Skip to content
← Back to browse
LUNG Publication-derived

Differentiated Human Airway Organoids for Influenza Virus Infectivity Assessment

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

👤 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 ⏱ 72 days 📋 9 phases 🧫 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

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

Full SOP

🔬

Create a free account to access this protocol

Join OrganMatch to unlock step-by-step procedures, reagent concentrations, QC checklists, and downloadable batch record templates.

Create free account

Already registered? Log in

Attribution

This SOP was authored by Organthis based on the published method in Zhou et al., 2018. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

This wording is awaiting legal review.

Something wrong with this entry? Report an issue with this protocol

Need a commercial licence?
Use this protocol in your therapeutic or diagnostic pipeline.
Request Commercial Licence