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LUNG Publication-derived

Tropism, Replication Competence, and Innate Immune Responses of Influenza Virus in Human Airway Organoids and Ex-Vivo Bronchus Cultures

Source Hui et al., 2018 · Li Ka Shing Faculty of Medicine, The University of Hong Kong · 10.1101/318444

👤 Kenrie P Y Hui, Rachel H H Ching, Stan K H Chan, John M Nicholls, Norman Sachs, Hans Clevers, J S Malik Peiris, Michael C W Chan ⏱ 72 days 📋 10 phases 🧫 Human lung stem cells derived from non-tumour lung tissue

Abstract

This protocol describes the derivation of human airway organoids from lung stem cells and their use to model influenza A virus infection and innate immune responses. The three-dimensional organoid cultures contain ciliated cells, goblet cells, club cells, and basal cells arranged in physiologically relevant architecture similar to human airways. The protocol enables comparative analysis of viral tropism, replication competence, and cytokine/chemokine induction between human and avian influenza strains in organoids and ex-vivo human bronchus explants.

Cell source
Human lung stem cells derived from non-tumour lung tissue
Application
Disease modeling and pandemic risk assessment of influenza viruses

Protocol overview

41 steps across 10 phases

Isolation and Preparation of Human Lung Cells Day 0
  1. 1 Lung Tissue Collection
  2. 2 Tissue Mincing
  3. 3 Enzymatic Digestion
  4. 4 Tissue Shearing and Filtration
  5. 5 Cell Collection and Concentration
  6. 6 Red Blood Cell Lysis
  7. 7 Cell Pellet Resuspension and Centrifugation
Human Airway Organoid Culture Initiation Day 0–14
  1. 1 Cell Resuspension in Matrigel
  2. 2 Organoid Seeding in Suspension Culture Plates
  3. 3 Addition of Organoid Medium
  4. 4 Initial Culture Incubation
  5. 5 Regular Medium Changes
  6. 6 Organoid Passaging
  7. 7 Organoid Maturation After Passaging
Preparation of Influenza Viruses and Ex-Vivo Bronchus Explants Before infection
  1. 1 Influenza Virus Preparation
  2. 2 Fresh Human Bronchus Tissue Collection
  3. 3 Bronchus Explant Preparation
Influenza Virus Infection of Human Airway Organoids Day of infection
  1. 1 Organoid Extraction from Matrigel
  2. 2 Mechanical Disruption of Organoids
  3. 3 Virus Inoculation
  4. 4 Washing and Re-embedding
  5. 5 Post-Infection Culture
Influenza Virus Infection of Ex-Vivo Human Bronchus Explants Day of infection
  1. 1 Bronchus Fragment Infection Setup
  2. 2 Washing
  3. 3 Air–Liquid Interface Culture Setup
  4. 4 Explant Incubation
Viral Titre Assessment and Sample Collection Hours 1, 24, 48, 72 post-infection
  1. 1 Collection of Culture Supernatants
  2. 2 Viral Titre Determination by TCID50 Assay
Sample Collection for Molecular and Immunohistochemical Analysis 24 h post-infection
  1. 1 Collection of Cell Lysates for mRNA Expression
  2. 2 Fixation of Organoids for Immunohistochemistry
  3. 3 Fixation of Bronchus Explants for Immunohistochemistry
Immunohistochemistry, Immunofluorescence, and Lectin Staining After fixation
  1. 1 Organoid Preparation for Staining
  2. 2 Staining with Cell-Type Specific Markers
  3. 3 Lectin Histochemical Staining for Sialic Acid Distribution
Real-Time PCR Analysis of Cytokine and Chemokine Expression After RNA extraction
  1. 1 RNA Extraction
  2. 2 Reverse Transcription
  3. 3 Real-Time PCR Setup and Amplification
  4. 4 Data Normalization and Analysis
Transmission Electron Microscopy (Optional) During characterization or detailed tropism studies
  1. 1 Sample Preparation and Fixation
  2. 2 Embedding and Sectioning
  3. 3 Negative Staining and TEM Imaging

Full SOP

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Attribution

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

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