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LUNG Publication-derived

Development of a Novel Air–Liquid Interface Airway Tissue Equivalent Model for In Vitro Respiratory Modeling Studies

Source Leach et al., 2023 · Wake Forest School of Medicine, Wake Forest Institute for Regenerative Medicine · 10.1038/s41598-023-36863-1

👤 Timothy Leach, Uma Gandhi, Kimberly D. Reeves, Kristina Stumpf, Kenichi Okuda, Frank C. Marini, Stephen J. Walker, Richard Boucher, Jeannie Chan, Laura A. Cox, Anthony Atala, Sean V. Murphy ⏱ 30 days 📋 7 phases 🧫 Human iPSC-derived bronchial epithelial cells (HBE) and primary human lung fibroblasts

Abstract

This protocol describes the development of a 3D organ tissue equivalent (OTE) model of the human airway using an air–liquid interface culture system. The model integrates native lung fibroblasts, solubilized human lung extracellular matrix, and a photocrosslinkable hydrogel substrate with tunable stiffness and porosity to recapitulate the physiological microenvironment of bronchial tissue. The OTE model supports well-differentiated human bronchial epithelial (HBE) cultures with mature cell phenotypes including ciliated, goblet, club, and basal cells.

Cell source
Human iPSC-derived bronchial epithelial cells (HBE) and primary human lung fibroblasts
Application
Disease modeling and developmental study of airway epithelium

Protocol overview

42 steps across 7 phases

Lung Tissue Decellularization and Solubilized ECM Preparation Days -30 to 0 (prior to culture)
  1. 1 Source non-diseased human lung tissue
  2. 2 Decellularize lung tissue
  3. 3 Lyophilize and cryomill decellularized tissue
  4. 4 Enzymatic digestion of cryomilled tissue
  5. 5 Quantify solubilized ECM components
  6. 6 Characterize growth factors in sECM
  7. 7 Aliquot and freeze sECM
Hydrogel Synthesis and Characterization Days -14 to -1 (prior to culture)
  1. 1 Prepare photoinitiator solution
  2. 2 Mix hydrogel base components with sECM and photoinitiator
  3. 3 Add crosslinker to achieve desired stiffness
  4. 4 Measure hydrogel elastic modulus via rheometry
  5. 5 Measure hydrogel pore size via scanning electron microscopy
  6. 6 Verify stiffness consistency across batches
Cell Culture Preparation Days -7 to 0
  1. 1 Culture native human lung fibroblasts
  2. 2 Culture primary human bronchial epithelial cells
  3. 3 Expand cells to required density
  4. 4 Prepare fibroblast-hydrogel mixture for OTE fabrication
Three-Dimensional Organ Tissue Equivalent (OTE) Fabrication Day 0
  1. 1 Prepare culture inserts
  2. 2 Pipette hydrogel with fibroblasts onto inserts
  3. 3 UV crosslink hydrogel
  4. 4 Add basal media post-crosslinking
  5. 5 Incubate OTEs for 2 days post-crosslinking
Epithelial Cell Seeding and Liquid Culture Phase (Days 2–4) Day 2 to Day 4
  1. 1 Seed HBEs onto OTE hydrogel surface
  2. 2 Maintain HBE cultures in liquid phase
  3. 3 Monitor cell confluency and TEER
  4. 4 Assess epithelial confluence readiness
Air–Liquid Interface (ALI) Culture Phase (Days 4–28) Day 4 to Day 28
  1. 1 Transition cultures to air–liquid interface
  2. 2 Maintain ALI cultures with basal media only
  3. 3 Weekly apical surface washing
  4. 4 Measure TEER throughout ALI phase
  5. 5 Assess epithelial morphology and confluency
  6. 6 Culture termination and sample collection at Day 28
Sample Preparation and Analysis Day 28 (endpoint)
  1. 1 Prepare samples for histology and immunofluorescence
  2. 2 Perform hematoxylin and eosin staining
  3. 3 Perform multispectral immunofluorescence staining
  4. 4 Quantify differentiation markers via Nuance software
  5. 5 Prepare samples for RNA extraction
  6. 6 Extract total RNA
  7. 7 Prepare cDNA libraries
  8. 8 Perform RNA-sequencing
  9. 9 Process and analyze sequencing data
  10. 10 Perform pathway enrichment analysis

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Leach et al., 2023. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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