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LIVER Publication-derived

Development of NAFLD-Specific Human Liver Organoid Models on a Microengineered Array Chip for Semaglutide Efficacy Evaluation

Source You et al., 2025 · Henan University of Science and Technology; Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences · 10.1111/cpr.70118

👤 Xiao-yan You, Xiang-yang Li, Hui Wang, Guo-ping Zhao ⏱ 22 days 📋 14 phases 🧫 Human iPSC

Abstract

This protocol describes the generation of human liver organoids (hLOs) from induced pluripotent stem cells (hiPSCs) using microengineered array chips, followed by induction of non-alcoholic fatty liver disease (NAFLD) via free fatty acid exposure and evaluation of semaglutide efficacy as a therapeutic intervention. The system recapitulates NAFLD pathology including steatosis, inflammatory responses, and fibrogenic activation, enabling robust preclinical drug evaluation.

Cell source
Human iPSC
Application
Disease modeling; Drug screening

Protocol overview

63 steps across 14 phases

Microporous Array Chip Fabrication and Preparation Preparation (pre-experiment)
  1. 1 Prepare PDMS prepolymer mixture
  2. 2 Cast PDMS into mold and degas
  3. 3 Thermal curing of PDMS chips
  4. 4 Sterilize chips and combine with culture plates
  5. 5 UV treatment
  6. 6 Surface functionalization with PF127
hiPSC Culture and Expansion Days -7 to 0 (pre-differentiation)
  1. 1 Coat culture plates with Matrigel
  2. 2 Equilibrate plates in mTeSR1 medium
  3. 3 Seed hiPSCs
  4. 4 Maintain hiPSC cultures with daily medium changes
  5. 5 Passage hiPSCs at 80–90% confluence
  6. 6 Post-dissociation viability enhancement
Liver Organoid Differentiation in Array Chips (Days 0–20) Days 0–20
  1. 1 Prepare and seed hiPSCs into array chips (Day 0, Stage 0)
  2. 2 Stage 1: Definitive Endoderm Induction (Days 0–5)
  3. 3 Stage 2: Hepatic Progenitor Differentiation (Days 5–10)
  4. 4 Stage 3: Hepatocyte Maturation Phase 1 (Days 10–15)
  5. 5 Stage 4: Hepatocyte Maturation Phase 2 (Days 15–20)
  6. 6 Daily monitoring of hLO maturation
NAFLD Induction via Free Fatty Acid (FFA) Exposure (Days 18–20) Days 18–20
  1. 1 Prepare FFA mixture
  2. 2 FFA treatment of hLOs
  3. 3 Prepare control organoids
Drug Intervention and NAFLD Model Validation (Days 20–22) Days 20–22
  1. 1 Replace FFA medium with normal culture medium
  2. 2 Prepare semaglutide working solutions
  3. 3 Treat NAFLD hLOs with semaglutide or positive control
Sample Collection and Immunohistochemical Analysis Days 0–22 (at each differentiation stage and after drug treatment)
  1. 1 Harvest and fix organoid spheroids
  2. 2 Rinse fixed samples with DPBS
  3. 3 Cryoprotect samples in sucrose solution
  4. 4 Embed samples in OCT compound
  5. 5 Flash-freeze in liquid nitrogen and store
  6. 6 Prepare cryosections
Immunofluorescence Staining Protocol After cryosectioning (variable timing)
  1. 1 Permeabilize and block sections
  2. 2 Apply primary antibodies
  3. 3 Wash sections after primary antibody incubation
  4. 4 Apply secondary antibodies
  5. 5 Counterstain nuclei with DAPI
  6. 6 Mount slides in glycerol-DPBS and image
Lipid Droplet Visualization (BODIPY and Oil Red O Staining) After cryosectioning
  1. 1 Block sections for lipid staining
  2. 2 BODIPY staining for lipid droplets
  3. 3 Oil Red O staining for lipid droplets
Histological Staining (PAS and H&E) After cryosectioning
  1. 1 Periodic Acid-Schiff (PAS) staining
  2. 2 Haematoxylin and eosin (H&E) staining
  3. 3 Image stained sections
RNA Extraction and Real-Time Quantitative PCR (qRT-PCR) Days 0–22 (at each differentiation stage and post-treatment)
  1. 1 Extract total RNA
  2. 2 Synthesize cDNA
  3. 3 Perform qRT-PCR
Transcriptomic Analysis (RNA Sequencing) Days 20–22 (post-drug treatment)
  1. 1 Extract RNA from FFA-treated and semaglutide-treated hLOs
  2. 2 Prepare RNA for sequencing
  3. 3 Perform paired-end RNA sequencing
Viability Assessment (Live-Dead Staining) Day 20
  1. 1 Wash organoids with DPBS
  2. 2 Apply Live-Dead staining
  3. 3 Image live-dead stained organoids
Biochemical Analysis (Albumin, Urea, Lipids, Protein) Days 10 and 20 (media); Day 22 (lysates for lipids)
  1. 1 Collect media for albumin and urea quantification
  2. 2 Measure albumin secretion
  3. 3 Measure urea synthesis
  4. 4 Lyse organoids for lipid quantification
  5. 5 Measure total cholesterol (TC)
  6. 6 Measure triglycerides (TG)
  7. 7 Normalize lipid data to total protein
Data Analysis and Statistical Evaluation Post-experiment (variable timing)
  1. 1 Compile and organize experimental data
  2. 2 Perform statistical testing
  3. 3 Calculate descriptive statistics
  4. 4 Perform transcriptomic analysis (KEGG and GO enrichment)
  5. 5 Generate visualizations

Full SOP

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Attribution

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

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