Dispase

Mouse lungs were perfused with HBSS (Gibco) followed by instillation of dispase
(BD Biosciences) into the lung through the trachea and incubation in dispase for
40 minutes as previously described56 (link). Trachea and
large airways were dissected and the remaining distal lung tissue was
homogenized (GentleMACS, MACS Miltenyi Biotech) in DMEM/2.5% HEPES with 0.01%
DNase (Serva) and filtered through 100 μm and
40 μm nylon filters. Cell suspensions were incubated
with biotinylated rat anti-mouse CD45, CD16/32 and CD31 mAb (BD
Biosciences) for 30 minutes at 37 °C
followed by incubation with biotin-binding magnetic beads and magnetic
separation to deplete leukocytes and endothelial cells prior to flow cytometric
analysis.

Alveolar epithelial cells were isolated from the lungs of Sox2^SPC-rtTA mice by Dispase (BD, Pharmingen) digestion as described previously with few modifications [43] (link). Lungs were exsanguinated by perfusing through the right ventricle with 4 ml PBS after opening the peritoneum, clipping the vena cava inferior and removing the ribcage. 1 ml Dispase (BD, Pharmingen) was instilled over a tracheal cannula into the lung, immediately a sterilized suture (Braun) was used to tighten a node around the cannulised trachea. Lungs were isolated, incubated for 45 minutes in 1 ml Dispase at room temperature and transferred to a culture dish containing 5 ml DMEM/F12 medium (Gibco) supplemented with 0.04 mg/ml DNase I (AppliChem), 3.6 mg/ml D-(+)-Glucose (AppliChem) and 1% Penicillin/Streptomycin (P/S). The small airways were gently removed and the obtained cell suspension was serially filtered through 100, 70 and 40 µm nylon meshes and centrifuged at 200 g for 10 minutes at 15°C. The supernatant was discarded and the cell pellet was resuspended in 500 µl DMEM/F12 (Gibco) medium supplemented with 3.6 mg/ml D-(+)-Glucose (AppliChem), 1% P/S and 2% FCS. AVTII cells were cultured in DMEM/F10 containing 10% FCS and 1% P/S in tissue culture cover slip immersed in 12 well plates (Corning, NY) previously coated with collagen (Inamed); cultures were maintained in a 5% CO₂/air incubator.

Three allanto-amniotic membranes were obtained at the term of a normal and physiological pregnancy, transported at 4 °C in phosphate-buffered saline solution (PBS) supplemented with 4 mg/mL amphotericin B (Euroclone), 100 U/mL penicillin (Euroclone) and 100 mg/mL streptomycin (Euroclone), and were processed within 8 h of collection. Amniotic membranes were mechanically separated from the overlaying allantois and cut into small sections of 9 cm² each, for a total weight of approximately 12 g and an extension of 630 cm². Then, amniotic fragments were incubated for 9 min at 38.5 °C in PBS containing 2.4 U/mL dispase (Becton Dickinson & Company, Milan, Italy). After a final incubation of 5–10 min at room temperature in HG-DMEM (high-glucose Dulbecco’s modified Eagle’s medium, Euroclone) supplemented with 10% fetal bovine serum (FBS) and 2 mM L-glutamine, fragments were digested with 1 mg/mL collagenase type I and 20 µg/mL DNase (Roche, Mannheim, Germany) for 3 h at 38.5 °C.
Fragments were then removed, and the products of enzymatic digestion were passed through a 100 µm filter. Dissociated cells were collected through centrifugation at 250× g for 10 min.
Isolated cells were defined as AMSCs.