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LIVER Publication-derived

3D Multi-Cell-Type Liver Organoids: A Model of Non-Alcoholic Fatty Liver Disease for Drug Safety Assessments

Source Bronsard et al., 2024 · INSERM, Université de Rennes, INRAE, Institut NuMeCan UMR1317 · 10.1016/j.tiv.2023.105728

👤 J. Bronsard, C. Savary, J. Massart, R. Viel, L. Moutaux, D. Catheline, V. Rioux, B. Clement, A. Corlu, B. Fromenty, P.J. Ferron ⏱ 14 days 📋 6 phases 🧫 Human iPSC-derived HepaRG cells, Primary Human Macrophages, LX-2 hepatic stellate cells

Abstract

This protocol describes the generation of three-dimensional multi-cell-type liver organoids (HML organoids) composed of HepaRG cells, primary human macrophages, and LX-2 stellate cells. HML organoids maintain differentiated hepatocyte and cholangiocyte phenotypes with high expression of xenobiotic-metabolizing enzymes. NAFLD-like features are induced through exposure to stearic and oleic acids, creating a model suitable for evaluating drug-induced liver injury in the context of fatty liver disease.

Cell source
Human iPSC-derived HepaRG cells, Primary Human Macrophages, LX-2 hepatic stellate cells
Application
Disease modeling and drug screening

Protocol overview

26 steps across 6 phases

Cell Culture and Differentiation D0 to D14
  1. 1 HepaRG Cell Culture and Progenitor Seeding
  2. 2 HepaRG Cell Differentiation with DMSO
  3. 3 Human Macrophage Differentiation from Peripheral Blood Mononuclear Cells
  4. 4 LX-2 Hepatic Stellate Cell Culture
HML Organoid Generation D0 to D14
  1. 1 Prepare Mixed Cell Suspension and Seed into Micromolds
  2. 2 Early-Stage Culture (D0 to D3)
  3. 3 Serum Reduction (D3 onwards)
NAFLD Induction via Fatty Acid Treatment D5 to D14
  1. 1 Prepare Fatty Acid Mixture
  2. 2 Initiate Fatty Acid Treatment on D5
Characterization and Analysis D5 to D14
  1. 1 RNA Extraction at Multiple Timepoints
  2. 2 cDNA Synthesis and RT-qPCR
  3. 3 Histological Processing and Staining
  4. 4 Immunohistochemical Staining on Automated Stainer
  5. 5 Image Analysis using QuPath
  6. 6 Lipid Extraction and Fatty Acid Analysis by GC-MS
  7. 7 Lipid Staining with BODIPY
  8. 8 Quantification of Secreted Cytokines by ELISA
Drug Toxicity Assessment D5 to D14
  1. 1 Prepare Drug Compounds and Serial Dilutions
  2. 2 Seed Organoids in 96-Well Ultra-Low Attachment Plates
  3. 3 Initiate Fatty Acid Treatment (Control vs. FA-Exposed Groups)
  4. 4 Begin Drug Exposure (D7 to D14)
  5. 5 Measure Intracellular ATP (CellTiter-Glo Assay)
Data Analysis and Statistical Evaluation Post-Experiment
  1. 1 Calculate IC50 Values Using Dose-Response Modeling
  2. 2 Determine Benchmark Dose (BMD) Using PROAST
  3. 3 Perform Statistical Analysis
  4. 4 Correlate IC50/BMD with Published Cmax Values

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Bronsard et al., 2024. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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