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LUNG Publication-derived

Airway Organoids for Infection Studies

Full SOP with Academic or a plan

Source Zhou et al., 2018 · The University of Hong Kong · 10.1073/pnas.1806308115

Authors: Jie Zhou, Cun Li, Norman Sachs, Man Chun Chiu, Bosco Ho-Yin Wong, Hin Chu, Vincent Kwok-Man Poon, Dong Wang, Xiaoyu Zhao, Lei Wen, Wenjun Song, Shuofeng Yuan, Kenneth Kak-Yuen Wong, Jasper Fuk-Woo Chan, Kelvin Kai-Wang To, Honglin Chen, Hans Clevers, Kwok-Yung Yuen Duration: 72 days 9 phases Cell source: Human ASC-derived airway organoids from normal lung tissue

Abstract

This protocol describes the generation and proximal differentiation of long-term expanding human airway organoids (AOs) from adult stem cells and their conversion to 2D monolayers. The differentiated AOs recapitulate human airway epithelium morphology and function with elevated ciliated cells (~40%), synchronously beating cilia, and elevated serine protease expression. These models enable rapid assessment of emerging influenza virus infectivity in humans.

Cell source
Human ASC-derived airway organoids from normal lung tissue
Application
Disease modeling and viral infectivity assessment

Protocol overview

38 steps across 9 phases

Phase 1 · Establishment and Maintenance of ASC-Derived Human Airway Organoids Ongoing (passages every 2-3 weeks)
  1. a Tissue collection and dissociation
  2. b Cell straining and preparation
  3. c Matrigel embedding and culture initiation
  4. d Organoid passage
Phase 2 · Proximal Differentiation of Airway Organoids Day 0-16
  1. a Transition to proximal differentiation medium
  2. b Maintain organoids in PD medium for 16 days
  3. c Monitor ciliated cell differentiation
Phase 3 · Generation of 2D Differentiated Airway Monolayers Day 0-14 post-seeding
  1. a Dissociate organoids into single-cell suspension
  2. b Seed cells into Transwell inserts
  3. c Transition to proximal differentiation medium
  4. d Measure transepithelial electrical resistance (TEER)
  5. e Assess epithelial barrier integrity by dextran penetration assay
Phase 4 · Influenza Virus Preparation Varies (72 hours for MDCK; 36 hours for embryonated eggs)
  1. a Propagation of H1N1pdm and H7N9 viruses in MDCK cells
  2. b Propagation of avian viruses in embryonated chicken eggs
  3. c Determine viral titer by plaque assay
Phase 5 · Influenza Virus Infection of 3D Differentiated Airway Organoids Day 0-4 post-inoculation
  1. a Prepare organoids for viral inoculation
  2. b Inoculate organoids with virus
  3. c Re-embed organoids in Matrigel
  4. d Optional serine protease inhibitor treatment
  5. e Harvest samples for viral load quantitation
Phase 6 · Influenza Virus Infection of 2D Differentiated Airway Monolayers Day 0-3 post-inoculation
  1. a Prepare 2D monolayers for apical viral inoculation
  2. b Inoculate monolayers from the apical side
  3. c Harvest apical and basolateral media
  4. d Prepare samples for immunofluorescence staining
Phase 7 · Viral Load Quantitation Concurrent with infection experiments
  1. a Extract RNA from organoid and monolayer samples
  2. b Synthesize cDNA
  3. c Perform quantitative PCR (qPCR)
  4. d Perform viral titration by plaque assay
Phase 8 · Immunofluorescence Staining and Characterization Concurrent with other phases
  1. a Fix organoids or monolayers
  2. b Permeabilize and block
  3. c Incubate with primary antibodies
  4. d Incubate with secondary antibodies and counterstains
  5. e Acquire confocal images
Phase 9 · Flow Cytometry Analysis of Cell-Type Composition Concurrent with characterization phases
  1. a Dissociate organoids
  2. b Fix cells
  3. c Permeabilize cells
  4. d Stain with primary and secondary antibodies
  5. e Analyze by flow cytometry

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Attribution

This SOP was written by Organthis based on the published method in Zhou et al., 2018. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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