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LIVER Publication-derived

Chronic Hepatitis D Liver Organoids

Full SOP with Academic or a plan

Source Shen et al., 2025 · Shanghai Sixth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine · 10.1016/j.antiviral.2025.106266

Authors: Leer Shen, Luying Tian, Qingxin Guo, Rongli He, Yi Zhang, Siyuan Ma, Weiwei Hu, Jie Chen, Soon Seng Ng, Jinmei Chen, Xiaohua Chen Duration: 28 days 13 phases Cell source: Human iPSC

Abstract

This protocol establishes HBV/HDV superinfected liver organoids derived from human iPSCs differentiated into hepatocyte-like cells and seeded on inverted colloidal crystal scaffolds. The protocol demonstrates that ubiquitinated small hepatitis D antigen (Ub-S-HDAg) transduced into dendritic cells promotes CD8+ T cell activation via the JAK/STAT pathway, leading to significant reduction in HDV viral load and providing a platform for immunotherapy screening.

Cell source
Human iPSC
Application
Disease modeling; Antiviral immunotherapy development

Protocol overview

60 steps across 13 phases

Phase 1 · iPSC Culture and Hepatocyte Differentiation Days 0-21
  1. a iPSC Culture on Laminin-coated Plates
  2. b Initiate Endoderm Formation
  3. c Promote Hepatoblast Formation
  4. d Differentiate into Hepatocyte-like Cells
Phase 2 · Inverted Colloidal Crystal Scaffold Preparation and Organoid Culture Days 15-28
  1. a Construct Inverted Colloidal Crystal Scaffold
  2. b Remove Polystyrene Microspheres
  3. c Coat Scaffold with Collagen I
  4. d Seed Hepatocyte-like Cells onto Scaffold
Phase 3 · HBV and HDV Production and Infection Days 0-21
  1. a Produce HBV in HepAD38 Cells
  2. b Produce HDV Particles
  3. c Purify and Concentrate HBV/HDV Particles
  4. d Infect Liver Organoids with HBV
  5. e Superinfect HBV-infected Organoids with HDV
Phase 4 · Dendritic Cell and T Cell Isolation and Culture Days 0-14
  1. a Differentiate PBMCs into Dendritic Cells
  2. b Isolate and Culture T Cells
Phase 5 · Lentiviral Vector Construction and DC Transduction Days 0-10
  1. a Synthesize and Clone Ub-S-HDAg Sequence
  2. b Produce Lentiviral Particles
  3. c Transduce DCs with Lentiviral Vector
  4. d Culture Transduced DCs
  5. e Establish Experimental Groups
Phase 6 · DC and T Cell Co-culture Days 5-8
  1. a Co-culture DCs and T Cells
  2. b Harvest CD8+ T Cells
Phase 7 · Flow Cytometry Analysis of DC Maturation Days 3-5
  1. a Prepare Cells for Flow Cytometry
  2. b Stain with DC Maturation Markers
  3. c Assess Cell Viability
  4. d Analyze via Flow Cytometry
Phase 8 · Cytokine Measurement and Immune Function Assays Days 5-8
  1. a Culture CD8+ T Cells Post-isolation
  2. b Collect Supernatants
  3. c Measure Cytokine Levels by ELISA
  4. d Perform IFN-γ ELISPOT Assay
Phase 9 · Gene Expression Analysis and JAK/STAT Pathway Evaluation Days 5-10
  1. a Extract Total RNA
  2. b Assess RNA Integrity
  3. c Synthesize cDNA
  4. d Perform qRT-PCR for JAK/STAT Genes
  5. e Prepare Libraries for Whole Transcriptome Sequencing
  6. f Perform Whole Transcriptome Sequencing and Analysis
Phase 10 · Cell Proliferation and Cytotoxicity Evaluation Days 5-8
  1. a Assess Cell Proliferation via CCK-8 Assay
  2. b Measure Absorbance at 450 nm
  3. c Prepare Effector and Target Cells for Cytotoxicity Assay
  4. d Measure Lactate Dehydrogenase Activity
Phase 11 · Western Blot Analysis of JAK/STAT Pathway Components Days 5-8
  1. a Extract Total Protein
  2. b Clarify Protein Extract
  3. c Determine Protein Concentration
  4. d Prepare Protein Samples
  5. e Run SDS-PAGE Gel Electrophoresis
  6. f Transfer Proteins to Membrane
  7. g Probe with Primary Antibodies
  8. h Incubate with Secondary Antibodies
  9. i Visualize and Analyze Blots
Phase 12 · HDV Infection and Antiviral Treatment Assessment Days 0-21
  1. a Establish Five Experimental Treatment Groups
  2. b Perform Interventions on HDV-infected Organoids
  3. c Collect Culture Supernatants
  4. d Extract Viral Nucleic Acids
  5. e Quantify Viral Load via qRT-PCR
Phase 13 · Immunofluorescence and Immunohistochemistry Analysis Throughout organoid culture
  1. a Prepare Samples for Immunofluorescence
  2. b Incubate with Primary Antibodies
  3. c Incubate with Secondary Antibodies
  4. d Mount Coverslips
  5. e Image and Analyze Samples
  6. f Perform Immunohistochemical Staining

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Attribution

This SOP was written by Organthis based on the published method in Shen et al., 2025. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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