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LIVER Publication-derived

Alcohol-induced CYP2E1, mitochondrial dynamics and retrograde signaling in human hepatic 3D organoids

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

👤 Rajesh Angireddy, Anindya Roy Chowdhury, Jacek Zielonka, Gordon Ruthel, Balaraman Kalyanaraman, Narayan G. Avadhani ⏱ 21 days 📋 9 phases 🧫 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

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

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Angireddy et al., 2020. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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