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LUNG Publication-derived

Differentiation and characterization of human airway organoids for assessing influenza virus infectivity

Source Zhou et al., 2018 · The University of Hong Kong, Department of Microbiology · 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 📋 5 phases 🧫 Adult stem cell-derived human airway organoids from normal lung tissue adjacent to resected diseased tissue

Abstract

This protocol describes the establishment and proximal differentiation of human airway organoids (AOs) derived from adult stem cells, which develop into pseudostratified ciliated epithelium containing ciliated, goblet, basal, and club cells with elevated serine protease expression. Both 3D differentiated organoids and 2D monolayer variants are utilized to assess the infectivity of emerging respiratory viruses, specifically distinguishing human-infective influenza strains from poorly human-infective strains.

Cell source
Adult stem cell-derived human airway organoids from normal lung tissue adjacent to resected diseased tissue
Application
Disease modeling and infectivity assessment of emerging influenza viruses

Protocol overview

31 steps across 5 phases

Establishment of adult stem cell-derived airway organoids 0-21
  1. 1 Tissue collection and initial preparation
  2. 2 Enzymatic tissue digestion
  3. 3 Mechanical dissociation and cell straining
  4. 4 Cell embedding in Matrigel
  5. 5 Initial organoid culture maintenance
Proximal differentiation of airway organoids 0-16
  1. 1 Organoid splitting and pre-differentiation culture
  2. 2 Media switch to proximal differentiation medium
  3. 3 Proximal differentiation culture period
  4. 4 Capture of cilia beating and morphological assessment
  5. 5 Harvest organoids for analysis at day 16
Establishment of 2D differentiated airway organoid monolayers 0-14
  1. 1 Dissociation of 3D organoids into single-cell suspension
  2. 2 Seeding cells into Transwell inserts
  3. 3 Initial culture in standard AO medium
  4. 4 Switch to proximal differentiation medium
  5. 5 Culture maintenance and medium replacement
  6. 6 Measure transepithelial electrical resistance (TEER)
  7. 7 Assess epithelial barrier integrity at day 12
Influenza virus infection of differentiated airway organoids 0-72
  1. 1 Preparation of 3D organoids for virus inoculation
  2. 2 Virus inoculation of 3D organoids
  3. 3 Post-inoculation washing of 3D organoids
  4. 4 Re-embedding of inoculated 3D organoids
  5. 5 Harvest infected organoids and supernatants at specified timepoints
  6. 6 Serine protease inhibitor treatment (optional validation)
  7. 7 Virus inoculation of 2D organoid monolayers
  8. 8 Harvest media from 2D monolayers
  9. 9 Immunofluorescence staining for virus-infected cells
Viral load detection and quantification 0-3
  1. 1 RNA extraction from organoids and supernatants
  2. 2 cDNA synthesis
  3. 3 Quantitative RT-PCR for viral gene copy number
  4. 4 RT-qPCR for cellular gene expression
  5. 5 Viral titration by plaque assay

Full SOP

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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.

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