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LUNG Publication-derived

Alveolar Organoids for Disease and Development

Full SOP with Academic or a plan

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

Authors: 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 Duration: 17 days 6 phases Cell source: 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

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

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Attribution

This SOP was written by Organthis based on the published method in Barkauskas et al., 2013. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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