 h
  3. c Assess vascular barrier integrity using FITC-dextran permeability
  4. d Collect perfusate for proteomic analysis
  5. e Measure ALT and AST in OOC perfusates
Phase 11 · Drug Screening Using ALI Model in 3HLOs-on-Chip Day 1-3 post-ethanol exposure
  1. a Initiate drug treatment on day 1 of ALI induction
  2. b Assess hepatoprotective efficacy using ROS, JC-10, and viability assays
  3. c Measure ALT and AST in drug-treated perfusates
Phase 12 · In Vivo ALI Model and Therapeutic Validation Day 0-26 (NIAAA protocol)
  1. a Establish ALI in C57BL/6J mice using NIAAA protocol
  2. b Monitor disease progression and determine transplantation timing
  3. c Perform subcutaneous organoid transplantation on day 5
  4. d Monitor survival and body weight recovery
  5. e Assess hepatic lipid accumulation via H&E staining
  6. f Measure liver-to-body weight ratio
  7. g Measure serum liver function markers
  8. h Assess organoid vascularization and anastomosis in vivo
  9. i Perform IHC for human vascular markers and host anastomosis
Phase 13 · Drug Validation in Vivo ALI Model Day 1-26 (NIAAA protocol with drug treatment)
  1. a Establish drug treatment groups in ALI mice
  2. b Monitor body weight and clinical signs
  3. c Assess liver morphology at endpoint
  4. d Perform H&E staining and quantify steatosis reduction
  5. e Measure serum biomarkers in drug-treated groups
  6. f Compare in vivo drug efficacy with 3HLOs-on-chip predictions
Phase 14 · Multi-Omics Analysis of 3HLOs Therapeutic Mechanisms Day 5 (organoid collection) to endpoint analysis
  1. a Perform proteomic profiling of organoid secretome
  2. b Analyze human proteome in mouse plasma
  3. c Perform transcriptomic analysis of organoid-derived immune modulation
  4. d Validate key therapeutic proteins (A1AT, AACT)

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Attribution

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

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