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INTESTINAL Publication-derived

COVID-19 Intestinal Organoids

Full SOP with Academic or a plan

Source Lamers et al. · Hubrecht Institute and Erasmus MC, Utrecht/Rotterdam, Netherlands · 10.1126/science.abc1669

Authors: Lamers MM, Beumer J, van der Vaart J, Knoops K, Puschhof J, Breugem TI, Ravelli RBG, van Schayck JP, Mykytyn AZ, Duimel HQ, van Donselaar E, Riesber S, Kuijpers J, Peters PJ, Haagmans BL, Clevers H Duration: 35 days 14 phases Cell source: Primary (Human Intestinal Biopsy-Derived Organoids)

Abstract

Demonstrated productive SARS-CoV-2 infection of human intestinal organoids targeting ACE2-expressing mature enterocytes. The study explained gastrointestinal symptoms in COVID-19 patients and established organoids as a BSL-3 platform for studying intestinal coronavirus infection and screening antivirals.

Cell source
Primary (Human Intestinal Biopsy-Derived Organoids)
Application
COVID-19 Intestinal Research

Protocol overview

77 steps across 14 phases

Phase 1 · Vero E6 Cell Maintenance and Virus Propagation Ongoing (for viral stock preparation)
  1. a Maintain Vero E6 cells in culture
  2. b Propagate SARS-CoV and SARS-CoV-2 on Vero E6 cells
  3. c Clear and store viral stocks
  4. d Determine viral stock titers by TCID50
Phase 2 · Human Small Intestinal Organoid Isolation and Expansion Days 0-7
  1. a Obtain and process intestinal tissue
  2. b Isolate human small intestinal cells
  3. c Maintain organoids in expansion medium
Phase 3 · Organoid Differentiation to Specific Cell Lineages Days 7-21
  1. a Generate enteroendocrine cells (EECs) via doxycycline induction
  2. b Perform general differentiation (DIF)
  3. c Activate BMP signaling for enhanced differentiation
Phase 4 · SARS-CoV and SARS-CoV-2 Infection of Small Intestinal Organoids Days 1-3 post-infection
  1. a Prepare organoids for infection
  2. b Wash organoids before viral inoculation
  3. c Infect organoids with virus
  4. d Remove unbound virus
  5. e Re-embed organoids in Matrigel
  6. f Culture infected organoids
  7. g Sample infected organoids at indicated timepoints
  8. h Store samples
Phase 5 · Human Airway Organoid Isolation, Culture, and Differentiation Days 1-35
  1. a Obtain and prepare lung tissue
  2. b Enzymatically digest bronchial tissue
  3. c Shear and filter digested tissue
  4. d Prepare Matrigel droplets
  5. e Solidify Matrigel and add medium
  6. f Maintain airway organoid cultures
  7. g Dissociate organoids into single cells
  8. h Seed cells on Transwell membranes
  9. i Establish air-liquid interface culture
  10. j Change medium during air-liquid interface culture
  11. k Prepare differentiated cultures for infection
Phase 6 · SARS-CoV and SARS-CoV-2 Infection of Differentiated Airway Cultures Days 1-3 post-infection
  1. a Prepare airway cultures for infection
  2. b Inoculate airway cultures from apical side
  3. c Allow virus adsorption
  4. d Remove unbound virus
  5. e Sample airway cultures at indicated timepoints
  6. f Prepare samples for viral titer determination
Phase 7 · Viral Titer Determination by TCID50 and qRT-PCR Days 1-7 post-infection
  1. a Determine live virus titers by TCID50
  2. b Lyse and extract RNA from samples for qRT-PCR
  3. c Purify viral RNA using magnetic beads
  4. d Perform qRT-PCR to detect viral RNA
Phase 8 · Immunofluorescence Microscopy on Differentiated Airway Cultures Days 1-3 post-infection
  1. a Fix airway cultures
  2. b Permeabilize fixed cultures
  3. c Block non-specific binding
  4. d Incubate with primary antibodies
  5. e Wash and incubate with secondary antibodies
  6. f Wash and stain nuclei
  7. g Mount samples for microscopy
  8. h Image on confocal microscope
Phase 9 · Immunostaining of Human Small Intestinal Organoids Days 1-3 post-infection
  1. a Stain organoids as previously described
  2. b Incubate with secondary antibodies
  3. c Mount organoid sections
  4. d Image organoids on confocal microscope
Phase 10 · Quantitative PCR Analysis of Gene Expression Days 1-3 post-infection
  1. a Design and validate qPCR primers
  2. b Perform qPCR analysis of ACE2
  3. c Perform qPCR analysis of interferon-stimulated genes
Phase 11 · Bulk RNA Sequencing Days 1-14 (library prep and sequencing)
  1. a Extract total RNA
  2. b Perform library preparation using CEL-seq protocol
  3. c Perform paired-end sequencing
  4. d Process and align sequencing reads
  5. e Perform differential gene expression analysis
  6. f Perform functional enrichment analysis
  7. g Deposit sequencing data to GEO
Phase 12 · Transmission Electron Microscopy Days 1-5 (fixation through imaging)
  1. a Chemically fix organoids
  2. b Wash fixed organoids
  3. c Post-fix in osmium tetroxide
  4. d Rinse and dehydrate organoids
  5. e Embed in Epon resin
  6. f Polymerize Epon
  7. g Cut ultrathin sections
  8. h Mount sections on grids
  9. i Post-stain sections
  10. j Acquire TEM images
Phase 13 · Multiplex Cytokine ELISA Days 1-3 post-infection
  1. a Prepare organoid supernatant samples
  2. b Determine cytokine concentrations
  3. c Inactivate capture beads
  4. d Wash inactivated beads
  5. e Complete staining protocol
Phase 14 · Statistical Analysis Days 1-7 (data analysis)
  1. a Analyze viral titer and cytokine data

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Attribution

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

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