Culture of human alveolar epithelial type II cells by sprouting

Human lung tissue was obtained with approval of the Human Ethics Committee of the University of Basel (EKBB 05/06) and written informed consent was obtained from all patients who underwent lung biopsy.

Epithelial cell cultures were established from lung tissue obtained from patients undergoing diagnostic or therapeutic video-assisted thoracoscopic surgery (VATS) performed at the Division of Thoracic Surgery or undergoing flexible bronchoscopy with transbronchial biopsy at the Clinics of Respiratory Medicine, University Hospital Basel, Switzerland. In patients with lung tumors, lung tissue for cell culture was obtained from the macroscopically normal part away from the tumor.

Lung tissue was cut into small pieces and those were placed into cell culture flasks for cell sprouting containing supplemented epithelial growth medium (Cnt-17) (CELLnTEC Advanced Cell System AB; Bern, Switzerland). AT2 cells were grown under standard conditions (37 °C, 21% O₂, 5% CO₂). Complete epithelial culture medium was replaced every fourth day. All experiments were performed using non-passaged cells. Primary human lung fibroblasts cultured in DMEM/10% FCS were used as negative controls [14].

Immunofluorescence analysis of AT2 cells or fibroblasts was performed as previously described [15]: cells were fixed with 4% formalin (10 min), and permeabilized with methanol/acetic acid (3:1, ice-cold, 10 min). Cells were blocked with 5% BSA (1 h), and then incubated with TRIC-Phalloidin (Sigma) for 1 h at room temperature or antibodies against E-cadherin (Abcam, Santa Cruz, LabForce AG), pan-cytokeratin (Sigma-Aldrich, USA), calponin (Abcam), surfactant protein C (SP-C) (ThermoFisher Scientific), overnight at 4 °C in moist box; for LysoTracker® Green DND-26 (Cell Signaling Technologies, USA) cells were incubated for 30 min at 37 degree. The primary antibodies were detected by addition of Alexa Fluor 488 donkey anti-rabbit, Alexa Fluor 594 goat anti-rabbit or Alexa Fluor donkey anti-mouse IgG (all ThermoFisher Scientific) for 1 h. To visualise the nuclei 4,6-diamido-2-phenylindole (Sigma) was added (5 min) and cells were subsequently examined on a fluorescence microscope Leica DMI4000B (Leica, Germany).

For live cell imaging of fluorescent surfactant associated protein B (SP-B)uptake and secretion, 10 μg of recombinant SP-B (Cloud-Clone Corp, USA) was labelled using BODIPY® FL NHS Ester (ThermoFischer, USA) as described [16] and incubated in a 5% CO2 environment. 8 μg/ml of the resulting fluorescent SP-B were added to each well for 30 min and then rinsed with PBS. The first baseline picture was taken. Later, 100 μM of a diacylglycerol analog (DAG; 1-Oleoyl-2-acetyl-sn-glycerol; Sigma Aldrich,USA) was added and pictures were taken after 5, 15 and 30 min using a Leica DMI4000B (Leica, Germany). The fluorescence intensity was quantified using Image J software (NIH, USA), and data is presented as mean fluorescence intensity.

Total RNA was extracted from AT2 cells with a Quick-RNA MiniPrep Kit (ZymoResearch, Orange, CA) from four different epithelial cell lines. RNA levels were determined by real-time PCR using an SP-A (Hs00831305_s1), SP-B (Hs01090667_m1), SP-C (Hs00161628), SP-D (Hs01108490_m1), or GAPDH (Hs03929097_g1) TaqMan® Gene Expression Assay (all Applied Biosystems, Foster City, CA). Samples were run at 50 °C for 2 min, 1 cycle; 95 °C for 10 min, 1 cycle; 95 °C for 15 s, 60 °C for 1 min, 40 cycle and quantified by the ΔΔCt calculation method.

Cells were grown to confluency in 6-well plates and were fixed with 2.5% glutaraldehyde in 0.15 M Hepes buffer (707 mOsm, pH 7.4) and stored in the fixative solution. The cells were further rinsed with the same fixative and postfixed for 1 h with a solution of 0.1 M sodium cacodylate buffer with 1% solution of osmium teratoxide (369 mOsm, pH 7.4). After rinsing with 0.05 M maleate buffer (pH 5.0), cells were again postfixed in a solution of 0.05 M maleate buffer containing 0.5% uranyl acetate. After 1 h of incubation, cells were dehydrated in consequently increasing concentrations of ethanol (70%, 80%, 96%, 100%) and then left in a mixture of ethanol and epon (1:1) overnight. On the following day, cells were embedded in epon resin and left to polymerize at 60 °C for 6 days. Ultrathin sections (70 nm) were cut with a Reichert-Jung Ultracut E microtome, put on formyar coated 2 mm × 1 mm single slot copper grids and double stained with 1% uranyl acetate and 3% lead citrate. EM pictures were taken with the Philips EM 400 transmission electron microscope, using Morada digital camera and iTEM/cell^F program.

Alveolar epithelial cells from two different donors were used (i.e. sample-1 and sample-2). The cells were lysed with Pierce IP lysis buffer (Thermo Fischer Scientific, Waltham, MA, USA) according to the manufacturer’s specifications. Protein content was analyzed with Bradford assay (BIO-RAD, Hercules, CA, USA) as described in the manufacturer’s protocol. 10 μL of loading buffer (LI-COR, Lincoln, NE, USA) containing 10% β-Mercapto-ethanol (Sigma-Aldrich, St Louis, MI, USA) were added to the samples and incubation at 95 °C for 5 min followed. Samples were shortly centrifuged and were loaded on 12% gels (BIO-RAD, Hercules, CA, USA). A protein molecular weight marker was added (LI-QOR, Lincoln, NE, USA). Gel electrophoresis was performed for 20 min at 80 V and 1 h at 150 V. Gels were transferred to Nitrocellulose membranes (BIO-RAD) by Trans-Blot Turbo Transfer System (BIO-RAD). B-actin (LI-QOR, Lincoln, NE, USA) was used as a control. SP-C (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and β–Actin were added at 1:1000 dilutions and gels were incubated overnight at 4 °C, protected from light with gentle shaking. Gels were washed and incubated with secondary antibodies for 1 h in the dark at room temperature. Secondary antibodies were purchased from LI-COR (IRDye-680RD and IRDye-800RD) and visualization was done with a LI-COR Odyssey scanner.

AT2 cells were serum deprived for 24 h. The cell supernatants were collected and centrifuged at 1200 rpm for 10 min. Epithelial growth media was used as control. Phospholipid secretion was measured by a Phospholipid Assay Kit (Sigma, Aldrich USA) according to the manufacturer’s instructions and data were expressed as concentration of phospholipids in μM.