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.

Expansion medium was based on F-medium [24] with modifications: 3:1 mixture of Ham's F-12 nutrient mix (Life Technologies) and DMEM supplemented with 5% fetal calf serum/0.4 μg/ml hydrocortisone (Sigma-Aldrich)/5 μg/ml recombinant human insulin (Sigma-Aldrich)/8.4 ng/ml cholera toxin (Sigma-Aldrich)/24 μg/ml adenine (Sigma-Aldrich)/10 ng/ml recombinant human epidermal growth factor (Life Technologies)/9 μM Y27632 (Miltenyi Biotec), supplemented with 10 μg/ml ciprofloxacin (Bayer Vital) and 10 μg/ ml vancomycin (Serva) for the first three days, and 2.5 μg/ml amphotericin B (Cayman Chemical, Ann Arbor, USA) for the first seven days. Flasks were maintained in a humidified incubator at 37°C with 5% CO 2 , and medium changed and 1.5-2 × 10 4 /cm 2 freshly irradiated feeders supplemented every 2-3 days.