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INTESTINAL Publication-derived

Derived Intestinal Organoids

Source Ettayebi et al. · Baylor College of Medicine, Houston, USA · 10.1126/science.aaf5211

👤 Ettayebi K, Crawford SE, Murakami K, Broughman JR, Karandikar U, Tenge VR, Neill FH, Blutt SE, Zeng XL, Qu L, Kou B, Opekun AR, Burrin D, Graham DY, Ramani S, Atmar RL, Estes MK ⏱ 6 days 📋 7 phases 🧫 Primary (Human Jejunal Biopsy-Derived Organoids)

Abstract

Achieved the first robust in vitro cultivation of human norovirus using stem cell-derived intestinal organoids, solving a decades-long challenge. Bile acids were identified as critical cofactors for viral entry, and FUT2 secretor status determined organoid susceptibility, matching known epidemiological patterns.

Cell source
Primary (Human Jejunal Biopsy-Derived Organoids)
Application
Norovirus Research

Protocol overview

28 steps across 7 phases

Preparation of Human Intestinal Enteroid (HIE) Monolayers Day −2 to Day 0
  1. 1 Generate or obtain HIE cultures from intestinal biopsies
  2. 2 Prepare monolayer cultures from HIEs
Preparation of Viral Inoculum and Bile Treatment (for GII.3, GII.17, GI.1) Day −2 to Day −1
  1. 1 Prepare stool filtrate inoculum
  2. 2 Prepare bile solution for use with the following strains: GII.3, GII.17, and GI.1.
  3. 3 Treat HIE monolayers with bile (GII.3, GII.17, GI.1 only)
Viral Infection of HIE Monolayers Day 0
  1. 1 Remove culture medium from HIE monolayers
  2. 2 Inoculate HIE monolayers with HuNoV stool filtrate
  3. 3 Allow viral adsorption
  4. 4 Remove inoculum and wash monolayers (for baseline sampling)
  5. 5 Add culture medium with or without bile
Viral Replication and Sample Collection Day 0 to Day 6
  1. 1 Incubate infected monolayers
  2. 2 Collect samples at defined timepoints
  3. 3 Prepare samples for RT-qPCR analysis
  4. 4 Monitor cytopathic effects (CPE)
  5. 5 Detect viral proteins by immunofluorescence
  6. 6 Detect viral particles by transmission electron microscopy (TEM)
  7. 7 Analyze viral protein synthesis by Western blot
Viral Passaging (Optional) Day 3 to Day 5
  1. 1 Harvest supernatant from primary infected culture
  2. 2 Inoculate fresh HIE monolayers with passaged virus
  3. 3 Monitor passaged virus replication
Viral Neutralization Studies (Optional) Day 0 to Day 4
  1. 1 Prepare serum samples
  2. 2 Pre-incubate virus with diluted serum
  3. 3 Inoculate HIE monolayers with virus-serum mixtures
  4. 4 Quantify viral replication and measure neutralization
Viral Inactivation Studies (Optional) Day 0 to Day 3
  1. 1 Gamma irradiation of virus
  2. 2 Heat inactivation of virus
  3. 3 Inoculate HIEs with inactivated virus
  4. 4 Assess viral replication

Full SOP

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Attribution

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

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