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LIVER Publication-derived

Fatty Liver Disease Organoids for Drug Screening

Full SOP with Academic or a plan

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

Authors: J. Bronsard, C. Savary, J. Massart, R. Viel, L. Moutaux, D. Catheline, V. Rioux, B. Clement, A. Corlu, B. Fromenty, P.J. Ferron Duration: 14 days 6 phases Cell source: 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

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

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Attribution

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

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