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LIVER Publication-derived

Modeling Human Nonalcoholic Fatty Liver Disease (NAFLD) with an Organoids-on-chip System

Source Wang et al., 2020 · Dalian Institute of Chemical Physics, Chinese Academy of Sciences

👤 Yaqing Wang, Hui Wang, Pengwei Deng, Tingting Tao, Haitao Liu, Shuo Wu, Wenwen Chen, Jianhua Qin ⏱ 35 days 📋 10 phases 🧫 Human iPSC

Abstract

This protocol describes the generation of human induced pluripotent stem cell (hiPSC)-derived liver organoids on a microfluidic chip system and their exposure to free fatty acids (FFAs) to model nonalcoholic fatty liver disease (NAFLD). The system enables characterization of NAFLD pathological features including lipid droplet formation, triglyceride accumulation, altered lipid metabolism, oxidative stress, inflammatory responses, and fibrogenic markers under perfused 3D culture conditions.

Cell source
Human iPSC
Application
Disease modeling

Protocol overview

37 steps across 10 phases

Chip Fabrication and Preparation Day -3 to Day 0
  1. 1 Fabricate PDMS chip layers
  2. 2 Assemble chip layers
  3. 3 Sterilize and treat chip with PF-127
iPSC Culture and Preparation Day -7 to Day 0
  1. 1 Culture hiPSCs in mTeSR1 medium
  2. 2 Passage hiPSCs
Hepatic Differentiation (2D Stage) Day 0 to Day 10
  1. 1 Definitive endoderm differentiation
  2. 2 Hepatic endoderm differentiation
  3. 3 Hepatic progenitor expansion
  4. 4 Dissociate and resuspend hepatic progenitors
3D Liver Organoid Formation on Chip Day 10 to Day 23
  1. 1 Seed cells on micropillar chip
  2. 2 Allow 3D aggregate formation overnight
  3. 3 Initiate perfusion culture
  4. 4 Facilitate hepatocyte differentiation (days 13-18)
  5. 5 Mature organoid culture (days 18+)
Free Fatty Acid (FFA) Exposure for NAFLD Modeling Day 23 to Day 30
  1. 1 Prepare FFA-supplemented medium
  2. 2 Expose organoids to FFAs
  3. 3 Collect samples for analysis
Lipid Accumulation and Triglyceride Analysis Day 23 to Day 30
  1. 1 Oil Red O staining
  2. 2 Image lipid droplets
  3. 3 Measure triglyceride accumulation
Gene Expression Analysis Day 15 to Day 30
  1. 1 Collect samples and extract mRNA
  2. 2 Synthesize cDNA
  3. 3 Perform real-time quantitative PCR
  4. 4 Data analysis and fold-change calculation
Immunohistochemical Staining and Analysis Day 15 to Day 30
  1. 1 Prepare cryosections
  2. 2 Incubate with primary antibodies
  3. 3 Incubate with secondary antibodies
  4. 4 Stain nuclei with DAPI
  5. 5 Visualize and acquire confocal images
  6. 6 Analyze images
Oxidative Stress and Cytotoxicity Assessment Day 23 to Day 30
  1. 1 Measure ROS levels with DCFH-DA
  2. 2 Acquire ROS fluorescence images
  3. 3 Quantify ROS levels
  4. 4 Measure LDH release (cytotoxicity marker)
Albumin Secretion Assay Day 11 to Day 35
  1. 1 Collect culture supernatant
  2. 2 Measure albumin with ELISA
  3. 3 Analyze albumin secretion data

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Wang et al., 2020. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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