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LUNG Publication-derived

Airway Bioprinted and Engineered Tissues for Disease Modelling

Full SOP with Academic or a plan

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

Authors: 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 Duration: 30 days 7 phases Cell source: 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

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

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Attribution

This SOP was written by Organthis based on the published method in Leach et al., 2023. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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