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INTESTINAL Publication-derived

Decoding the role of the intestinal epithelium in hepatitis E virus infection using a human organoid prototype of "gut-liver" axis

Source Liu et al., 2025 · Erasmus MC-University Medical Center, Rotterdam, Netherlands · 10.1016/j.virol.2025.110615

👤 Kuan Liu, Yang Wang, Jiangrong Zhou, Joy Joan van der Meij, Luc J.W. van der Laan, Pengfei Li, Qiuwei Pan ⏱ 11 days 📋 12 phases 🧫 Human primary intestinal and liver organoids

Abstract

This protocol describes the establishment and use of a primary human intestinal organoid (HIO) model for studying hepatitis E virus (HEV) replication and transmission to liver-derived organoids in a transwell-based gut-liver axis model. The protocol demonstrates robust HEV replication in differentiated intestinal organoids and evaluates the role of bile acids in modulating viral infection through the farnesoid X receptor (FXR) signaling pathway.

Cell source
Human primary intestinal and liver organoids
Application
Disease modeling; Viral infection study; Antiviral drug screening

Protocol overview

50 steps across 12 phases

Human intestinal organoid (HIO) culture and maintenance Ongoing, medium changes every 2-3 days
  1. 1 Prepare organoid expansion medium (OEM)
  2. 2 Culture primary human intestinal organoids
  3. 3 Perform medium changes
HIO differentiation into specific cell types 5 days post-initiation of differentiation medium
  1. 1 Differentiate organoids into enterocytes
  2. 2 Differentiate organoids into goblet cells
  3. 3 Differentiate organoids into enteroendocrine cells
  4. 4 Replace differentiation media and monitor morphology
Intrahepatic cholangiocyte organoid (ICO) culture Ongoing, medium changes every 2-3 days
  1. 1 Prepare ICO expansion medium (EM)
  2. 2 Culture human intrahepatic cholangiocyte organoids (ICOs)
  3. 3 Perform medium changes for ICOs
HEV RNA preparation and electroporation Day 0 (electroporation)
  1. 1 Linearize HEV plasmid constructs
  2. 2 Transcribe linearized DNA into genomic RNA
  3. 3 Prepare organoids for electroporation
  4. 4 Perform electroporation
  5. 5 Re-culture electroporated organoids
HEV particle inoculation and incubation Day 0 (inoculation)
  1. 1 Prepare HEV particle inoculum
  2. 2 Fragment intestinal organoids
  3. 3 Inoculate organoids with HEV particles
  4. 4 Remove inoculum and wash organoids
  5. 5 Re-culture inoculated organoids
Bile acid and antiviral treatment Days 1-5 post-infection/electroporation
  1. 1 Prepare bile acid stock solutions
  2. 2 Add bile or bile acids to infected organoids
  3. 3 Treat organoids with antiviral agents
  4. 4 Perform TGR5 inhibitor studies
Transwell co-culture gut-liver axis model construction Days 1-7 post-infection
  1. 1 Prepare transwell co-culture system
  2. 2 Seed intrahepatic cholangiocyte organoids (ICOs)
  3. 3 Monitor viral transmission over time
  4. 4 Add bile or bile acids to the co-culture system
  5. 5 Assess viral transmission and replication
HEV genome quantification by qRT-PCR As indicated throughout the infection study
  1. 1 Extract total RNA from organoids
  2. 2 Assess RNA quality and concentration
  3. 3 Prepare standard curve plasmid dilutions
  4. 4 Perform qRT-PCR
  5. 5 Calculate HEV copy numbers
Luciferase reporter assay for viral replication Days 1-11 post-electroporation (for subgenomic GT3 HEV with Gaussia luciferase reporter)
  1. 1 Collect culture supernatants
  2. 2 Measure Gaussia luciferase activity
  3. 3 Normalize and plot results
Immunofluorescence assay for viral detection and cell typing As indicated (typically at 5-7 days post-infection/electroporation)
  1. 1 Prepare organoid samples
  2. 2 Permeabilize and block
  3. 3 Incubate with primary antibodies
  4. 4 Wash and incubate with secondary antibodies
  5. 5 Counterstain nuclei and mount
  6. 6 Image and analyze
Viral infectivity assessment by re-infection assay Days 5-7 post-infection/electroporation
  1. 1 Collect viral supernatants from organoids
  2. 2 Inoculate human liver Huh7 cells
  3. 3 Culture infected Huh7 cells and assess infection
AlamarBlue assay for organoid viability As needed during infection or treatment studies
  1. 1 Remove culture supernatant
  2. 2 Incubate organoids with AlamarBlue
  3. 3 Measure fluorescence
  4. 4 Analyze results

Full SOP

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Attribution

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

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