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INTESTINAL Publication-derived

Infection of bat and human intestinal organoids by SARS-CoV-2

Source Zhou et al., 2020 · The University of Hong Kong · 10.1038/s41591-020-0912-6

👤 Jie Zhou, Cun Li, Xiaojuan Liu, Man Chun Chiu, Xiaoyu Zhao, Dong Wang, Yuxuan Wei, Andrew Lee, Anna Jinxia Zhang, Hin Chu, Jian-Piao Cai, Cyril Chik-Yan Yip, Ivy Hau-Yee Chan, Kenneth Kak-Yuen Wong, Owen Tak-Yin Tsang, Kwok-Hung Chan, Jasper Fuk-Woo Chan, Kelvin Kai-Wang To, Honglin Chen, Kwok Yung Yuen ⏱ 72 days 📋 8 phases 🧫 Human iPSC-derived (pediatric donors)

Abstract

This protocol establishes bat intestinal organoids (enteroids) derived from Rhinolophus sinicus and evaluates their susceptibility to SARS-CoV-2 infection alongside human intestinal organoids. The protocol demonstrates robust viral replication in both bat and human enteroids and enables isolation of infectious SARS-CoV-2 from patient clinical specimens, providing an in vitro model for studying enteric coronavirus infection.

Cell source
Human iPSC-derived (pediatric donors)
Application
Disease modeling and virus isolation/susceptibility testing

Protocol overview

35 steps across 8 phases

Bat intestinal tissue collection and crypt isolation Day 0
  1. 1 Bat euthanasia and intestinal tissue harvesting
  2. 2 Intestinal tissue chopping and chelation buffer incubation
  3. 3 Crypt pellet isolation by centrifugation
Matrigel embedding and expansion phase Days 0–7
  1. 1 Matrigel droplet formation in 24-well plate
  2. 2 Addition of expansion medium
  3. 3 Culture maintenance in expansion phase
  4. 4 Organoid passaging (1:2 ratio every 7 days)
Organoid differentiation phase Days 0–4 (from start of differentiation)
  1. 1 Medium replacement with differentiation medium
  2. 2 Differentiation culture incubation
  3. 3 Verification of differentiation
Virus isolation and inoculation Day 1 (of infection experiment)
  1. 1 Preparation of clinical specimens
  2. 2 Mechanical shearing of differentiated organoids
  3. 3 Organoid-specimen co-incubation
  4. 4 Re-embedding in Matrigel and culture in differentiation medium
  5. 5 Incubation and observation
Viral load quantification by RT-qPCR At designated timepoints during infection
  1. 1 Collection of cell-free culture media
  2. 2 Viral RNA extraction
  3. 3 One-step RT-qPCR assay setup
  4. 4 RT-qPCR cycling protocol
  5. 5 Data analysis and viral load calculation
Viral titration by TCID50 assay At designated timepoints during infection
  1. 1 Preparation of serial virus dilutions
  2. 2 Inoculation of Vero cell monolayers
  3. 3 Culture incubation and CPE observation
  4. 4 TCID50 calculation
Immunofluorescence staining for viral nucleocapsid protein (NP) At designated timepoints (e.g., 24, 48, or 72 hpi)
  1. 1 Fixation of infected organoids
  2. 2 Washing and permeabilization
  3. 3 Blocking and primary antibody incubation
  4. 4 Secondary antibody incubation
  5. 5 Counterstaining and mounting
  6. 6 Confocal microscopy imaging
Gene expression analysis by RT-qPCR At designated timepoints (e.g., 24 or 48 hpi)
  1. 1 Organoid harvesting and RNA extraction
  2. 2 Reverse transcription
  3. 3 qPCR setup and amplification (individual targets)
  4. 4 Multiplex PCR array (optional, for comprehensive cytokine profiling)
  5. 5 Data analysis and fold-change calculation

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Zhou et al., 2020. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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