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LIVER Publication-derived

Alcohol-Related Liver Disease Organoids

Full SOP with Academic or a plan

Source Angireddy et al., 2020 · University of Pennsylvania, School of Veterinary Medicine, Department of Biomedical Sciences · 10.1016/j.freeradbiomed.2020.06.030

Authors: Rajesh Angireddy, Anindya Roy Chowdhury, Jacek Zielonka, Gordon Ruthel, Balaraman Kalyanaraman, Narayan G. Avadhani Duration: 21 days 9 phases Cell source: Human HepaRG hepatocyte cell line (derived from hepatic tumors but differentiated to functional hepatocytes)

Abstract

This protocol describes a method to generate and characterize 3D hepatic organoids from differentiated HepaRG cells to model alcohol-induced liver toxicity. The organoids recapitulate the induction of CYP2E1, increased reactive oxygen species production, mitochondrial dysfunction, retrograde signaling activation, and hepatic steatosis markers observed in alcohol-related liver disease.

Cell source
Human HepaRG hepatocyte cell line (derived from hepatic tumors but differentiated to functional hepatocytes)
Application
Disease modeling

Protocol overview

33 steps across 9 phases

Phase 1 · HepaRG Cell Differentiation (Monolayer) Days 0-21
  1. a Obtain and maintain HepaRG cells
  2. b Induce hepatocyte differentiation
Phase 2 · 3D Organoid Formation (AggreWell™800 Method) Days 0-17
  1. a Prepare AggreWell™800 plates
  2. b Seed differentiated HepaRG cells
  3. c Maintain organoid cultures (first 5 days)
  4. d Replace medium and continue culture
Phase 3 · Alcohol Treatment Days 14-17 (after organoid maturation)
  1. a Prepare organoids for treatment
  2. b Treat organoids with ethanol
  3. c Optional: Co-treat with inhibitors
Phase 4 · Analysis Phase 1: Microscopy and Live Cell Imaging During or after treatment
  1. a Assess organoid viability with live/dead staining
  2. b Measure mitochondrial mass
  3. c Measure mitochondrial membrane potential
  4. d Quantify ROS using fluorescent imaging
  5. e Stain for lipid accumulation
Phase 5 · Analysis Phase 2: RNA and Gene Expression After treatment completion
  1. a Harvest organoids for RNA extraction
  2. b Extract total RNA
  3. c Reverse transcription to cDNA
  4. d Perform RT-qPCR
Phase 6 · Analysis Phase 3: ROS Quantification (HPLC and Amplex Red Methods) After treatment completion
  1. a ROS analysis using hydroethidine (HE) - HPLC method
  2. b Profile HE products by HPLC
  3. c Measure H2O2 using Amplex UltraRed™ assay
Phase 7 · Analysis Phase 4: Metabolic and Biochemical Assays After treatment completion
  1. a Seahorse XF analysis - oxygen consumption and acidification rates
  2. b Metabolomic analysis
  3. c Measure triglyceride levels
  4. d Quantify NAD+ and NADH levels
Phase 8 · Analysis Phase 5: Histology and Immunohistochemistry After treatment completion
  1. a Fix organoids for histological analysis
  2. b Perform H&E staining
  3. c Perform immunohistochemistry (IHC)
  4. d Perform confocal fluorescence microscopy
  5. e Perform Oil O Red staining for lipids
Phase 9 · Analysis Phase 6: Mitochondrial Isolation and Enzyme Assays After treatment completion (optional - for monolayer cultures)
  1. a Isolate mitochondria from cells
  2. b Measure Complex I activity
  3. c Measure cytochrome c oxidase (CcO) activity

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Attribution

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

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