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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)|
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