Lung epithelial A549 cells (ATCC CCL-185) were grown in Roswell Park Memorial Institute (RPMI) 1640 Medium (Gibco 21875) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 10 mM HEPES (Sigma), 100 U mL-1 penicillin, and 0.1 mg mL-1 streptomycin (Gibco) at 37 °C in a humidified 5% CO2 incubator. Human airway epithelial cells NuLi-1 (ATCC CRL-4011) were grown in PromoCell Small Airway Epithelial Cell Growth Medium (Promocell, C-21070) at 37 °C in a humidified 5% CO2 incubator. Flasks and plates were coated with collagen type IV from placenta (Sigma C-7521). Cells were routinely tested for Mycoplasma contamination.
Small airway epithelial cell growth medium
Manufactured by PromoCell
Sourced in Germany, Switzerland
Small Airway Epithelial Cell Growth Medium is a cell culture medium specifically formulated to support the growth and maintenance of human small airway epithelial cells in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Hsaec, by Lonza (2 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Hepes, by Merck Group (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Hsaepc, by PromoCell (1 mentions)
Pc 3 cells crl 1435, by American Type Culture Collection (1 mentions)
Hacat cells, by Addexbio (1 mentions)
Pneumocult ali medium, by STEMCELL (1 mentions)
206 il 010 cf, by R&D Systems (1 mentions)
Smooth muscle cell growth medium 2, by PromoCell (1 mentions)
6 protocols using small airway epithelial cell growth medium
1
Cultivating Lung and Airway Epithelial Cells
2
SARS-CoV-2 Infection of Airway Epithelial Cells
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Small airway epithelial cells immortalized via expression of telomerase reverse transcriptase (HSAEC-KT CRL-4050, American Type Culture Collection, Manassas, VA) were expanded in culture, using small airway epithelial cell growth medium (PromoCell, Heidelberg, Germany) and infected with SARS-CoV-2 or mock. These distal airway epithelial cells demonstrated gene expression consistent with distal respiratory/alveolar epithelial cells. Additional data on SARS-CoV-2 infection of human epithelial cells can be found in the eMethods (http://links.lww.com/CCX/B117 ).
3
Culturing Human Small Airway Epithelial Cells
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Human small airway epithelial cells (HSAEpCs) were purchased from PromoCell (Heidelberg, Germany). HSAEpCs were cultured in small airway epithelial cell growth medium (PromoCell) in a humidified atmosphere containing 5% CO2 at 37℃.
4
Cell Lines and Culture Conditions
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The following cell lines were utilized in this study pHLF (Lonza Biosciences), hSAEC (Lonza Biosciences), MEF (BNCC100518, ATCC), PC3 cells (CRL-1435™, ATCC), HaCat cells (AddexBio T0020001). Primary human lung fibroblast cells (pHLF) and human small airway epithelium cells (hSAECs) were cultured according to supplier’s details (Lonza, Basel, Switzerland), Small airway epithelial cell growth medium (PromoCell, C-21070), respectively. Ogg1+/+ and Ogg1−/− mouse fibroblast (MF) cells were kindly provided by Dr. Deborah E. Barnes (Imperial Cancer Research Fund, Clare Hall Labs, United Kingdom). MF cells were maintained in DMEM/Ham’s F-12 (3:1) supplemented with 10% fetal bovine serum (FBS), glutamine, penicillin (100 U), and streptomycin (100 µg/mL). PC3 cells (CRL-1435™) maintained in F-12K medium, supplemented with 10% FBS, glutamine, penicillin (100 U), and streptomycin (100 µg/mL). HaCat cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS, glutamine, penicillin (100 U), and streptomycin (100 µg/mL). Cells were routinely tested for mycoplasma contamination. Ogg1 expression in Ogg1+/+ and Ogg1−/− MF cells were characterized by qRT-PCR. Exon 3-4F: 5′-TGGACCTCGACTCATTCAGC-3′; R: 5′-CTTCGAGGATGGCTTTGGCA-3′; Exon 5-6F: 5′-TATGGCAGATTGCCCATCGT-3′; R: 5′-CCAGCATAAGGTCCCCACAG-3′.
5
Differentiation of Bronchial Stem Cells
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Differentiation of BSCs in the ALI was previously described (21 (link), 22 (link)). Briefly, BSCs were seeded on 0.4-μm Transwell membranes in Small Airway Epithelial Cell Growth Medium (PromoCell, catalog no. C-21070). After the BSCs reached confluency, the medium was removed from the top compartment, and Pneumocult-ALI medium (StemCell Technology, catalog no. 05001) was applied in the bottom compartment. The medium was changed every 2 days. For cytokine pretreatment, BSCs in Small Airway Epithelial Cell Growth Medium were treated for 7 days with IL6 alone (10 ng/ml; R&D Systems, catalog no. 206-IL-010/CF) or a cocktail containing IL6, IFN-γ (R&D Systems, catalog no. 285-IF-100/CF), and IFNλ2 (Sigma-Aldrich, catalog no. SRP3060), each cytokine at 5 and 10 ng/ml. After treatment, BSCs were passaged (splitting ratio, 1:4) followed by differentiation in the ALI. For treatment with S3I-201 (20 μM; Selleck Chemicals, catalog no. S1155), the ALI culture was treated from Day 0 to Day 7, fixed on Day 21 when epithelial differentiation was deemed complete, and processed for cryosection and antibody staining (18 (link), 27 (link)).
6
Airway Cell Culture and Stimulation
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Small airway epithelial cells (SAECs) and bronchial smooth muscle cells (BSMCs) from healthy subjects were purchased from Lonza. SAEC were cultured in Small Airway Epithelial Cell Growth Medium (Promocell) as described by the provider. BSMCs were cultured in Smooth Muscle Cell Growth Medium 2 (Promocell). Both media were supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin. All cultures were maintained at 37°C in an atmosphere containing 5% CO2. SAEC and BSMC were stimulated with IL‐13 (20 ng/ml) to induce the expression and secretion of periostin and RhoA.
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