3D Culture of Lung Alveolar Cells
In Brief
Youk et al. describe a protocol for the establishment of feeder-free, long-term 3D cultures of human lung alveolar type 2 (hAT2) cells in chemically-defined conditions and their use in the study of SARS-CoV-2 infection.
Distal parenchymal tissue from healthy donor lungs was dissociated and hAT2 cells were isolated. Isolated HTII-280+ cells were resuspended in a mixture of base media and growth factor-reduced (GFR) basement membrane extract (BME, e.g. Cultrex™) at a ratio of 1:1 in 24-well transwell inserts. Alveolar media was added to the lower chamber and cells were maintained under standard culture conditions with media changes every 2-3 day. ROCK inhibitor Y-27632 was included in the media for the first 48 h to promote cell survival. For 3D culture, cells were transferred to 48-well plates, and embedded in GFR-BME.
Under these conditions, hAT2 cells self-organize into 3D cellular structures with heterogeneous size and morphology by around day 35, including both folded and cystic-like structures consisting of mature hAT2 cells expressing pro-surfactant protein C (pro-SFTPC) HTII-280, and ABCA3. Cells in 3D cultures could be passaged multiple times and at different days depending on size.
Cocktails
| Base Media | Alveolar Media (Base Media + B27) | ||
|---|---|---|---|
| DMEM/F-12 (Cat.No. M23350) | human R-Spondin 1 (Cat.No. 4645RS) | 10% | |
| HEPES (Cat.No. 3173) | 10 mM | human EGF (Cat.No. 236-EG) | 50 ng/mL |
| Nicotinamide (Cat.No. 4106) | 10 mM | human KGF (Cat.No. 251-KG/CF) | 100 ng/mL |
| N-Acetylcysteine (Cat.No. 5619) | 1 mM | human FGF-10 (Cat.No. 345-FG/CF) | 100 ng/mL |
| Penicillin/Streptomycin (Cat.No. B21210) | 1 U/mL | human Noggin (Cat.No. 6057-NG/CF) | 100 ng/mL |
| SB 431542 (Cat.No. 1614) | 10 μM | ||
| CHIR 99021 (Cat.No. 4423) | 3 μM | ||
| Y-27632 (Cat.No. 1254) | 10 μM (day 1 and 2 only) | ||
| Amphotericin B (Cat.No. 6930) | 250 ng/mL (days 1 -5) | ||
| Gentamicin (Cat.No. 6442) | 50 μg/mL (days 1 -5) | ||
Reference
Youk et al. (2020) Three-dimensional human alveolar stem cell culture models reveal infection response to SARS CoV- 2. Cell Stem Cell 27 1.
PMID: 33142113