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INTESTINAL Publication-derived

Human Intestinal Organoids as Model for Rotavirus Infection

Source Finkbeiner et al. · Baylor College of Medicine, Houston, USA · 10.1128/mbio.00159-12

👤 Finkbeiner SR, Zeng XL, Utama B, Atmar RL, Shroyer NF, Estes MK ⏱ 60 days 📋 7 phases 🧫 iPSC-Derived (Human iPSC-Derived Intestinal Organoids)

Abstract

Demonstrated productive human rotavirus infection in iPSC-derived intestinal organoids with enterocyte-specific tropism and viral replication. This established organoids as a platform for studying human-specific rotavirus strains and screening antiviral compounds.

Cell source
iPSC-Derived (Human iPSC-Derived Intestinal Organoids)
Application
Rotavirus Research

Protocol overview

35 steps across 7 phases

iHIO Generation and Preparation Days 1-60 (approximately 1-2 months)
  1. 1 Culture WA09 stem cells under feeder-free conditions
  2. 2 Induce definitive endoderm differentiation
  3. 3 Generate hindgut spheroids
  4. 4 Embed spheroids in matrigel
  5. 5 Culture organoids with intestinal growth factors
  6. 6 Characterize iHIO cell types by immunofluorescence
iHIO Preparation for Viral Infection Day of infection
  1. 1 Remove iHIOs from matrigel
  2. 2 Wash organoids with PBS
  3. 3 Physically open iHIOs using tungsten needles
Rotavirus Infection of iHIOs 6 hours (inoculation period)
  1. 1 Prepare infection medium
  2. 2 Add viral inoculum
  3. 3 Perform 6-hour inoculation with manual agitation
  4. 4 Remove virus inoculum and wash organoids
  5. 5 Re-embed organoids in matrigel with growth medium
  6. 6 Add pancreatin for optimal virus replication (optional optimization)
Analysis of Viral Replication — Immunofluorescence (24 hpi) 24 hours post-inoculation
  1. 1 Fix organoids with paraformaldehyde
  2. 2 Stain and embed organoids
  3. 3 Section organoids using cryostat
  4. 4 Perform immunofluorescence staining
  5. 5 Examine by standard and confocal microscopy
Quantitative Analysis of Viral Replication — qRT-PCR (1, 24, 48 hpi) 1, 24, and 48 hours post-inoculation
  1. 1 Extract RNA from infected and mock organoids
  2. 2 Perform TaqMan qRT-PCR for rotavirus VP7
  3. 3 Calculate fold increases in VP7 RNA
Infectious Virus Yield Assay — Fluorescent Focus Assay (1 and 48 hpi) 1 and 48 hours post-inoculation
  1. 1 Harvest infected organoids and freeze lysates
  2. 2 Thaw and prepare organoid lysates
  3. 3 Activate virus with trypsin
  4. 4 Clarify lysate and prepare dilutions
  5. 5 Perform fluorescent focus assay (FFA) on MA104 cells
  6. 6 Calculate infectious virus titer
Clinical Rotavirus Isolate Preparation and Infection 1 day preparation; infection per Phase 3
  1. 1 Select and prepare stool samples
  2. 2 Clarify stool suspensions by centrifugation and filtration
  3. 3 Extract RNA for genotyping
  4. 4 Infect iHIOs with clinical isolate filtrates
  5. 5 Assess infection by immunofluorescence at 24 hpi
  6. 6 Quantify RNA levels by qRT-PCR

Full SOP

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Attribution

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

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