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LIVER Publication-derived

Novel Vascularized Human Liver Organoids for Modeling Alcohol-Induced Liver Injury

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

👤 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 ⏱ 26 days 📋 14 phases 🧫 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

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

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Yang et al., 2025. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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