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LIVER Publication-derived

Fatty Liver Disease Organoids for Developmental Studies

Full SOP with Academic or a plan

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

Authors: Xiao-yan You, Xiang-yang Li, Hui Wang, Guo-ping Zhao Duration: 22 days 14 phases Cell source: 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

Phase 1 · Microporous Array Chip Fabrication and Preparation Preparation (pre-experiment)
  1. a Prepare PDMS prepolymer mixture
  2. b Cast PDMS into mold and degas
  3. c Thermal curing of PDMS chips
  4. d Sterilize chips and combine with culture plates
  5. e UV treatment
  6. f Surface functionalization with PF127
Phase 2 · hiPSC Culture and Expansion Days -7 to 0 (pre-differentiation)
  1. a Coat culture plates with Matrigel
  2. b Equilibrate plates in mTeSR1 medium
  3. c Seed hiPSCs
  4. d Maintain hiPSC cultures with daily medium changes
  5. e Passage hiPSCs at 80-90% confluence
  6. f Post-dissociation viability enhancement
Phase 3 · Liver Organoid Differentiation in Array Chips (Days 0-20) Days 0-20
  1. a Prepare and seed hiPSCs into array chips (Day 0, Stage 0)
  2. b Stage 1: Definitive Endoderm Induction (Days 0-5)
  3. c Stage 2: Hepatic Progenitor Differentiation (Days 5-10)
  4. d Stage 3: Hepatocyte Maturation Phase 1 (Days 10-15)
  5. e Stage 4: Hepatocyte Maturation Phase 2 (Days 15-20)
  6. f Daily monitoring of hLO maturation
Phase 4 · NAFLD Induction via Free Fatty Acid (FFA) Exposure (Days 18-20) Days 18-20
  1. a Prepare FFA mixture
  2. b FFA treatment of hLOs
  3. c Prepare control organoids
Phase 5 · Drug Intervention and NAFLD Model Validation (Days 20-22) Days 20-22
  1. a Replace FFA medium with normal culture medium
  2. b Prepare semaglutide working solutions
  3. c Treat NAFLD hLOs with semaglutide or positive control
Phase 6 · Sample Collection and Immunohistochemical Analysis Days 0-22 (at each differentiation stage and after drug treatment)
  1. a Harvest and fix organoid spheroids
  2. b Rinse fixed samples with DPBS
  3. c Cryoprotect samples in sucrose solution
  4. d Embed samples in OCT compound
  5. e Flash-freeze in liquid nitrogen and store
  6. f Prepare cryosections
Phase 7 · Immunofluorescence Staining Protocol After cryosectioning (variable timing)
  1. a Permeabilize and block sections
  2. b Apply primary antibodies
  3. c Wash sections after primary antibody incubation
  4. d Apply secondary antibodies
  5. e Counterstain nuclei with DAPI
  6. f Mount slides in glycerol-DPBS and image
Phase 8 · Lipid Droplet Visualization (BODIPY and Oil Red O Staining) After cryosectioning
  1. a Block sections for lipid staining
  2. b BODIPY staining for lipid droplets
  3. c Oil Red O staining for lipid droplets
Phase 9 · Histological Staining (PAS and H&E) After cryosectioning
  1. a Periodic Acid-Schiff (PAS) staining
  2. b Haematoxylin and eosin (H&E) staining
  3. c Image stained sections
Phase 10 · RNA Extraction and Real-Time Quantitative PCR (qRT-PCR) Days 0-22 (at each differentiation stage and post-treatment)
  1. a Extract total RNA
  2. b Synthesize cDNA
  3. c Perform qRT-PCR
Phase 11 · Transcriptomic Analysis (RNA Sequencing) Days 20-22 (post-drug treatment)
  1. a Extract RNA from FFA-treated and semaglutide-treated hLOs
  2. b Prepare RNA for sequencing
  3. c Perform paired-end RNA sequencing
Phase 12 · Viability Assessment (Live-Dead Staining) Day 20
  1. a Wash organoids with DPBS
  2. b Apply Live-Dead staining
  3. c Image live-dead stained organoids
Phase 13 · Biochemical Analysis (Albumin, Urea, Lipids, Protein) Days 10 and 20 (media); Day 22 (lysates for lipids)
  1. a Collect media for albumin and urea quantification
  2. b Measure albumin secretion
  3. c Measure urea synthesis
  4. d Lyse organoids for lipid quantification
  5. e Measure total cholesterol (TC)
  6. f Measure triglycerides (TG)
  7. g Normalize lipid data to total protein
Phase 14 · Data Analysis and Statistical Evaluation Post-experiment (variable timing)
  1. a Compile and organize experimental data
  2. b Perform statistical testing
  3. c Calculate descriptive statistics
  4. d Perform transcriptomic analysis (KEGG and GO enrichment)
  5. e Generate visualizations

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Attribution

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

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