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LUNG Publication-derived

Non-Small Cell Lung Cancer Airway Organoids

Full SOP with Academic or a plan

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

Authors: Norman Sachs, Angelos Papaspyropoulos, Domenique D Zomer-van Ommen, Inha Heo, Lena Böttinger, Hans Clevers Duration: 21 days 8 phases 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)

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

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

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Attribution

This SOP was written by Organthis based on the published method in Sachs et al., 2019. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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