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INTESTINAL Publication-derived

Rotavirus Infection Intestinal Organoids

Full SOP with Academic or a plan

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

Authors: Finkbeiner SR, Zeng XL, Utama B, Atmar RL, Shroyer NF, Estes MK Duration: 60 days 7 phases Cell source: 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

Phase 1 · iHIO Generation and Preparation Days 1-60 (approximately 1-2 months)
  1. a Culture WA09 stem cells under feeder-free conditions
  2. b Induce definitive endoderm differentiation
  3. c Generate hindgut spheroids
  4. d Embed spheroids in matrigel
  5. e Culture organoids with intestinal growth factors
  6. f Characterize iHIO cell types by immunofluorescence
Phase 2 · iHIO Preparation for Viral Infection Day of infection
  1. a Remove iHIOs from matrigel
  2. b Wash organoids with PBS
  3. c Physically open iHIOs using tungsten needles
Phase 3 · Rotavirus Infection of iHIOs 6 hours (inoculation period)
  1. a Prepare infection medium
  2. b Add viral inoculum
  3. c Perform 6-hour inoculation with manual agitation
  4. d Remove virus inoculum and wash organoids
  5. e Re-embed organoids in matrigel with growth medium
  6. f Add pancreatin for optimal virus replication (optional optimization)
Phase 4 · Analysis of Viral Replication — Immunofluorescence (24 hpi) 24 hours post-inoculation
  1. a Fix organoids with paraformaldehyde
  2. b Stain and embed organoids
  3. c Section organoids using cryostat
  4. d Perform immunofluorescence staining
  5. e Examine by standard and confocal microscopy
Phase 5 · Quantitative Analysis of Viral Replication — qRT-PCR (1, 24, 48 hpi) 1, 24, and 48 hours post-inoculation
  1. a Extract RNA from infected and mock organoids
  2. b Perform TaqMan qRT-PCR for rotavirus VP7
  3. c Calculate fold increases in VP7 RNA
Phase 6 · Infectious Virus Yield Assay — Fluorescent Focus Assay (1 and 48 hpi) 1 and 48 hours post-inoculation
  1. a Harvest infected organoids and freeze lysates
  2. b Thaw and prepare organoid lysates
  3. c Activate virus with trypsin
  4. d Clarify lysate and prepare dilutions
  5. e Perform fluorescent focus assay (FFA) on MA104 cells
  6. f Calculate infectious virus titer
Phase 7 · Clinical Rotavirus Isolate Preparation and Infection 1 day preparation; infection per Phase 3
  1. a Select and prepare stool samples
  2. b Clarify stool suspensions by centrifugation and filtration
  3. c Extract RNA for genotyping
  4. d Infect iHIOs with clinical isolate filtrates
  5. e Assess infection by immunofluorescence at 24 hpi
  6. f Quantify RNA levels by qRT-PCR

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Attribution

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

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