Long-Term Culture of Distal Airway Epithelial Cells Allows Differentiation Towards Alveolar Epithelial Cells Suited for Influenza Virus Studies

Imai-Matsushima, Aki; Martin-Sancho, Laura; Karlas, Alexander; Imai, Seiichiro; Zoranovic, Tamara; Hocke, Andreas C.; Mollenkopf, Hans-Joachim; Berger, Hilmar; Meyer, Thomas F.

Four days after passaging, DAECs in co-culture were treated with small molecules and growth factors to induce differentiation toward alveolar epithelial cells: 3 μM CHIR99021 (Merck Millipore)/10 ng/ml keratinocyte growth factor (Peprotech)/100 μM 3-isobutyl-1-methylxanthine (Sigma-Aldrich)/100 μM 8-bromoadenosine 3′,5′-cyclic monophosphate (Sigma-Aldrich)/25 nM dexamethasone (Sigma-Aldrich)/10 ng/ml fibroblast growth factor-10 (Peprotech) [22] and 20 μM DBZ. Three days later cells were separated from feeders and subjected to downstream experiments. For further details see Supplementary Materials and Methods. A/Vietnam/1203/2004 (H5N1) was tested in a BSL3 laboratory. For titration of viral progeny 50 μl supernatant was transferred to MDCK cells seeded in 96-well plates. After 6 h cells were fixed with 3.7% formaldehyde, immunolabeled with anti-influenza A nucleoprotein (NP) antibody (BIO-RAD) followed by anti-mouse IgG secondary antibody (Dianova). Images were acquired using automated microscopy and analyzed by ScanR Analysis Software (Olympus).