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LIVER Publication-derived

Steatohepatitis Liver Organoids for Developmental Studies

Full SOP with Academic or a plan

Source Ouchi et al., 2019 · Cincinnati Children's Hospital Medical Center · 10.1016/j.cmet.2019.05.007

Authors: Rie Ouchi, Shodai Togo, Masaki Kimura, Tadahiro Shinozawa, Masaru Koido, Hiroyuki Koike, Wendy Thompson, Rebekah A. Karns, Christopher N. Mayhew, Patrick S. McGrath, Heather A. McCauley, Ran-Ran Zhang, Kyle Lewis, Shoyo Hakozaki, Autumn Ferguson, Norikazu Saiki, Yosuke Yoneyama, Ichiro Takeuchi, Yo Mabuchi, Chihiro Akazawa, Hiroshi Y. Yoshikawa, James M. Wells, Takanori Takebe Duration: 30 days 5 phases Cell source: Human iPSC, Human ESC

Abstract

This protocol describes the generation of multi-cellular human liver organoids (HLOs) from pluripotent stem cells, containing hepatocyte-like, stellate-like, and Kupffer-like cells. The organoids recapitulate progressive features of steatohepatitis including steatosis, inflammation, and fibrosis when exposed to free fatty acids, and can model genetic liver diseases such as Wolman disease.

Cell source
Human iPSC, Human ESC
Application
Disease modeling

Protocol overview

22 steps across 5 phases

Phase 1 · Definitive Endoderm Induction Day 0-6
  1. a Cell dissociation and plating
  2. b Day 1 endoderm induction
  3. c Day 2 endoderm induction
  4. d Day 3 endoderm induction
  5. e Days 4-6 foregut specification
Phase 2 · Liver Organoid (HLO) Induction Day 6-20
  1. a Spheroid collection and embedding
  2. b Retinoic acid treatment
  3. c Switch to hepatocyte maturation medium
  4. d HLO maturation culture
Phase 3 · Free Fatty Acid (FFA) Exposure and Steatohepatitis Induction Day 20-27
  1. a Prepare organoids for FFA treatment
  2. b Oleic acid treatment initiation
  3. c Steatohepatitis phenotype assessment (Day 3)
  4. d Fibrosis marker assessment (Day 5-7)
Phase 4 · Treatment with FXR Agonist (Obeticholic Acid / OCA) and FGF19 Day 20-27
  1. a Prepare Wolman disease organoids
  2. b FGF19 treatment
  3. c Assess FGF19 rescue at day 7
  4. d Viability assessment (Day 12)
Phase 5 · Organoid Stiffness Measurement by Atomic Force Microscopy (AFM) Day 20-30
  1. a Prepare fibronectin-coated AFM substrate
  2. b Transfer organoids to AFM substrate
  3. c AFM cantilever setup
  4. d AFM indentation measurement
  5. e Young's modulus calculation

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Attribution

This SOP was written by Organthis based on the published method in Ouchi et al., 2019. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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