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LIVER Publication-derived

Development of a Representative 3D Human Model Derived from iPSC to Study Hepatitis B Virus In Vitro

Source Eugène, 2025 · Université de Montréal · 10.3389/fimmu.2019.02694

👤 Nedley Eugène ⏱ 25 days 📋 10 phases 🧫 Human iPSC

Abstract

This protocol describes the differentiation of human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) and the generation of 3D liver organoids incorporating iPSC-derived hepatic progenitor cells, endothelial cells, and mesenchymal cells. The resulting model is used to study hepatitis B virus infection, viral lifecycle, and optimize infection conditions for both 2D and 3D culture systems.

Cell source
Human iPSC
Application
Disease modeling

Protocol overview

44 steps across 10 phases

iPSC Maintenance and Preparation for Differentiation Prior to differentiation
  1. 1 Culture iPSCs in hypoxic conditions
  2. 2 Passage iPSCs at appropriate confluence
  3. 3 Prepare for single-cell passaging
Hepatocyte-Like Cell (HLC) Differentiation – Days 1–15 (Hepatic Progenitor Cell Stage) Days 1–15
  1. 1 Seed iPSCs for differentiation
  2. 2 Days 1–2: Activate Wnt and Nodal signaling
  3. 3 Days 3–5: Specify definitive endoderm with reduced Activin A
  4. 4 Days 6–10: Direct to ventral posterior foregut with growth factors
  5. 5 Days 11–15: Hepatic progenitor cell (HPC) maturation
  6. 6 Days 11–15: Sample cells for quality control
Liver Organoid Generation (Starting from Day 15 HPCs) Days 15–25
  1. 1 Prepare cells for organoid assembly on Day 15
  2. 2 Seed organoid cultures
  3. 3 Days 1–3: Daily partial medium changes
  4. 4 Days 4–10: Complete medium changes every 2 days
  5. 5 Assess organoid maturation
Viral Production from HepAD38 Cells As needed (typically 4–6 weeks)
  1. 1 Culture HepAD38 cells under tetracycline control
  2. 2 Induce viral production
Viral Concentration and Titration As needed after production
  1. 1 Concentrate virus by PEG precipitation
  2. 2 Titrate viral stock by ddPCR
  3. 3 Characterize viral quality by iodixanol ultracentrifugation
Cell Infection – 2D Culture (HepG2-NTCP and HPCs) As needed for infection studies
  1. 1 Pre-treat cells with DMSO (optional for enhanced infection)
  2. 2 Prepare cells for infection
  3. 3 Prepare infection inoculum
  4. 4 Perform infection
  5. 5 Collect samples during infection time course
3D Organoid Infection As needed for infection studies
  1. 1 Pre-formation infection (PreF)
  2. 2 Post-formation infection (PostF)
  3. 3 Post-infection culture and sampling
Nucleic Acid Extraction from Infected Cells and Organoids Post-infection (as needed)
  1. 1 Extract extracellular viral DNA from supernatant
  2. 2 Simultaneous extraction of intracellular DNA and RNA from 2D cultures
  3. 3 Isolate RNA from PK-treated lysate
  4. 4 Isolate rcDNA from PK-treated lysate
  5. 5 Isolate cccDNA from non-PK-treated lysate (genomic DNA must be removed)
  6. 6 Extract nucleic acids from organoids
Quantification of Viral Components by qPCR and ddPCR Post-extraction (as needed)
  1. 1 Quantify HBV RNA by qPCR
  2. 2 Quantify rcDNA by SYBR Green qPCR
  3. 3 Quantify rcDNA by SYBR Green qPCR (extracellular, from supernatant)
  4. 4 Quantify cccDNA by digital droplet PCR (ddPCR)
HBeAg Detection by ELISA Post-infection (as needed)
  1. 1 Prepare ELISA plate and samples
  2. 2 Add detection antibody
  3. 3 Incubate with detection antibody
  4. 4 Wash plate
  5. 5 Develop substrate reaction
  6. 6 Read optical density
  7. 7 Interpret results

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Eugène, 2025. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

This wording is awaiting legal review.

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