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LIVER Publication-derived

Fatty Liver Disease Organ-on-Chip Models

Full SOP with Academic or a plan

Source Wang et al., 2024 · Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences · 10.1016/j.engreg.2024.06.002

Authors: Hui Wang, Yue Zhu, Pengcheng Shi, Xiangyang Li, Qingyun Bu, Yachun Li, Xiaoyan You, Guoping Zhao Duration: 30 days 6 phases Cell source: Human iPSC

Abstract

This protocol describes the generation of hiPSC-derived liver organoids (LOs) on a microfluidic chip platform, modeling free fatty acid (FFA)-induced nonalcoholic fatty liver disease (NAFLD), and evaluating the therapeutic effects of ginsenoside Rb1. The LOs-on-a-chip system recapitulates key pathological features of NAFLD including steatosis, inflammation, and fibrosis, and allows high-throughput assessment of drug efficacy.

Cell source
Human iPSC
Application
Disease modeling and drug screening

Protocol overview

34 steps across 6 phases

Phase 1 · Microfluidic chip fabrication and preparation Pre-culture
  1. a PDMS mixture preparation
  2. b PDMS curing
  3. c Layer bonding via oxygen plasma treatment
  4. d Chip sterilization option 1: Autoclave
  5. e Chip sterilization option 2: UV exposure
  6. f Chip surface hydrophobization
Phase 2 · HiPSC culture and expansion Days -5 to 0
  1. a Matrigel coating plate preparation
  2. b Matrigel coating incubation
  3. c HiPSC seeding on Matrigel-coated plates
  4. d Daily medium changes
  5. e HiPSC passage at 80-90% confluence
  6. f HiPSC subculture at dilution ratio
  7. g Y27632 treatment post-passage
  8. h Medium replacement after Y27632 treatment
Phase 3 · HiPSC differentiation to liver organoids (Days 0-10) Days 0-10
  1. a Initial hiPSC seeding at 1:6 dilution
  2. b Definitive endoderm induction (Days 0-5)
  3. c Liver-specific differentiation (Days 5-8)
  4. d Hepatic progenitor amplification (Days 8-10)
  5. e Cell dissociation and harvesting (Day 10)
Phase 4 · Liver organoid formation and maturation on chip (Days 10-30) Days 10-30
  1. a Cell seeding into microfluidic chip
  2. b 3D spheroid formation (Day 10-11, overnight)
  3. c Perfusion initiation (Days 10-13)
  4. d Hepatocyte maturation medium introduction (Day 13)
  5. e OSM removal (Day 18)
  6. f Physiological LO maturation (Days 18-30)
Phase 5 · NAFLD disease modeling with free fatty acids (Days 20-25) Days 20-25
  1. a FFA preparation
  2. b FFA exposure initiation (Day 20)
  3. c Control group preparation
  4. d FFA exposure duration (Days 20-25)
Phase 6 · Drug treatment with ginsenoside Rb1 and controls (Days 25-30) Days 25-30
  1. a Drug treatment initiation (Day 25)
  2. b Positive control drug treatment
  3. c Control groups during drug treatment
  4. d Drug exposure duration (Days 25-30)
  5. e Organoid harvest (Day 30)

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Attribution

This SOP was written by Organthis based on the published method in Wang et al., 2024. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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