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INTESTINAL Publication-derived

Human Intestinal Organoid Culture and Enteroviral Infection Model

Source Li et al., 2025 · Department of Immunology and Microbiology, College of Life Science and Technology, Jinan University, Guangzhou, China · 10.1080/21505594.2025.2542455

👤 Chengcheng Li, Yanxi Chen, Xing Zhou, Jiuxiu Lin, Zhen Luo ⏱ 21 days 📋 9 phases 🧫 Fetal Human Colon (FHC)

Abstract

This protocol describes the generation of human intestinal organoids (HIOs) from fetal human colon cells using 3D Matrigel culture, and their characterization and use as a physiologically relevant model for studying enteroviral infections (EV71, CVB3, ECHO6). HIOs support robust enteroviral replication with higher viral titers compared to traditional 2D monolayer cultures.

Cell source
Fetal Human Colon (FHC)
Application
Disease modeling; Viral infection modeling

Protocol overview

49 steps across 9 phases

Cell Culture and Virus Propagation Setup Day 0 (preparation)
  1. 1 Culture FHC cells in standard 2D monolayer
  2. 2 Culture RD cells for virus propagation
  3. 3 Prepare virus stocks for infection
Matrigel Coating and Single-Cell Suspension Preparation Day 0–1 (HIO initiation)
  1. 1 Prepare diluted Matrigel for well coating
  2. 2 Coat low-adhesion 24-well plates with diluted Matrigel
  3. 3 Harvest and prepare FHC single-cell suspension
  4. 4 Mix cells with Matrigel and Rho kinase inhibitor
  5. 5 Seed cell-Matrigel mixture into coated plates
  6. 6 Add maintenance medium to wells
Human Intestinal Organoid Formation and Maturation Day 1–21 (culture period)
  1. 1 Culture FHC cells in Matrigel with differentiation medium
  2. 2 Monitor organoid morphology and size
  3. 3 Passage organoids at 1:3 ratio (if continuing culture beyond 10 days)
  4. 4 Maintain organoids in differentiation medium for 7–10 days post-passage
  5. 5 Perform quality control assays on organoid batches
Viral Infection of Organoids Day 7, 14, or 21 (infection initiation)
  1. 1 Prepare 2D monolayer FHC cells for comparison infection
  2. 2 Infect 2D monolayer FHC cells with virus
  3. 3 Count organoids in one well for normalization
  4. 4 Infect organoids with virus
  5. 5 Collect samples for viral titer and RNA quantification
RNA Extraction and Quantitative PCR (qPCR) Analysis Same day as sample collection or after (virus quantification)
  1. 1 Extract total RNA from infected cells/organoids
  2. 2 Measure RNA concentration and purity
  3. 3 Reverse-transcribe RNA to cDNA
  4. 4 Set up qPCR reactions for viral and cellular genes
  5. 5 Perform real-time qPCR cycling
  6. 6 Calculate relative viral RNA levels and fold-change expression
Viral Titer Determination (TCID50 Assay) Same day as sample collection or after (virus quantification)
  1. 1 Seed RD cells into 96-well plates
  2. 2 Prepare tenfold serial dilutions of viral samples
  3. 3 Infect RD cells with diluted virus samples
  4. 4 Monitor cytopathic effects (CPE) daily
  5. 5 Calculate TCID50 using Reed and Muench method
Immunofluorescence Staining and Microscopy Concurrent with infection studies (Day 7, 14, 21 post-organoid formation + infection)
  1. 1 Collect and fix organoid samples
  2. 2 Process samples for paraffin embedding and H&E staining (optional histology)
  3. 3 Permeabilize organoid samples for immunofluorescence
  4. 4 Block and incubate with primary antibodies
  5. 5 Incubate with secondary antibodies
  6. 6 Stain cell membranes with DiI
  7. 7 Stain cell nuclei with DAPI
  8. 8 Mount organoids for confocal microscopy
  9. 9 Perform multiple-protein detection (TSA-RM) if needed
  10. 10 Acquire and analyze confocal images
Antiviral Drug Screening (Optional Extension) Day 14 (post-organoid formation) + infection
  1. 1 Prepare antiviral compound dilutions
  2. 2 Co-infect 14-day organoids with virus and antiviral compounds
  3. 3 Measure viral RNA levels at defined time point
  4. 4 Calculate IC50 (half-maximal inhibitory concentration)
Statistical Analysis Throughout protocol (data analysis)
  1. 1 Organize and compile experimental data
  2. 2 Assess data normality and homogeneity of variance
  3. 3 Perform Student's t-test for pairwise comparisons
  4. 4 Report statistical significance levels
  5. 5 Perform multi-group comparisons if applicable

Full SOP

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Attribution

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

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