Trachea-Derived Progenitor Cell Isolation

Two fresh tracheas were used for cell isolation. Samples were washed with 1X PBS + 0.5% ATB and transferred to a petri dish for tissue digestion (collagenase, Type I, 1 mg/m, #17018029, ThermoFisher) for 1 hour and a half at 37°C. Following, they were centrifuged (1200 rpm, clinical centrifuge) and culture medium (α-MEM, LGC Biotechnology) was added, supplemented with 10% fetal bovine serum (FBS) and 1% ATB, and incubated at 37°C in a humid atmosphere. YS cells were previously described by the group [21 (link),22 (link)]. Both lineage were cultured in 35 mm (Corning) petri dish with α-MEM medium at same conditions as above mentioned. At 80% confluence, cells were trypsinized (0,25%, LGC Bio) for subsequent freezing in complete medium (FBS) containing dimethylsulfoxide (10% DMSO, (CH₃)₂SO, LGC Bio) and preserved in liquid nitrogen (NL₂).