3D Culture of Lung Alveolar Cells

This is intended as a guide only; for full experimental details please read the reference provided.

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.



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)

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