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LUNG Publication-derived

3D Alveolosphere Culture and Clonal Expansion of Type 2 Alveolar Epithelial Cells (AEC2s)

Source Barkauskas et al., 2013 · Duke University Medical Center · 10.1172/jci68782

👤 Christina E. Barkauskas, Michael J. Cronce, Craig R. Rackley, Emily J. Bowie, Douglas R. Keene, Barry R. Stripp, Scott H. Randell, Paul W. Noble, Brigid L.M. Hogan ⏱ 17 days 📋 6 phases 🧫 Mouse AEC2 (lineage-labeled SFTPC-positive); Human AEC2 (HTII-280+)

Abstract

This protocol describes the isolation and 3D culture of type 2 alveolar epithelial cells (AEC2s) to form self-renewing alveolospheres containing both mature AEC2s and AEC1 (type 1 alveolar epithelial cell) markers. AEC2s are cocultured with PDGFRA+ lung mesenchymal cells (primarily lipofibroblasts) in Matrigel-based Transwell inserts to generate organoid-like structures demonstrating long-term self-renewal and differentiation capacity.

Cell source
Mouse AEC2 (lineage-labeled SFTPC-positive); Human AEC2 (HTII-280+)
Application
Developmental study; Stem cell characterization; Disease modeling

Protocol overview

17 steps across 6 phases

Cell Isolation and Preparation Day 0
  1. 1 Lung Dissociation and Enzymatic Processing
  2. 2 FACS-Based Cell Sorting for Tomato+ (Tm+) AEC2s
Mesenchymal Cell Isolation (PDGFRA-GFP+ Stromal Cells) Day 0
  1. 3 Isolation of PDGFRA-GFP+ Stromal Cells
3D Coculture Seeding in Transwell Inserts Day 0
  1. 4 Prepare Matrigel-Medium Mixture
  2. 5 Seed AEC2s and Stromal Cells into Transwell Inserts
  3. 6 Add Culture Medium to Lower Chamber and Incubate
Alveolosphere Growth and Differentiation Days 1–17
  1. 7 Monitor Sphere Formation
  2. 8 Change Medium Every Other Day
Harvest and Analysis of Alveolospheres Days 14–17
  1. 9 Fix Cultures for Histology
  2. 10 Dehydration and Paraffin Embedding
  3. 11 Sectioning and Immunohistochemistry
  4. 12 Microscopy and Cell Characterization
  5. 13 Transmission Electron Microscopy (TEM) for Ultrastructure Validation
Passaging and Serial Culture (Optional) Day 14 onwards
  1. 14 Dissociation of Spheres from Matrigel
  2. 15 Trypsin Digestion to Single Cells
  3. 16 FACS Sorting of Tm+ Epithelial Cells
  4. 17 Replating of Passaged Cells

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Barkauskas et al., 2013. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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