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LIVER Publication-derived

Human Liver Organoids as Models for Investigating Drug-Induced Liver Injury

Source Don, 2021 · University of Cambridge, Department of Physiology, Development and Neuroscience · 10.1186/s13036019-0148-5

👤 Mewanthi Flaminia Kaluthantrige Don ⏱ 35 days 📋 4 phases 🧫 Human hepatic ductal cells and primary human hepatocytes

Abstract

This protocol describes the differentiation of human liver organoids from ductal/progenitor cells into functional hepatocyte organoids using chemically defined culture conditions. The differentiated organoids form bile canalicular structures and express hepatocyte markers, enabling their use as an in vitro system to model drug-induced liver injury including cholestasis and steatosis.

Cell source
Human hepatic ductal cells and primary human hepatocytes
Application
Drug-induced liver injury modeling and toxicity screening

Protocol overview

25 steps across 4 phases

Human Hepatic Ductal Cell Isolation and Organoid Formation Days 0-7
  1. 1 Tissue preparation and mincing
  2. 2 Enzymatic digestion
  3. 3 Hand-picking ductal fragments
  4. 4 Embedding in BME and seeding
  5. 5 Organoid culture initiation
  6. 6 Organoid growth and passaging
  7. 7 Organoid maintenance and cryopreservation
Hepatocyte Organoid Differentiation Days 5-20
  1. 1 Pre-differentiation preparation
  2. 2 Mechanical dissociation and splitting
  3. 3 DM medium differentiation (Standard protocol)
  4. 4 DM+ medium differentiation (Enhanced protocol with WNT modulation)
  5. 5 DM+VP medium differentiation (Enhanced protocol with YAP inhibition)
  6. 6 Organoid assessment and imaging
Characterization of Hepatocyte Organoid Function Days 15-30
  1. 1 Immunofluorescence staining for hepatocyte markers
  2. 2 Bile acid transport assay (CMFDA secretion)
  3. 3 Cell viability assay (Live/Dead staining)
  4. 4 3D cell viability assay (CellTiter-Glo)
  5. 5 Total bile acid measurement
  6. 6 Albumin secretion ELISA
  7. 7 CYP3A4 activity assay
Drug Toxicity Testing (DILI Modeling) Days 20-35
  1. 1 Organoid drug treatment preparation
  2. 2 Cholestasis phenotype assessment - CMFDA intracellular accumulation
  3. 3 Steatosis phenotype assessment - lipid staining
  4. 4 Cell viability assessment after drug treatment
  5. 5 Gene expression analysis of drug response

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Don, 2021. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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