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INTESTINAL Publication-derived

Hepatitis E Intestinal Organoids

Full SOP with Academic or a plan

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

Authors: Kuan Liu, Yang Wang, Jiangrong Zhou, Joy Joan van der Meij, Luc J.W. van der Laan, Pengfei Li, Qiuwei Pan Duration: 11 days 12 phases Cell source: 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

Phase 1 · Human intestinal organoid (HIO) culture and maintenance Ongoing, medium changes every 2-3 days
  1. a Prepare organoid expansion medium (OEM)
  2. b Culture primary human intestinal organoids
  3. c Perform medium changes
Phase 2 · HIO differentiation into specific cell types 5 days post-initiation of differentiation medium
  1. a Differentiate organoids into enterocytes
  2. b Differentiate organoids into goblet cells
  3. c Differentiate organoids into enteroendocrine cells
  4. d Replace differentiation media and monitor morphology
Phase 3 · Intrahepatic cholangiocyte organoid (ICO) culture Ongoing, medium changes every 2-3 days
  1. a Prepare ICO expansion medium (EM)
  2. b Culture human intrahepatic cholangiocyte organoids (ICOs)
  3. c Perform medium changes for ICOs
Phase 4 · HEV RNA preparation and electroporation Day 0 (electroporation)
  1. a Linearize HEV plasmid constructs
  2. b Transcribe linearized DNA into genomic RNA
  3. c Prepare organoids for electroporation
  4. d Perform electroporation
  5. e Re-culture electroporated organoids
Phase 5 · HEV particle inoculation and incubation Day 0 (inoculation)
  1. a Prepare HEV particle inoculum
  2. b Fragment intestinal organoids
  3. c Inoculate organoids with HEV particles
  4. d Remove inoculum and wash organoids
  5. e Re-culture inoculated organoids
Phase 6 · Bile acid and antiviral treatment Days 1-5 post-infection/electroporation
  1. a Prepare bile acid stock solutions
  2. b Add bile or bile acids to infected organoids
  3. c Treat organoids with antiviral agents
  4. d Perform TGR5 inhibitor studies
Phase 7 · Transwell co-culture gut-liver axis model construction Days 1-7 post-infection
  1. a Prepare transwell co-culture system
  2. b Seed intrahepatic cholangiocyte organoids (ICOs)
  3. c Monitor viral transmission over time
  4. d Add bile or bile acids to the co-culture system
  5. e Assess viral transmission and replication
Phase 8 · HEV genome quantification by qRT-PCR As indicated throughout the infection study
  1. a Extract total RNA from organoids
  2. b Assess RNA quality and concentration
  3. c Prepare standard curve plasmid dilutions
  4. d Perform qRT-PCR
  5. e Calculate HEV copy numbers
Phase 9 · Luciferase reporter assay for viral replication Days 1-11 post-electroporation (for subgenomic GT3 HEV with Gaussia luciferase reporter)
  1. a Collect culture supernatants
  2. b Measure Gaussia luciferase activity
  3. c Normalize and plot results
Phase 10 · Immunofluorescence assay for viral detection and cell typing As indicated (typically at 5-7 days post-infection/electroporation)
  1. a Prepare organoid samples
  2. b Permeabilize and block
  3. c Incubate with primary antibodies
  4. d Wash and incubate with secondary antibodies
  5. e Counterstain nuclei and mount
  6. f Image and analyze
Phase 11 · Viral infectivity assessment by re-infection assay Days 5-7 post-infection/electroporation
  1. a Collect viral supernatants from organoids
  2. b Inoculate human liver Huh7 cells
  3. c Culture infected Huh7 cells and assess infection
Phase 12 · AlamarBlue assay for organoid viability As needed during infection or treatment studies
  1. a Remove culture supernatant
  2. b Incubate organoids with AlamarBlue
  3. c Measure fluorescence
  4. d Analyze results

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Attribution

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

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