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INTESTINAL Publication-derived

COVID-19 Small Intestinal Organoids

Full SOP with Academic or a plan

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

Authors: 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 Duration: 72 days 8 phases Cell source: 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

Phase 1 · Bat intestinal tissue collection and crypt isolation Day 0
  1. a Bat euthanasia and intestinal tissue harvesting
  2. b Intestinal tissue chopping and chelation buffer incubation
  3. c Crypt pellet isolation by centrifugation
Phase 2 · Matrigel embedding and expansion phase Days 0-7
  1. a Matrigel droplet formation in 24-well plate
  2. b Addition of expansion medium
  3. c Culture maintenance in expansion phase
  4. d Organoid passaging (1:2 ratio every 7 days)
Phase 3 · Organoid differentiation phase Days 0-4 (from start of differentiation)
  1. a Medium replacement with differentiation medium
  2. b Differentiation culture incubation
  3. c Verification of differentiation
Phase 4 · Virus isolation and inoculation Day 1 (of infection experiment)
  1. a Preparation of clinical specimens
  2. b Mechanical shearing of differentiated organoids
  3. c Organoid-specimen co-incubation
  4. d Re-embedding in Matrigel and culture in differentiation medium
  5. e Incubation and observation
Phase 5 · Viral load quantification by RT-qPCR At designated timepoints during infection
  1. a Collection of cell-free culture media
  2. b Viral RNA extraction
  3. c One-step RT-qPCR assay setup
  4. d RT-qPCR cycling protocol
  5. e Data analysis and viral load calculation
Phase 6 · Viral titration by TCID50 assay At designated timepoints during infection
  1. a Preparation of serial virus dilutions
  2. b Inoculation of Vero cell monolayers
  3. c Culture incubation and CPE observation
  4. d TCID50 calculation
Phase 7 · Immunofluorescence staining for viral nucleocapsid protein (NP) At designated timepoints (e.g., 24, 48, or 72 hpi)
  1. a Fixation of infected organoids
  2. b Washing and permeabilization
  3. c Blocking and primary antibody incubation
  4. d Secondary antibody incubation
  5. e Counterstaining and mounting
  6. f Confocal microscopy imaging
Phase 8 · Gene expression analysis by RT-qPCR At designated timepoints (e.g., 24 or 48 hpi)
  1. a Organoid harvesting and RNA extraction
  2. b Reverse transcription
  3. c qPCR setup and amplification (individual targets)
  4. d Multiplex PCR array (optional, for comprehensive cytokine profiling)
  5. e Data analysis and fold-change calculation

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Attribution

This SOP was written by Organthis based on the published method in Zhou et al., 2020. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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