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LUNG Publication-derived

Long-term expanding human airway organoids for disease modeling

Source Sachs et al., 2019 · Oncode Institute, Hubrecht Institute-KNAW and UMC Utrecht, Utrecht, The Netherlands · 10.15252/embj.2018100300

👤 Norman Sachs, Angelos Papaspyropoulos, Domenique D Zomer-van Ommen, Inha Heo, Lena Böttinger, Hans Clevers ⏱ 21 days 📋 8 phases 🧫 Human adult lung epithelial cells (from broncho-alveolar lavage or resection tissue); Patient-derived iPSC (Cystic Fibrosis); Patient-derived lung cancer cells (NSCLC)

Abstract

This protocol describes methods to establish long-term-expanding human airway organoids (AOs) from broncho-alveolar resections or lavage material. The pseudostratified airway organoids contain basal cells, multi-ciliated cells, mucus-producing secretory cells, and club cells, enabling modeling of hereditary (cystic fibrosis), malignant (lung cancer), and infectious (RSV) pulmonary diseases with potential for personalized drug screening.

Cell source
Human adult lung epithelial cells (from broncho-alveolar lavage or resection tissue); Patient-derived iPSC (Cystic Fibrosis); Patient-derived lung cancer cells (NSCLC)
Application
Disease modeling and drug screening for cystic fibrosis, lung cancer, and respiratory viral infections

Protocol overview

46 steps across 8 phases

Tissue Processing Day 0
  1. 1 Solid lung tissue mincing and initial digestion
  2. 2 Sequential shearing and filtration
  3. 3 First wash and centrifugation
  4. 4 Red blood cell lysis (if visible red pellet)
  5. 5 Broncho-alveolar lavage fluid processing
  6. 6 BAL fluid mild shearing and filtration
  7. 7 BAL fluid wash and centrifugation
Organoid Culture Initiation and Expansion Day 0–14 (first passage cycle)
  1. 1 Cell resuspension in BME
  2. 2 BME drop seeding
  3. 3 Culture medium addition
  4. 4 Incubation in humidified CO₂ incubator
  5. 5 Medium changes every 4 days
  6. 6 Organoid passaging every 2 weeks
  7. 7 Post-dissociation washing and reseeding
  8. 8 Analysis after passage
Lung Cancer Tumoroid Selection (Optional) Day 0–7 (enrichment phase)
  1. 1 Nutlin-3a selection for TP53 wild-type elimination
  2. 2 Manual separation of tumor from normal organoids
Cystic Fibrosis Functional Assay (Organoid Swelling) Day 7–10
  1. 1 Organoid preparation for swelling assay
  2. 2 CFTR modulator pre-incubation (optional)
  3. 3 CFTR inhibitor pre-treatment
  4. 4 Calcein loading and stimulation
  5. 5 Live-cell confocal imaging and analysis
Air–Liquid Interface (ALI) Differentiation Day 0–21
  1. 1 Organoid dissociation to single cells
  2. 2 Transwell seeding
  3. 3 Air–liquid interface exposure
  4. 4 Differentiation phase
  5. 5 CFTR function measurement by Ussing chamber
RSV Infection Protocol Day 0–7 (infection and monitoring)
  1. 1 Organoid preparation for infection
  2. 2 Virus adsorption
  3. 3 Virus removal and washing
  4. 4 Infected organoid reseeding
  5. 5 Palivizumab control (optional)
  6. 6 RSV replication monitoring by qPCR
  7. 7 Morphological analysis by light and electron microscopy
  8. 8 Time-lapse microscopy for cell motility and organoid rotation
Neutrophil Co-Culture with RSV-Infected Organoids Day 3–4 (post-RSV infection)
  1. 1 Neutrophil isolation from whole blood
  2. 2 Red blood cell lysis
  3. 3 Co-seeding with RSV-infected AOs
  4. 4 Real-time imaging of neutrophil recruitment
  5. 5 Quantification of neutrophil attachment
Drug Screening on Tumor Organoids Day 5–10 (assay period)
  1. 1 Organoid dissociation and resuspension
  2. 2 384-well seeding
  3. 3 Drug dispensing
  4. 4 Incubation with drugs
  5. 5 Cell viability assay
  6. 6 Data analysis and IC50 calculation

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Sachs et al., 2019. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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