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LIVER Publication-derived

Steatohepatitis Liver Organoids for Drug Screening

Full SOP with Academic or a plan

Source Bin Ramli et al., 2020 · Genome Institute of Singapore · 10.1053/j.gastro.2020.06.010

Authors: Muhammad Nadzim Bin Ramli, Yee Siang Lim, Chwee Tat Koe, Deniz Demircioglu, Weiquan Tng, Kevin Andrew Uy Gonzales, Cheng Peow Tan, Iwona Szczerbinska, Hongqing Liang, Einsi Lynn Soe, Zhiping Lu, Chaiyaboot Ariyachet, Ka Man Yu, Shu Hui Koh, Lai Ping Yaw, Nur Halisah Binte Jumat, John Soon Yew Lim, Graham Wright, Asim Shabbir, Yock Young Dan, Huck-Hui Ng, Yun-Shen Chan Duration: 57 days 12 phases Cell source: Human ESC (WA-01) and Human iPSC (GM23338)

Abstract

This protocol describes a stepwise, chemically defined approach to generate functional hepatic organoids from human pluripotent stem cells comprising hepatocytes and cholangiocytes organized with a functional bile canaliculi network. The organoids enable modeling of complex liver diseases including nonalcoholic fatty liver disease and drug-induced cholestasis.

Cell source
Human ESC (WA-01) and Human iPSC (GM23338)
Application
Disease modeling (NAFLD/NASH, drug-induced cholestasis)

Protocol overview

35 steps across 12 phases

Phase 1 · Pluripotent Stem Cell Culture and Expansion Ongoing maintenance
  1. a Culture H1 hESCs and iPSCs
  2. b Passage cells using Dispase
Phase 2 · Definitive Endoderm Induction Days 0-4
  1. a Seed hESCs in 12-well plate
  2. b Change to definitive endoderm differentiation medium
Phase 3 · Posterior Foregut Induction Days 4-10
  1. a Culture in posterior foregut induction medium
Phase 4 · Hepatic Endoderm Spheroid Formation Days 10-18
  1. a Harvest and prepare PFG cells
  2. b Culture in hepatic endoderm induction medium
Phase 5 · Hepatoblast Spheroid Differentiation Days 18-26
  1. a Replace valproic acid with growth factors
  2. b Maintain hepatoblast spheroid culture
Phase 6 · Hepatic Organoid Formation and Maturation Days 26-50
  1. a Harvest and dissociate hepatoblast spheroids
  2. b Prepare cells for organoid formation
  3. c First differentiation phase (12 days)
  4. d Second differentiation phase (14 days) - final maturation
  5. e Final organoid assessment
Phase 7 · Functional Characterization - Bile Canaliculi Visualization Days 36-50+
  1. a Prepare organoids for bile canaliculi imaging
  2. b Snapshot imaging of bile canaliculi
  3. c Time-lapse imaging of bile canaliculi
Phase 8 · Drug-Induced Cholestasis Modeling Days 36-50+
  1. a Prepare organoids for troglitazone treatment
  2. b Visualize bile flow disruption
  3. c Assess chronic cholestatic response
Phase 9 · Fatty Liver Disease Modeling Days 36-50+
  1. a Incubate organoids with free fatty acids
  2. b Visualize lipid droplet formation
  3. c Measure oxidative stress and lipid peroxidation
  4. d Assess bile canaliculi disruption in FFA-treated organoids
Phase 10 · Functional Assays - Hepatocyte Function Days 26-57
  1. a Measure albumin secretion
  2. b Measure apolipoprotein B secretion
  3. c Measure CYP3A4 activity
Phase 11 · Functional Assays - Cholangiocyte Function Days 36-57
  1. a Measure gamma glutamyl transferase activity
  2. b Measure alkaline phosphatase activity
  3. c Assess secretin-induced biliary proliferation
Phase 12 · Gene Expression Profiling and Analysis Variable (performed on day 4 for FFA experiments; day 50 for standard organoids)
  1. a Extract RNA
  2. b Prepare sequencing library
  3. c Perform quantitative polymerase chain reaction validation
  4. d Analyze differential gene expression
  5. e Perform gene ontology enrichment analysis

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Attribution

This SOP was written by Organthis based on the published method in Bin Ramli et al., 2020. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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